This publication was supported by Programa de Apoyo a Proyectos de Investigaci&#243;n e Innovaci&#243;n Tecnol&#243;gica (PAPIIT), UNAM [IN223623 to XP-M]. UPD is a CONAHCYT fellow (CVU:883299). We want to thank Dr. Ariann Mendoza-Mart&#237;nez for technical help with the light microscope images. Biorender licenses: DU26OMVLUU (Figure 2); BK26TH9GXH (Figure 3); GD26TH80R5 (Figure 4); PU26THARYD (Figure 7); ML26THAIFG (Figure 9).

NOTE: We recommend making six transformations for each construct since mitochondrial transformation efficiency is usually low. The composition of the different growth media is shown in Table 2.
1. Tungsten particle preparation
<ol>
	<li>Weigh 30 mg of 0.7 &#181;m tungsten particles (WPs, microcarriers) in a microtube. Add 1.5 mL of 70% ethanol (EtOH) to sterilize. Vortex the WPs and let them rest for 10 min at room temperature.</li>
	<li>Centrifuge at 13,200 x g for 15 min at room temperature. Wash the WPs by adding 1.5 mL of sterile water to the particles, vortexing them, and centrifuging at 13,200 &#215; g for 15 min at room temperature. Discard the supernatant.</li>
	<li>Add 500 &#181;L of sterile 50% glycerol (final concentration is 60 &#181;g of WPs/&#181;L). 
	NOTE: WPs can be stored at -20 &#176;C for at least 6 months. From this stock, take the required amount for six transformations (100 &#181;L).</li>
</ol>
2. WPs DNA coating
<ol>
	<li>Add 5 &#181;g of the 2 &#181; plasmid and 15 &#181;g of the bacterial plasmid containing the mitochondrial sequence to a microtube on ice. 
	NOTE: The final volume must not exceed 50 &#181;L to avoid further diluting the reagents needed for DNA coating.</li>
	<li>Add 100 &#181;L of the WP suspension and vortex for 5 s (6 mg of WPs).</li>
	<li>Add 4 &#181;L of 1 M spermidine. Briefly vortex.</li>
	<li>Add 100 &#181;L of ice-cold 2.5 M CaCl2, briefly vortex. Incubate on ice for 10 min; briefly vortex the mix every 3 min. 
	NOTE: The CaCl2 solution is filter-sterilized and must not be autoclaved.</li>
	<li>Centrifuge the WPs for 30 s at 13,200 &#215; g at 4 &#176;C and discard the supernatant. Wash the particles by gently resuspending them in 200 &#181;L of 100% EtOH at -20 &#176;C.</li>
	<li>Centrifuge the WPs for 30 s at 13,200 &#215; g at 4 &#176;C and discard the supernatant. Repeat the wash.</li>
	<li>Resuspend the WPs in 60-70 &#181;L of room temperature 100% EtOH using a pipette tip; do not vortex. 
	NOTE: To ensure the quality of the plasmids, DNA coating should be performed on the same day as biolistic transformation.</li>
</ol>
3. Biolistic material preparation
<ol>
	<li>Before bombardment, sterilize six macrocarrier holders (Figure 5) in an autoclave and thoroughly dry them. Alternatively, soak them in EtOH and sterilize them using a flame with the help of forceps. Place them on a sterile glass plate.</li>
	<li>During DNA precipitation (10 min incubation on ice in step 2.4), insert six macrocarriers (or flying discs) in each one of the six macrocarrier holders with the help of sterile forceps. 
	NOTE: Do not sterilize the macrocarriers.</li>
	<li>Mix the DNA-coated WPs with the pipette. Add 10 &#181;L of the WPs to each macrocarrier surface and distribute over the entire surface with the pipette tip. If necessary, add a few extra microliters of 100% EtOH to the macrocarrier surface to make an even spreading of the WPs. Let the suspension dry at room temperature for ~20 min.</li>
</ol>
4. Preparation of yeast cells and bombardment plates
<ol>
	<li>Inoculate the rho0 receptor strain on 2 mL of YPR medium (Table 2). Grow for one night at 30 &#176;C in a rotating wheel.</li>
	<li>Dilute 300 &#181;L of the culture in 30 mL of fresh YPR medium. Grow at 30 &#176;C and 200 rpm on a rotary shaker for two nights until the culture is saturated.</li>
	<li>Centrifuge the yeast cells at 600 &#215; g for 5 min at room temperature. Discard the supernatant and resuspend the yeast cells in 600 &#181;L of YPD medium (Table 2).</li>
	<li>Using a sterile glass handle, spread 100 &#181;L of the yeast cell suspension on a solid bombardment medium plate. 
	NOTE: The bombardment medium must lack the selection marker of the 2 &#181; plasmid that will be used for transformation.</li>
	<li>Repeat step 4.4 for the other five plates.</li>
	<li>Incubate for 1-3 h at room temperature before bombardment to ensure the liquid medium is completely absorbed on the plate&#39;s surface.</li>
</ol>
5. Generation of a synthetic rho- strain by biolistic transformation
NOTE: Before beginning, be sure to read the user&#39;s manual for the biolistic equipment. We will describe the protocol of the referenced equipment (Table of Materials and Figure 5).
<ol>
	<li>Clean the chamber of the biolistic particle delivery system with 70% ethanol to prevent contamination and let it stand for at least 10 min. Just before bombardment, wipe the surface with a clean paper towel.</li>
	<li>Open the helium tank valves to set around 2,000 psi.</li>
	<li>Power on the equipment and turn on the vacuum source.</li>
	<li>Using forceps, place the macrocarrier facing down on the macrocarrier holder. 
	NOTE: Do not use the stopping screen that comes with the equipment.</li>
	<li>Screw the macrocarrier lid on top of the macrocarrier. All parts together constitute the microcarrier launch system (Figure 5).</li>
	<li>Place the microcarrier launch system inside the vacuum chamber at the fifth level from the bottom.</li>
	<li>Place one of the plates containing the rho0 strain lawn on the second level from the bottom of the chamber. Ensure that the plate faces up and is without the lid.</li>
	<li>Using sterile forceps, put one rupture disk on the retaining cap. Screw the retaining cap on the acceleration tube; make sure that the retaining cap is snug. 
	NOTE: Do not treat the ruptured discs with isopropanol as suggested in the manual since we have observed that transformation efficiency decreases.</li>
	<li>Close the door of the vacuum chamber.</li>
	<li>Put the VAC/VENT/HOLD switch in the VAC position. When 25-28 inHg vacuum is reached, change the VAC/VENT/HOLD switch to the HOLD position. 
	NOTE: Be aware that achievable vacuum can vary according to sea level. We found success reaching 25 inHg.</li>
	<li>Press the FIRE switch to shoot the WPs. Wait for the pressure to build in the acceleration tube until it reaches 1,300 psi when the rupture disk will break, making a pop sound.</li>
	<li>Immediately after the rupture disk breaks, the FIRE switch and the vacuum are released by changing the VAC/VENT/HOLD switch to the VENT position.</li>
	<li>Carefully, with sterile forceps, remove any debris of the rupture disk present on the surface of the plate and cover the plate with its lid.</li>
	<li>Repeat the procedure for the remaining five plates, placing a new rupture disk and macrocarrier in each round.</li>
	<li>At the end, close the main valve of the helium tank.</li>
	<li>Close the chamber and build a vacuum by taking the VAC/VENT/HOLD switch to the VAC position. Release the system from any helium pressure by pressing and releasing the FIRE switch a few times. Repeat with the door open.</li>
	<li>Turn off the vacuum pump and the bombardment equipment.</li>
	<li>Clean the delivery system with 70% EtOH. Wash the macrocarrier supports with a 0.1% SDS solution; rinse with sterile distilled water and 100% EtOH.</li>
</ol>
6. Synthetic rho- colony selection
<ol>
	<li>Incubate the bombardment plates for 5-7 nights at 30 &#176;C. 
	NOTE: It is important to check the plates daily for contamination. If necessary, the contaminated region can be cut out with a sterile spatula to avoid further contamination.</li>
	<li>Inoculate the tester strain of the opposite mating type in 2 mL of YPD medium overnight (see the specific example in Figure 2D,E).</li>
	<li>Inoculate 150 &#181;L of the tester strain culture on a YPD plate. Using a sterile glass handle, spread the tester evenly on the plate surface. Let the plate dry for at least 5 min.</li>
	<li>Make replicas of the bombardment plates using velvet replicating pads and a replicator stamp.
	<ol>
		<li>Replicate on a dropout medium plate corresponding to the transformed 2 &#181; plasmid auxotrophy marker. Growth takes one overnight incubation at 30 &#176;C. Afterward, store at 4 &#176;C until further use. This plate is the master plate.</li>
		<li>Replicate on the YPD plate containing the tester&#180;s lawn from step 6.3. Incubate at 30 &#176;C for two nights. Afterward, replicate this plate on the selective medium to detect the presence of the mitochondrial gene of interest, for example, respiratory medium or a medium lacking arginine, and incubate overnight at 30 &#176;C. Only the cells that harbored the synthetic bacterial plasmid in mitochondria will grow on the selective medium after mating with the tester strain.</li>
	</ol>
	</li>
	<li>Identify on the master plate (step 6.4.1) the corresponding colonies that grew in step 6.4.2. 
	NOTE: Since the bombardment plate (and thus, the master plate) is crowded with transformant colonies, it can be difficult to identify those positive colonies observed on the tester&#39;s mating plate. For this reason, it is highly recommended to draw a couple of small lines on each side of all plates with a permanent marker. Draw similar lines on the ring of the replicating stamp. These lines will be helpful in orienting and locating positive colonies on the master plate (see the example in Figure 2D).</li>
	<li>Purify the positive colonies by streaking them from the master plate to a second selective dropout plate, as in step 6.4.1. Incubate for two nights at 30 &#176;C. 
	NOTE: It is essential that streaking allows the growth of single colonies to ensure a pure strain.</li>
	<li>Repeat the selection process (steps 6.2-6.5) to confirm the presence of the bacterial plasmid in mitochondria. 
	NOTE: Usually, this process can be repeated two more times to select stable synthetic rho- strains.</li>
	<li>Once the synthetic rho- strain stably maintains the plasmid DNA in mitochondria, select 3-5 different positive colonies and inoculate them in 2 mL of YPD. Use this culture to prepare a cryovial stock (by mixing 600 &#956;L of culture and 600 &#956;L of 50% glycerol to preserve at -70 &#176;C) and to insert the plasmid sequence into the mtDNA of interest by cytoduction (step 6.7). 
	NOTE: Since there is no selective pressure to maintain the transformed plasmid in the mitochondria from the synthetic rho- strain, the plasmid can be easily lost. Therefore, we recommend making the cytoduction as soon as possible.</li>
</ol>
7. Integration of the construction present in the synthetic rho- strain into the target site in the mitochondrial genome by cytoduction
<ol>
	<li>Inoculate two tubes with 3 mL of YPD media, one with the donor cells (synthetic rho- cells) and one with the acceptor cells (yeast cell where the final construction is expected). Incubate overnight at 30 &#176;C.</li>
	<li>Mix 750 &#181;L of the donor strain and 250 &#181;L of the receptor strain in a sterile microtube (3:1 ratio).</li>
	<li>Centrifuge the mixture for 1 min at 13,200 &#215; g; discard the supernatant. Using a 1 mL pipette tip with the end cut off, take one drop of the cell pellet and place it on a YPD plate. Incubate at 30 &#176;C for 2-4 h. 
	NOTE: To avoid cross-contamination, do not put more than two cell suspensions in a single plate (see the example in Figure 4A).</li>
	<li>Take a small amount of the cell mixture, resuspend it in 15 &#181;L of sterile water on a slide, and place a cover slip. Check under a light microscope for the presence of shmoos with a 40x or 100x objective; this characteristic shape shows up when two mating types are present (Figure 3B).</li>
	<li>If shmoos are present, resuspend a small amount (~0.5 mm3) of the cell mass in 2 mL of YPD medium. Incubate for 2 h at 30 &#176;C in a rotating wheel to allow the binuclear zygotes to recover.</li>
	<li>Vortex the mix and dilute 10 &#181;L of the cell suspension in 990 &#181;L of sterile water (1:100 dilution). Use glass beads or a sterile glass handle to spread 100 &#181;L of the cell dilution on a dropout medium where only the acceptor strain (and sporadic diploids) grow. Incubate for 2-3 nights at 30 &#176;C.</li>
	<li>Replicate the resulting colonies on the necessary selective media as explained in the introduction (stage 4; see Figure 4C for a specific example).</li>
	<li>After identification of positive colonies, restreak them on the same selective media to purify the haploid strain carrying the mtDNA of interest.</li>
	<li>Select at least two positive colonies and grow them on 2 mL of YPD medium at 30 &#176;C overnight. Make a cryogenic backup by mixing 600 &#181;L of 50% glycerol with 600 &#181;L of the saturated YPD culture. Store at -70 &#176;C.</li>
	<li>Isolate DNA of the resulting strains31,32. Amplify the desired region by PCR and sequence to confirm the presence of the mitochondrial mutation. 
	NOTE: Each positive colony is a technical replicate that must be confirmed independently to ensure the correct sequence in the mitochondrial genome.</li>
</ol>

Biolistic Transformation of Yeast Mitochondria for Mutagenetic Analysis

<ol>
	<li>Bonnefoy, N., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+mutated+initiation+codons+in+the+mitochondrial+COX2+gene+of+Saccharomyces+cerevisiae+fused+to+the+reporter+gene+ARG8m+reveals+lack+of+downstream+reinitiation.">In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation.</a> Mol Gen Genet. 262 (6), 1036-1046 (2000).</li><li>Franco, L. V. R., Su, C. H., McStay, G. P., Yu, G. J., Tzagoloff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cox2p+of+yeast+cytochrome+oxidase+assembles+as+a+stand-alone+subunit+with+the+Cox1p+and+Cox3p+modules.">Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules.</a> J Biol Chem. 293 (43), 16899-16911 (2018).</li><li>Flores-Mireles, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytochrome+b+carboxyl+terminal+region+is+necessary+for+mitochondrial+complex+III+assembly.">The cytochrome b carboxyl terminal region is necessary for mitochondrial complex III assembly.</a> Life Sci Alliance. 6 (7), (2023).</li><li>Rubalcava-Gracia, D., V&#225;zquez-Acevedo, M., Funes, S., P&#233;rez-Mart&#237;nez, X., Gonz&#225;lez-Halphen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+versus+nuclear+gene+expression+and+membrane+protein+assembly:+the+case+of+subunit+2+of+yeast+cytochrome.">Mitochondrial versus nuclear gene expression and membrane protein assembly: the case of subunit 2 of yeast cytochrome.</a> Mol Biol Cell. 29 (7), 820-833 (2018).</li><li>Garc&#237;a-Villegas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cox1+C-terminal+domain+is+a+central+regulator+of+cytochrome+c+oxidase+biogenesis+in+yeast+mitochondria.">The Cox1 C-terminal domain is a central regulator of cytochrome c oxidase biogenesis in yeast mitochondria.</a> J Biol Chem. 292 (26), 10912-10925 (2017).</li><li>Rak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+cells+lacking+the+mitochondrial+gene+encoding+the+ATP+synthase+subunit+6+exhibit+a+selective+loss+of+complex+IV+and+unusual+mitochondrial+morphology.">Yeast cells lacking the mitochondrial gene encoding the ATP synthase subunit 6 exhibit a selective loss of complex IV and unusual mitochondrial morphology.</a> J Biol Chem. 282 (15), 10853-10864 (2007).</li><li>Perez-Martinez, X., Broadley, S. A., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mss51p+promotes+mitochondrial+Cox1p+synthesis+and+interacts+with+newly+synthesized+Cox1p.">Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p.</a> EMBO J. 22 (21), 5951-5961 (2003).</li><li>Sanchirico, M. E., Fox, T. D., Mason, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+mitochondrially+synthesized+Saccharomyces+cerevisiae+Cox2p+and+Cox3p+depends+on+targeting+information+in+untranslated+portions+of+their+mRNAs.">Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs.</a> EMBO J. 17 (19), 5796-5804 (1998).</li><li>Saracco, S. A., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cox18p+is+required+for+export+of+the+mitochondrially+encoded+Saccharomyces+cerevisiae+Cox2p+C-tail+and+interacts+with+Pnt1p+and+Mss2p+in+the+inner+membrane.">Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail and interacts with Pnt1p and Mss2p in the inner membrane.</a> Mol Biol Cell. 13 (4), 1122-1131 (2002).</li><li>McStay, G. P., Su, C. H., Thomas, S. M., Xu, J. T., Tzagoloff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+assembly+intermediates+containing+subunit+1+of+yeast+cytochrome+oxidase.">Characterization of assembly intermediates containing subunit 1 of yeast cytochrome oxidase.</a> J Biol Chem. 288 (37), 26546-26556 (2013).</li><li>Golik, P., Bonnefoy, N., Szczepanek, T., Saint-Georges, Y., Lazowska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rieske+FeS+protein+encoded+and+synthesized+within+mitochondria+complements+a+deficiency+in+the+nuclear+gene.">The Rieske FeS protein encoded and synthesized within mitochondria complements a deficiency in the nuclear gene.</a> Proc Natl Acad Sci U S A. 100 (15), 8844-8849 (2003).</li><li>Franco, L. V. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allotopic+expression+of+COX6+elucidates+Atco-driven+co-assembly+of+cytochrome+oxidase+and+ATP+synthase.">Allotopic expression of COX6 elucidates Atco-driven co-assembly of cytochrome oxidase and ATP synthase.</a> Life Sci Alliance. 6 (11), e202301965 (2023).</li><li>Mireau, H., Arnal, N., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Barstar+as+a+selectable+marker+in+yeast+mitochondria.">Expression of Barstar as a selectable marker in yeast mitochondria.</a> Mol Genet Genomics. 270 (1), 1-8 (2003).</li><li>Cohen, J. S., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+green+fluorescent+protein+from+a+recoded+gene+inserted+into+Saccharomyces+cerevisiae+mitochondrial+DNA.">Expression of green fluorescent protein from a recoded gene inserted into Saccharomyces cerevisiae mitochondrial DNA.</a> Mitochondrion. 1 (2), 181-189 (2001).</li><li>Suhm, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+system+to+monitor+mitochondrial+translation+in+yeast.">A novel system to monitor mitochondrial translation in yeast.</a> Microb Cell. 5 (3), 158-164 (2018).</li><li>Rzepka, M., Suhm, T., Ott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+reporter+genes+into+mitochondrial+DNA+in+budding+yeast.">Incorporation of reporter genes into mitochondrial DNA in budding yeast.</a> STAR Protoc. 3 (2), 101359 (2022).</li><li>Steele, D. F., Butler, C. A., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+recoded+nuclear+gene+inserted+into+yeast+mitochondrial+DNA+is+limited+by+mRNA-specific+translational+activation.">Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation.</a> Proc Natl Acad Sci U S A. 93 (11), 5253-5257 (1996).</li><li>Flores-Mireles, D., Camacho-Villasana, Y., P&#233;rez-Mart&#237;nez, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ARG8m+reporter+for+the+study+of+yeast+mitochondrial+translation.">The ARG8m reporter for the study of yeast mitochondrial translation.</a> Methods Mol Biol. 2661, 281-301 (2023).</li><li>Perez-Martinez, X., Butler, C. A., Shingu-Vazquez, M., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+functions+of+Mss51+couple+synthesis+of+Cox1+to+assembly+of+cytochrome+c+oxidase+in+Saccharomyces+cerevisiae+mitochondria.">Dual functions of Mss51 couple synthesis of Cox1 to assembly of cytochrome c oxidase in Saccharomyces cerevisiae mitochondria.</a> Mol Biol Cell. 20 (20), 4371-4380 (2009).</li><li>Bonnefoy, N., Remacle, C., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Saccharomyces+cerevisiae+and+Chlamydomonas+reinhardtii+mitochondria.">Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria.</a> Methods Cell Biol. 80, 525-548 (2007).</li><li>Veloso Ribeiro Franco, L., Barros, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biolistic+transformation+of+the+yeast+Saccharomyces+cerevisiae+mitochondrial+DNA.">Biolistic transformation of the yeast Saccharomyces cerevisiae mitochondrial DNA.</a> IUBMB Life. 75 (12), 972-982 (2023).</li><li>Bonnefoy, N., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+alteration+of+Saccharomyces+cerevisiae+mitochondrial+DNA+by+biolistic+transformation+and+homologous+recombination.">Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination.</a> Methods Mol Biol. 372, 153-166 (2007).</li><li>Butow, R. A., Henke, R. M., Moran, J. V., Belcher, S. M., Perlman, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Saccharomyces+cerevisiae+mitochondria+using+the+biolistic+gun.">Transformation of Saccharomyces cerevisiae mitochondria using the biolistic gun.</a> Methods Enzymol. 264, 265-278 (1996).</li><li>Bonnefoy, N., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Saccharomyces+cerevisiae+mitochondria.">Genetic transformation of Saccharomyces cerevisiae mitochondria.</a> Methods Cell Biol. 65, 381-396 (2001).</li><li>Shing&#250;-V&#225;zquez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carboxyl-terminal+end+of+Cox1+is+required+for+feedback+assembly+regulation+of+Cox1+synthesis+in+Saccharomyces+cerevisiae+mitochondria.">The carboxyl-terminal end of Cox1 is required for feedback assembly regulation of Cox1 synthesis in Saccharomyces cerevisiae mitochondria.</a> J Biol Chem. 285 (45), 34382-34389 (2010).</li><li>Bonnefoy, N., Bsat, N., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+translation+of+Saccharomyces+cerevisiae+COX2+mRNA+is+controlled+by+the+nucleotide+sequence+specifying+the+pre-Cox2p+leader+peptide.">Mitochondrial translation of Saccharomyces cerevisiae COX2 mRNA is controlled by the nucleotide sequence specifying the pre-Cox2p leader peptide.</a> Mol Cell Biol. 21 (7), 2359-2372 (2001).</li><li>Meunier, B., Lemarre, P., Colson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screening+in+Saccharomyces+cerevisiae+for+large+numbers+of+mitochondrial+point+mutations+which+affect+structure+and+function+of+catalytic+subunits+of+cytochrome-c+oxidase.">Genetic screening in Saccharomyces cerevisiae for large numbers of mitochondrial point mutations which affect structure and function of catalytic subunits of cytochrome-c oxidase.</a> Eur J Biochem. 213 (1), 129-135 (1993).</li><li>Dorweiler, J. E., Manogaran, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoduction+and+plasmiduction+in+yeast.">Cytoduction and plasmiduction in yeast.</a> Bio Protoc. 11 (17), 4146 (2021).</li><li>Zakharov, I. A., Yarovoy, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoduction+as+a+new+tool+in+studying+the+cytoplasmic+heredity+in+yeast.">Cytoduction as a new tool in studying the cytoplasmic heredity in yeast.</a> Mol Cell Biochem. 14 (1-3), 15-18 (1977).</li><li>Conde, J., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutant+of+Saccharomyces+cerevisiae+defective+for+nuclear+fusion.">A mutant of Saccharomyces cerevisiae defective for nuclear fusion.</a> Proc Natl Acad Sci U S A. 73 (10), 3651-3655 (1976).</li><li>Burke, D., Dawson, D., Stearns, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harbor Laboratory. Methods in yeast genetics : a Cold Spring Harbor Laboratory manual. 2000 ed. , (2000).</li><li>Dunham, M. J., Gartenberg, M. R., Brown, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+:+a."> : a.</a> Methods in yeast genetics and genomics : a Cold Spring Harbor Laboratory course manual. 2015 edition. , (2015).</li><li>Ding, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27+An+improved+method+for+introducing+point+mutations+into+the+mitochondrial+cytochrome+B+gene+to+facilitate+studying+the+role+of+cytochrome+B+in+the+formation+of+reactive+oxygen+species.">Chapter 27 An improved method for introducing point mutations into the mitochondrial cytochrome B gene to facilitate studying the role of cytochrome B in the formation of reactive oxygen species.</a> Methods Enzymol. 456, 491-506 (2009).</li><li>Klein, C. J. L., Olsson, L., Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+control+in+Saccharomyces+cerevisiae:+the+role+of+Mig1+in+metabolic+functions.">Glucose control in Saccharomyces cerevisiae: the role of Mig1 in metabolic functions.</a> Microbiology (Reading). 144, 13-24 (1998).</li><li>Rolland, F., Winderickx, J., Thevelein, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-sensing+and+-signalling+mechanisms+in+yeast.">Glucose-sensing and -signalling mechanisms in yeast.</a> FEMS Yeast Res. 2 (2), 183-201 (2002).</li><li>Gruschke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cbp3-Cbp6+complex+coordinates+cytochrome+b+synthesis+with+bc(1)+complex+assembly+in+yeast+mitochondria.">The Cbp3-Cbp6 complex coordinates cytochrome b synthesis with bc(1) complex assembly in yeast mitochondria.</a> J Cell Biol. 199 (1), 137-150 (2012).</li><li>Seshadri, S. R., Banarjee, C., Barros, M. H., Fontanesi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translational+activator+Sov1+coordinates+mitochondrial+gene+expression+with+mitoribosome+biogenesis.">The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.</a> Nucleic Acids Res. 48 (12), 6759-6774 (2020).</li><li>Rak, M., Tzagoloff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F1-dependent+translation+of+mitochondrially+encoded+Atp6p+and+Atp8p+subunits+of+yeast+ATP+synthase.">F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase.</a> Proc Natl Acad Sci U S A. 106 (44), 18509-18514 (2009).</li><li>He, S., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+a+yeast+mitochondrial+inner+membrane+protein,+pnt1p,+block+export+of+a+mitochondrially+synthesized+fusion+protein+from+the+matrix.">Mutations affecting a yeast mitochondrial inner membrane protein, pnt1p, block export of a mitochondrially synthesized fusion protein from the matrix.</a> Mol Cell Biol. 19 (10), 6598-6607 (1999).</li><li>Rak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mitochondrial+translation+of+the+ATP8/ATP6+mRNA+by+Smt1p.">Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p.</a> Mol Biol Cell. 27 (6), 919-929 (2016).</li><li>Barrera-Paez, J. D., Moraes, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+genome+engineering+coming-of-age.">Mitochondrial genome engineering coming-of-age.</a> Trends Genet. 38 (8), 869-880 (2022).</li></ol>

The authors do not have any conflicts of interest to disclose.

The present work described how to transform mitochondria from the yeast S. cerevisiae successfully. The process, from high-velocity microprojectile bombardment until purification of the intended yeast strain, takes ~8-12 weeks, depending on how many rounds of purification of the synthetic rho- strain are necessary. Some of the critical steps of the method are as follows. First, the larger the flanking regions added around the mutation site in the mitochondrial gene construct, the higher the probability of successful integration of the target mutation. The minimum size of the flanking regions is ~100 nt; however, using more than 500 nt on each side of the mutation is highly recommended. Second, when selecting the synthetic rho-colonies after transformation, it is recommended that no more than 3-4 rounds of purification take place; otherwise, there is a high risk that eventually the mitochondrial construct will be lost, as there is no selective pressure to keep the plasmid in mitochondria. Third, to avoid harming the user or the equipment, you must carefully read the bombardment machine&#39;s manual before starting the procedure. This equipment is delicate and uses high pressure and vacuum.
One limitation of this procedure is that so far, only yeast and the green algae C. reinhardtii are candidates to transform mitochondria with this method. It would be relevant if we could introduce site-directed mutations on the mitochondrial genome from other organisms. Another limitation is that it is challenging to transform many different constructs in the same session. It is recommended to make six transformations for each construction, each of which must be tested, selected, and purified. This results in over 50 plates of media only to obtain pure rho- colonies of a single mitochondrial mutation.
Despite these limitations, the ability to manipulate the yeast mitochondrial genome has significantly contributed to understanding many pathways of respiratory complexes biogenesis. This approach can be used to introduce reporter genes to monitor and quantify mitochondrial translation1,7,36,37, interrupt mitochondrial genes38, identify assembly chaperones of respiratory complexes39, better understand the mechanisms of coupled mitochondrial translation and assembly7,11,12,36, and look for suppressors when following the expression of a mutated mitochondrial gene40. So far, although certain approaches to manipulate the mitochondrial genome in mammals have been reported earlier41, no widespread method to transform mitochondrial DNA exists for these organisms.

The yeast Saccharomyces cerevisiae is a widely recognized model used to study mitochondrial biogenesis. Since yeast is an anaerobic, facultative organism, it is possible to extensively study the causes and consequences of introducing mutations that impair respiration. In addition, this organism possesses friendly genetic and biochemical tools to study mitochondrial pathways. However, one of the most powerful resources to explore the mechanisms of respiratory complex assembly and mitochondrial protein synthesis is the ability to transform mitochondria and modify the organelle's genome. Previously, it has been helpful to introduce in the mitochondrial DNA (mtDNA) point mutations or small deletions/insertions1,2,3,4,5, delete genes6,7, make gene rearrangements7,8, add epitopes to mitochondrial proteins9,10, relocate genes from the nucleus to the mitochondria11,12, and introduce reporter genes like BarStar13, GFP14,15, luciferase16, and the most widely used ARG8m17,18. Mitochondrial genome modifications have allowed us to dissect and identify mechanisms that otherwise would have been difficult to comprehend. For example, the ARG8m reporter gene inserted at the COX1 locus in the mitochondrial DNA was crucial to understanding that Mss51 has a dual role in Cox1 biogenesis. First, it is a translational activator of the COX1 mRNA, and second, it is an assembly chaperone for the newly made Cox1 protein7,19. This work presents a detailed method to transform S. cerevisiae mitochondria. Although the mitochondrial transformation protocol was published earlier16,20,21,22,23, a visual approach through a video is essential to thoroughly understand the different stages and details of the method. The method consists of various steps and is divided into four general stages:
<img alt="Tungsten particle-DNA coating and bombardment diagram for mitochondrial genome integration." class="xfigimg" src="/files/ftp_upload/66856/66856fig01v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Overview of the mitochondrial transformation procedure by high-velocity microprojectile bombardment: 1) The tungsten particles are sterilized and prepared before DNA coating. 2) Two different plasmids are precipitated on the surface of the WPs. One is a bacterial plasmid containing the construct that will be directed to the mitochondrial matrix. The other is a nuclear yeast expression vector carrying an auxotrophy marker. 3) A receptor yeast strain lacking mitochondrial DNA (rho0) is grown on a fermentative carbon source like galactose or raffinose, which does not exert any glucose repression of mitochondrial gene expression34,35. The culture is spread on petri dishes containing the bombardment medium. 4) WPs coated with plasmids are shot to the receptor strain by high-velocity microprojectile bombardment. 5) Positive synthetic rho- colonies containing the mitochondrial plasmid are selected by mating to a tester strain. 6) The mitochondrial construct is integrated into the mitochondrial genome at the desired locus by mating the synthetic rho- strain (donor) with the acceptor strain by a process known as cytoduction. 7) The positive receptor, haploid strain carrying the mitochondrial construct is selected and purified in different media. Abbreviations: WPs = tungsten particles; mtDNA = mitochondrial DNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Transformation of cells with the DNA construct intended to integrate into the mitochondrial genome (Figure 1, steps 1-4) 
Although it is possible to directly transform a yeast containing a complete mitochondrial genome (rho+), the transformation is 10-20x more efficient if the cells lack mitochondrial DNA (rho0)24. Tungsten particles (WPs) are coated with two different plasmids that will be co-transformed. The first one is a yeast 2 µ expression vector carrying an auxotrophy marker, such as LEU2 or URA3 (e.g., YEp351 or YEp352, respectively). If the biolistic introduction of DNA into the yeast cells is successful, the transformants will grow on the auxotrophy medium (i.e., a medium lacking leucine or uracil). This plasmid is helpful in making the first selection of the cells that acquired the nuclear plasmid; otherwise, the number of resulting colonies would saturate the plate. The second plasmid is a bacterial plasmid (such as pBluescript or similar) containing the mitochondrial construction intended to be integrated into the mitochondrial genome. The construct must contain at least 100 nt of 5' and 3' flanking mitochondrial sequences for recombination with the mitochondrial region of interest. In our experience, larger flanking sequences are more likely to successfully recombine with the target locus in the mitochondrial genome.
A specific example is shown in Figure 225. In this example, the aim was to delete the region of the mitochondrial COX1 gene coding for the last 15 amino acids of the corresponding protein (Cox1ΔC15) (Figure 2A). The bacterial plasmid carrying the COX1ΔC15 mutation contained 395 nt and 990 nt of the gene's 5' and 3' untranslated regions, respectively. The plasmid was derived from pBluescript (pXPM61) and, together with the 2 µ plasmid YEp352, they were coprecipitated on the surface of WPs (Figure 2B). The WPs were then introduced by microprojectile bombardment into the selected recipient strain (Figure 2C). This strain, named NAB6926, is a MATa, rho0 strain bearing the kar1-1 and ade2 alleles (the importance of these characteristics is discussed below). The cells were plated on bombardment media lacking uracil to select those that acquired the 2 µ plasmid (Figure 2C). Cells were grown for 5-7 nights at 30 °C.
Selection of the cells that acquired the bacterial plasmid containing the mitochondrial construct and the nuclear 2 µ plasmid carrying the auxotrophy marker (Figure 1, step 5) 
The positive colonies will maintain many copies of the bacterial plasmid in their mitochondria22. Since the transformed cells are originally rho0, no mitochondrial sequences support the translation of the mitochondrial construct present in the plasmid; hence, the transformed mitochondrial gene will not be expressed. It is necessary to mate the transformants with a tester strain to detect the yeast colonies that acquired the mitochondrial plasmid. The mitochondrial genome of the tester strain contains a nonfunctional mutated version of the gene of interest. After mating, the mitochondria will fuse, and the mitochondrial sequence included in the transformed plasmid will recombine with the mutated mitochondrial gene from the tester strain; consequently, the recovery of the WT gene will reconstitute function. The resulting diploid will have a detectable positive phenotype (usually the capacity to grow in respiratory medium or a medium lacking arginine). The positive cells carrying the bacterial plasmid in mitochondria are named "synthetic rho- cells". In the specific example of Figure 2D, synthetic rho- cells carrying the COX1ΔC15 construct (named XPM199) were replica-plated on a medium lacking uracil (this is the master plate from which positive colonies were purified). They were also replica-plated on a tester strain L4527 lawn, which contains the nonfunctional mutation cox1D369N. After mating for two nights, the mitochondrial genome of L45 and XPM199 recombined, resulting in a functional COX1 gene; therefore, the diploids recovered the ability to grow on respiratory media. From the master plate, we picked the positive colonies. They were streaked on plates lacking uracil, and the selective tests were repeated to obtain pure synthetic rho- cells (Figure 2E). It is important to note that the testing plate is only for the identification of synthetic rho- colonies, and mutants cannot be recovered from these plates.
Integration of the construct into the mitochondrial genome of the intended strain 
This step is achieved by a process called cytoduction28,29 (Figure 1, step 6). In this approach, the synthetic rho- strain (donor strain) is mated to the intended acceptor strain of the opposite mating type. An essential requirement is that at least one of the two mating strains carries the kar1-1 mutation to impede nuclear fusion30. Hence, mating of the two cells produces a binucleated zygote (Figure 3). The mitochondrial network from the parental cells (donor and acceptor) fuse and the mitochondrial DNA molecules recombine. The binucleated zygotes are incubated/recovered to allow budding, which will produce haploid cells. These haploids carry the nuclear background of one of the parental cells. Similarly, haploids can carry the mitochondrial DNA from either the parental cell or the recombined mitochondrial DNA of interest. However, the kar1-1 mutation is not 100% effective, and some true diploids will be formed during the mating28,29. The synthetic rho- strain (donor, XPM199) carried the kar1-1 allele in the example. As a selective marker, it had the ade2 allele, making it an auxotroph for adenine (Figure 4). The acceptor strain was XPM10, a rho+ strain bearing the cox1Δ::ARG8m construct, where the reporter ARG8m replaced the COX1 codons in the mitochondrial genome7. The mixture of both cell cultures was added to a YPD plate as a drop to allow mating. After 3-5 h, the cells were observed under a light microscope to detect the formation of shmoos (Figure 3B), a cell shape associated with mating. After the incubation/recovery time, the binuclear zygotes were plated on a medium lacking adenine to prevent the growth of the donor cells.
Selection of the haploid strain carrying the mitochondrial genome of interest (Figure 1, step 7) 
A mixture of donor, acceptor, and diploid cells is present after cytoduction. Therefore, after the incubation/recovery of the binucleated zygotes, cells must be grown in different selective media to identify and purify the haploids of interest. The selective media depends on the genotypes of the donor, acceptor strains, and the intended mitochondrial construction. However, in general, the reasoning for choosing the selective media is: i) after incubation/recovery, the cytoduction mix is grown on selective medium where the donor parental strain cannot grow. In the specific example in Figure 4A,B, the donor (synthetic rho- strain) carries the nonfunctional ade2 allele. Thus, the cytoduction mix must be incubated on a medium plate lacking adenine. This is the master plate. ii) Once the colonies grow, the master plate is replicated again on a medium lacking adenine (to generate a new master plate from where the positive colonies of interest will be purified), and next, on a medium where only diploids can grow. This is necessary to avoid further inadvertent purification of diploid colonies. In the case of the example from Figure 4C, only diploids can grow on media lacking leucine. iii) The master plate is also replicated on media where only those acceptor haploids that incorporated the mitochondrial DNA of interest will grow. In the example of Figure 4C, haploids grow on respiratory media containing ethanol/glycerol as a carbon source since the Cox1ΔC15 protein is functional. The resulting rho+ strain bearing the mtDNA of interest was named XPM20925. The strains used in Figure 2 and Figure 4 are listed in Table 1.
<img alt="Mitochondrial DNA editing diagram; processes Cox1ΔC15, mtDNA recombination, and yeast transformation." class="xfigimg" src="/files/ftp_upload/66856/66856fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Diagram describing a specific example of mitochondrial transformation and selection of positive rho- cells. (A) The intended modification of the mitochondrial genome was a deletion of the region coding for the last 15 amino acids of the Cox1 subunit (Cox1ΔC15). (B) WPs were coated with two plasmids to transform yeast cells. One was the 2 µ plasmid Yep352 that was directed and expressed in the nucleus. The other was plasmid pXPM61, which contains the COX1ΔC15 allele plus 395 nt and 990 nt of the COX1 5' and 3'-UTRs, respectively. (C) The WPs were introduced into cells from the strain NAB69, which lacks mitochondrial DNA (rho0 strain). Transformant colonies obtained from the bombardment plates were replicated on a medium lacking uracil, the auxotrophic marker of the nuclear plasmid YEp352. (D) To select the positive rho- colonies, the -URA plate was replicated on a media lacking uracil. This was the master plate from which the positive colonies were picked and isolated. It was also replicated on plates with rich media (YPD) and a lawn of a tester strain. During mating, the synthetic rho- mitochondrial DNA was recombined with the mitochondrial DNA from the tester strain, L4527, which carries a mutation in the COX1 gene (D369N). The diploids that grew on respiratory media contained the transformed DNA. The corresponding colonies were picked from the master plate to restreak and purify. To help identify the positive colonies in the master plate throughout all replica plating, marks were made with a permanent marker on the edges of the plates (green lines). (E) The selected positive colonies were restreaked on a medium lacking uracil. This was the new master plate. As in D, two more rounds of testing were carried out to purify the synthetic rho- colonies. Abbreviations: WPs = tungsten particles; mtDNA = mitochondrial DNA; -URA/Sorb = lacking uracil/ containing Sorbitol; WT = wild type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Yeast cell mating process; diagram with haploids and microscope image of yeast cells at 10 µm scale." class="xfigimg" src="/files/ftp_upload/66856/66856fig03v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Diagram describing the cytoduction procedure. (A) General overview of how cells mate during cytoduction. During cytoduction, mitochondria from the donor and acceptor strains fuse so mitochondrial DNA from both strains recombine. Since nuclear fusion is reduced due to the kar1-1 mutation30, binuclear zygotes are formed. After some incubation/recovery time, the binuclear zygotes bud and haploid acceptors containing the intended mitochondrial mutation are selected. (B) Mating of the synthetic rho- strain (donor) and the acceptor strain through cytoduction allows the formation of shmoos (red arrows), a characteristic shape of mating yeast. The image was taken under a light microscope with a 100x objective. Scale bar = 10 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mitochondrial DNA recombination diagram with yeast strains; cell mix, selective plating steps." class="xfigimg" src="/files/ftp_upload/66856/66856fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Diagram describing a specific example of cytoduction to integrate the mutation COX1ΔC15 in the mitochondrial genome. (A) Liquid cultures of the synthetic rho- strain (donor, XPM199) and the strain of interest (acceptor, XPM10) were mixed to mate. After shmoo formation, the mix was recovered in a liquid culture for 2-4 h. Next, the cytoduction mix was spread on a plate with a medium lacking adenine to prevent the growth of the donor cells. (B) Schematic representation of the recombination events of the mitochondrial DNA from the donor (XPM199) and the acceptor (XPM10) during cytoduction. (C) The master plate was replicated on different selective media to identify and purify those haploids that integrated the intended mitochondrial gene in the organelle's DNA (XPM209)25. Abbreviations: -ADE = lacking adenine; -LEU = lacking leucine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>1 mL pipette tips</td><td>Axygen</td><td>T-1000-B</td><td></td></tr><tr><td>1.5 mL Microtube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>10 &amp;mu;L pipette tips</td><td>Axygen</td><td>T-10-C</td><td></td></tr><tr><td>15 mL conical bottom tube&amp;nbsp;</td><td>Axygen</td><td>SCT-25ML-25-S</td><td></td></tr><tr><td>200 &amp;mu;L pipette tips</td><td>Axygen</td><td>T-200-Y</td><td></td></tr><tr><td>50 mL conical bottom&amp;nbsp; tube</td><td>Axygen</td><td>SCT-50ML-25-S</td><td></td></tr><tr><td>AfiII</td><td>New England BioLabs</td><td>R0520S</td><td></td></tr><tr><td>Agarose</td><td>SeaKem</td><td>50004</td><td></td></tr><tr><td>Analytic balance</td><td>OHAUS</td><td>ARA520</td><td></td></tr><tr><td>Autoclave</td><td>TOMY</td><td>ES-315</td><td></td></tr><tr><td>Bacto agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Bacto peptone</td><td>BD</td><td>211677</td><td></td></tr><tr><td>Biolisitic Macrocarrier holder&amp;nbsp;</td><td>BIO-RAD</td><td>1652322</td><td></td></tr><tr><td>Bunsen burner</td><td>VWR</td><td>89038-528</td><td></td></tr><tr><td>Calcium chloride</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>CSM -ADE</td><td>Formedium</td><td>DCS0049</td><td></td></tr><tr><td>CSM -ARG</td><td>Formedium</td><td>DCS0059</td><td></td></tr><tr><td>CSM -LEU</td><td>Formedium</td><td>DCS0099</td><td></td></tr><tr><td>CSM -URA</td><td>Formedium</td><td>DCS0169</td><td></td></tr><tr><td>Culture glass flask</td><td>KIMAX KIMBLE</td><td>25615</td><td></td></tr><tr><td>Culture glass tube</td><td>Pyrex</td><td>9820</td><td></td></tr><tr><td>Dextrose</td><td>BD</td><td>215520</td><td></td></tr><tr><td>Ethanol</td><td>JT Baker&amp;nbsp;</td><td>9000</td><td></td></tr><tr><td>Forceps</td><td>Millipore</td><td>620006</td><td></td></tr><tr><td>Glass beads</td><td>Sigma</td><td>Z265926</td><td></td></tr><tr><td>Glass handle</td><td>Sigma</td><td>S4647</td><td></td></tr><tr><td>Glycerol</td><td>JT BAKER</td><td>2136-01</td><td></td></tr><tr><td>Helium tank grade 5 (99.99 %)</td><td>-</td><td>-</td><td></td></tr><tr><td>HSTaq&amp;nbsp; Kit</td><td>PCR BIO</td><td></td><td></td></tr><tr><td>Microcentrifugue</td><td>Eppendorf</td><td>022620100</td><td></td></tr><tr><td>NdeI</td><td>New England BioLabs</td><td>R0111L</td><td></td></tr><tr><td>Orbital shaker</td><td>New Brunswick scientific</td><td>NB-G25</td><td></td></tr><tr><td>PCR tubes</td><td>Axygen</td><td>PCR-02-C</td><td></td></tr><tr><td>PDS-1000/He TM Biolistic Particle Delivery System</td><td>BIO-RAD</td><td>165-2257</td><td></td></tr><tr><td>Petri dishes (100X10)</td><td>BD</td><td>252777</td><td></td></tr><tr><td>QIAprep Spin Miniprep</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>Raffinose</td><td>Formedium</td><td>RAF03</td><td></td></tr><tr><td>Replica plater</td><td>Scienceware</td><td>Z363391</td><td></td></tr><tr><td>Rupture discs 1350 Psi</td><td>BIO-RAD</td><td>1652330</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma</td><td>S7547</td><td></td></tr><tr><td>Spermidine</td><td>Sigma</td><td>S0266</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>Thermo Scientific</td><td>EL0011</td><td></td></tr><tr><td>Tissue Culture Rotator</td><td>Thermo Scientific</td><td>88882015</td><td></td></tr><tr><td>Tungsten microcarriers M10</td><td>BIO-RAD</td><td>1652266</td><td></td></tr><tr><td>Vaccum pump of 100L/min capacity</td><td>-</td><td>-</td><td></td></tr><tr><td>Velvet pads</td><td>Bel-Art</td><td>H37848-0002</td><td></td></tr><tr><td>Vortex&amp;nbsp;</td><td>Scientifc Industries</td><td>SI-0236</td><td></td></tr><tr><td>Wood aplicator stick</td><td>PROMA</td><td>1820060</td><td></td></tr><tr><td>Yeast extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>Yeast Nitrogen base without aminoacids</td><td>BD</td><td>291920</td><td></td></tr></tbody></table>

mitochondrial transformation in baker&#39;s yeast to study translation and respiratory complex assembly

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

This section presents some representative results from the different stages of mitochondrial transformation. Figure 6 shows a bombardment procedure. The synthetic rho- cells carried a bacterial plasmid with the reporter gene ARG8m, which will replace the coding sequence of a mitochondrial gene (Figure 6A). After bombardment, the plate was replicated on a medium lacking uracil (-URA); this is the master plate (Figure 6B). The growing colonies have the yeast expression vector with the URA3 auxotrophy marker. According to protocol section 6, the bombardment plate was also replicated on a YPD medium containing a lawn of the tester strain. In this case, the tester strain harbored a defective allele of the ARG8m gene in the mitochondrial genome (ARG8m-1)1. After 2 nights of incubation, the tester lawn and the transformant cells mated, and the mitochondria fused. This plate was replicated on a medium lacking arginine. Only those diploids containing the mitochondrial plasmid were able to grow on this selective medium because the ARG8m gene present in the synthetic rho- strain replaced the defective ARG8m-1 from the mitochondrial genome of the tester strain. 
The number of positive colonies generally varies from zero to 20 per plate. In the present example, four positive synthetic rho- colonies were obtained, which were purified by streaking them on medium lacking uracil. In the first round of purification, many negative colonies were obtained along with the positive ones. However, after two or three rounds of purification, it is possible to obtain the pure positive synthetic rho- strain. In Figure 6C, we showed a couple of positive synthetic rho- colonies after three rounds of purification and testing. Most colonies from the -URA master plate are positive for the tester probe. Interestingly, not all the cells of a positive colony contain the mitochondrial plasmid, indicating that the transformed plasmid was lost in a population of the colony. This result is expected since there is no selective pressure to keep the mitochondrial plasmid, making it unstable. However, a synthetic rho- strain like those shown in Figure 6C can be taken forward to the next step, cytoduction (protocol section 7), and used to prepare stock to be stored at-70 °C. Figure 6D also shows the third round of purification of a synthetic rho- strain. However, in this case, as the transformants were practically unable to maintain the mitochondrial plasmid, they were discarded.
We described one general strategy to select positive synthetic rho- colonies, which is presented in Figure 7A. In this case, the mitochondrial construct present in the synthetic rho- cells has several hundreds of nucleotides to recombine with the mitochondrial DNA from the tester strain (for example, the construct COX1ΔC15 in the synthetic rho- strain recombines with the mutated gene cox1D369N on the tester's mitochondrial DNA (Figure 2D); or the cox1Δ::ARG8m construct in the synthetic rho- strain with the mutated gene ARG8m-1 in the tester's mitochondrial DNA (Figure 6A). However, if there is no availability of a tester strain to select the positive synthetic rho- cells of interest, there is an indirect approach to select them33. In this case, the bacterial plasmid must also contain a fragment of a mitochondrial gene, like COX2 or COX3. The tester strain contains a nonfunctional copy of the mitochondrial cox2 or cox3 gene. In the given example, the sequence of the cox3 fragment present in the bacterial plasmid can complement the cox3 sequence present in the tester's mitochondria and, after recombination, provide a functional COX3 gene. After crossing the synthetic rho- cells with the tester strain, diploids can respire only if the synthetic rho- cells contain the bacterial plasmid inside mitochondria (Figure 7B).
Figure 8 exemplifies the cytoduction process. The donor and acceptor strains have leucine and adenine auxotrophy, respectively. Hence, after cytoduction, the mix was plated on a medium lacking leucine, preventing the growth of the donor cells. This is the master plate. At this point, only haploids with the nuclear background of the acceptor strain and sporadic diploids will grow. To rule out diploids, the master plate was replicated on media lacking adenine, where only diploids can grow. In this example, the resulting mitochondrial DNA after cytoduction is mutated, and the intended haploids cannot grow on the respiratory medium of ethanol/glycerol. Therefore, the master plate needs to be replicated on YPD plates with a lawn of a tester strain. The position of positive respiring diploids indicates which colonies to purify from the master plate. Two rounds of purification are necessary to obtain the final, pure strain. The mitochondrial integration is usually corroborated by PCR amplification of the surrounding region of interest and sequencing.
Finally, genes can be inserted in the specific locus in the mitochondrial genome. For example, reporter genes can be inserted at the 3' end of a mitochondrial gene's coding sequence or replace the mitochondrial gene's coding region, among other options (Figure 9A). However, genes can also be inserted at ectopic sites within noncoding regions of the mitochondrial genome. The commonly used site is 295 nt upstream of the start codon of the COX2 gene. The alteration at this noncoding region is innocuous13. To successfully express the gene in the ectopic site, it must be flanked by a mitochondrial promoter and terminator. The COX2 flanking sequences are the most optimal since COX2 is the mitochondria gene with the shortest 5' and 3' untranslated sequences (73 nt of the 5' UTR and 119 nt of the 3' UTR) (Figure 9B). For this construction, the bacterial plasmid has the following characteristics: the template is a plasmid that contains the COX2 coding region, with 1120 nt downstream of the stop codon and 670 nt upstream of the AUG start codon, named pXPM5025. The gene of interest is engineered to be flanked by 73 nt of the COX2 5'-UTR and 119 nt of the COX2 3'-UTR. Afterward, it was inserted, as previously indicated, at 295 nt upstream of the COX2 AUG start codon, which is a noncoding region (Figure 9B). Note that this plasmid, and consequently, in the resulting mitochondrial genome, the COX2 5' and 3' UTRs are duplicated. After the bombardment of this plasmid, the synthetic rho- cells are mated for cytoduction with a receptor strain that has an extensive deletion of the COX2 region (cox2-62 allele)1. During cytoduction, the mitochondrial plasmid of the synthetic rho- strain recombines with the mitochondrial DNA from the acceptor. The resulting mitochondrial genome has the ectopic construction and a WT COX2 gene. Therefore, the positive colonies will be able to grow in a respiratory medium (Figure 9B).
<img alt="Helium-driven microcarrier launch system diagram for gene delivery; includes acceleration tube setup." class="xfigimg" src="/files/ftp_upload/66856/66856fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Schematic representation of the high-velocity microprojectile bombardment equipment. Our lab uses the Biolistic-PDS-1000/He Particle Delivery System (Table of Materials). Abbreviation: WPs = tungsten particles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bacterial plasmid integration into mtDNA; yeast growth on selective media. Experimental results." class="xfigimg" src="/files/ftp_upload/66856/66856fig06v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Example of a high-velocity microprojectile transformation plate. (A,B) After bombardment, plates were incubated for 5-7 nights at 30 °C. If a contaminant grows (indicated by a blue arrow), it can be cut out with a sterile spatula. After replicating the plates on the selection marker medium (-URA in this case), the master plate was also tested for the presence of the mitochondrial plasmid by mating to a tester strain. In this case, the tester strain has the non-functional allele ARG8m-1 1. After mating for 2 nights and replicating the plate on a medium lacking arginine (-ARG), four positive colonies arose (indicated with red arrows). (C) The positive synthetic rho- colonies were purified by restreaking them on -URA medium, and replica testing was carried out as previously described. These plates represent the third round of purification. (D) Some positive synthetic rho- colonies lost the mitochondrial construct. Abbreviations: mtDNA = mitochondrial DNA; -URA = lacking uracil; -ARG = lacking arginine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Plasmid recombination diagram, gene assembly on bacterial plasmid, mtDNA integration process." class="xfigimg" src="/files/ftp_upload/66856/66856fig07.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: Diagram showing how to select positive synthetic rho- colonies after bombardment. (A) The bacterial plasmid contains a nonfunctional mutated version of a mitochondrial gene. For detection, the synthetic rho- colonies are mated to a tester strain containing a different nonfunctional mutation of the same gene in mitochondrial DNA. After mating, the mutant mtDNAs recombine, and a functional gene can be recovered by complementation. Here, the diploid grows on a selective medium where neither the synthetic rho- strain nor the tester can grow. (B) In this case, the plasmid contains an additional sequence of a mitochondrial gene (i.e., a piece of COX3). After transformation, the synthetic rho- strain is selected by its ability to complement a COX3 mutation in the tester strain, so only the diploids can grow on a selective medium (i.e., respiratory medium). This method is handy if a tester strain for the gene of interest is unavailable. Detection of positive synthetic rho- colonies in this case is indirect. Abbreviation: mtDNA = mitochondrial DNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cytoduction diagram: yeast growth on agar plates for genetic testing." class="xfigimg" src="/files/ftp_upload/66856/66856fig08.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8: Example of cytoduction to integrate a construct into the target locus in mitochondrial DNA. After mating and recovery, the mixed cells and binuclear zygotes were plated on a medium lacking leucine. In this case, as the donor strain has the leu2,3-112 allele, this medium prevents donor growth. This plate was replicated on the same medium (master plate), in a medium where only diploids can grow (-ADE) and in a medium where the acceptor with the intended mutation can grow (red arrows). In this case, neither the donor nor receptor cells can grow on respiratory media. Only the resulting diploid after mating with a lawn of a tester strain can grow on a respiratory medium. These respiring diploid colonies are a guide to selecting and purifying the positive colonies from the master plate. Abbreviations: -LEU = lacking leucine; -ADE = lacking adenine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Synthetic rho⁻ donor and mtDNA acceptor gene transfer diagram; mitochondrial gene editing method." class="xfigimg" src="/files/ftp_upload/66856/66856fig09.jpg" data-altupdatedat="1747911167000" data-alt="Figure 9"> 
Figure 9: Diagram showing how to integrate the mutated mitochondrial construct (present in the synthetic rho- strain) into the acceptor strain. (A) In this example, the acceptor strain carries the ARG8m reporter gene that replaces the codons from the gene of interest. The synthetic rho- DNA contains a mutated version of this gene. The plasmid and mitochondrial DNA recombine through the 5' and 3' flanking regions, and the ARG8m reporter is replaced with the mutated sequence of the gene of interest. (B) Foreign genes can be ectopically inserted in specific regions of the mitochondrial genome7,13,36. In this example, a gene can be introduced at a position 295 nt upstream of the COX2 gene. This is a noncoding region of mtDNA. A foreign gene of interest is flanked by 73 nt and 119 nt of the COX2 5' UTR and 3' UTR, respectively. This construction is inserted in a plasmid containing the COX2 gene plus 670 nt and 1120 nt of the 5' and 3' UTR regions. The synthetic rho- strain (donor) carries this construct in mitochondria. In this case, the acceptor strain (XPM13)7 has a significant deletion of the cox2 gene (allele cox2-62)1. After cytoduction, mitochondrial DNA from the donor and acceptor recombine. The COX2 gene is restored, and the gene of interest is inserted at the ectopic site (295 nt upstream of the COX2 gene). Abbreviations: mtDNA = mitochondrial DNA; nt = nucleotides. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66856/66856fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Yeast strain</td>
			<td colspan="3">Genotype</td>
			<td>REF</td>
		</tr>
		<tr>
			<td>NAB69</td>
			<td colspan="3">Mata,ade2-101, arg8-delta::hisG, ura3-52, kar1-1 (rho0)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>NB71 </td>
			<td colspan="3">Matα, ade2-101, ura3-52, leu2-delta, arg8-delta::URA3, kar1-1, (cox3Δ:ARG8m-1)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>XPM10b</td>
			<td colspan="3">Matα, lys2, leu2-3,112, arg8::hisG, ura3-52 (rho+ , cox1∆::ARG8m)</td>
			<td>7</td>
		</tr>
		<tr>
			<td>XPM209</td>
			<td colspan="3">Matα, lys2, arg8::hisG, ura3-52, leu2-3, 112, (rho+, ΔΣai, COX1ΔC15)</td>
			<td>26</td>
		</tr>
		<tr>
			<td>XPM199</td>
			<td colspan="3">Mata,ade2-101, arg8-delta::hisG, ura3-52, kar1-1 (rho-, pXPM61)</td>
			<td>26</td>
		</tr>
		<tr>
			<td>XPM13</td>
			<td colspan="3">Matα, lys2, leu2-3,112, arg8::hisG,ura3-52, his3-deltaHindIII (rho+,cox2-62, cox1∆::ARG8m)</td>
			<td>7</td>
		</tr>
		<tr>
			<td>L45</td>
			<td colspan="3">Matα, ade1, op1  (rho+, cox1-D369N)</td>
			<td>28</td>
		</tr>
	</tbody>
</table>
Table 1: Yeast strain examples used in biolistic transformation and cytoduction.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Growth media</td>
			<td colspan="3">Components</td>
		</tr>
		<tr>
			<td>YPD</td>
			<td colspan="3">2% Glucose, 2% bactopeptone, 1% yeast extract</td>
		</tr>
		<tr>
			<td>YPR</td>
			<td colspan="3">2% Raffinose, 2% bactopeptone, 1% yeast extract</td>
		</tr>
		<tr>
			<td>YPEG*</td>
			<td colspan="3">3% Glycerol, 3% ethanol*1, 2% bactopeptone, 1% yeast extract</td>
		</tr>
		<tr>
			<td>Drop-out media</td>
			<td colspan="3">2% Glucose, 6.8% Yeast nitrogen base with (NH4)2SO4, drop-out amino acid mix*2</td>
		</tr>
		<tr>
			<td>Bombardment media</td>
			<td colspan="3">5% Glucose, 6.8% yeast nitrogen base with (NH4)2SO4, 1 M sorbitol, drop-out amino acid mix*2,3.</td>
		</tr>
		<tr>
			<td colspan="4">*1  Add ethanol after autoclaving when the media is warm.</td>
		</tr>
		<tr>
			<td colspan="4">*2 The amount of amino acid mix may vary according to the stocks available, or the amount indicated by the manufacturer.</td>
		</tr>
		<tr>
			<td colspan="4">*3 For the bombardment media add the amino acids and nitrogen bases as needed depending on the yeast strain. Make sure the composition is specific for the auxotrophy on the 2μL plasmid used for the selection.</td>
		</tr>
	</tbody>
</table>
Table 2: Yeast growth media used in this protocol.

Author Spotlight: Advancing Techniques and Discoveries in Protein Synthesis and Assembly Through Innovative Mitochondrial Research

This work explains how to transform yeast mitochondria using a biolistic method. We also show how to select and purify the transformants and how to introduce the desired mutation in the target position within the mitochondrial genome.

Mitochondrial Transformation in Baker&#39;s Yeast to Study Translation and Respiratory Complex Assembly

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about ...

mitochondrial-transformation-baker-s-yeast-to-study-translation

Nanyang Technological University

Research

JoVE Journal

Genetics

861 Views.  Universidad Nacional Autónoma de México. Ons onderzoek verkent de regulatie van mitochondriale eiwitsynthese en het verband met de binnenste membraanassemblage, cruciaal voor oxidatieve fosforylering. Met behulp van het model bestuderen we het moleculaire mechanisme van de mitochondriale functie, waarbij we gebruik maken van de geschiktheid ervan voor gedetailleerd moleculair en genetisch onderzoek. Veel technologieën worden gebruikt voor onderzoek in ons vakgebied, zoals RNA deep sequencing, multi...

Video: Auteur in de schijnwerpers: Voortschrijdende technieken en ontdekkingen in eiwitsynthese en -assemblage door innovatief mitochondriaal onderzoek

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene&#39;s function in the living organism. In this protocol the OSCAR method of precise and rapid deletion plasmid construction is described. OSCAR relies on the cloning system in which a single recombinase reaction is carried out containing the purified PCR-amplified 5&#39; and 3&#39; flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. Agrobacterium tumefaciens is then used to mediate introduction of the deletion construct into fungal spores (referred to as ATMT). Finally, a PCR assay is described to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. This approach has been successfully used for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides among other species.

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Gene deletion mutants generated through homologous recombination are the gold standard for gene function studies. The OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) method for rapid generation of deletion constructs is described. Agrobacterium mediated fungal transformation follows. Finally, a PCR based confirmation method of gene deletions in fungal transformants is presented.

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Biochemistry

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Measuring Yeast Mitochondrial Respiration While Maintaining Cellular Context

Mitochondrial Respiration Quantification in Yeast Whole Cells

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to different nutritional and environmental conditions give valuable information to elucidate molecular mechanisms. Oxidative phosphorylation is the primary source of ATP in most cells, and mitochondrial respiration activity is a key component of oxidative phosphorylation, maintaining mitochondrial membrane potential for ATP synthesis. Mitochondrial respiration is often studied in isolated mitochondria that are missing the cellular context. Here, we present a method for quantifying mitochondrial respiration in yeast-intact cells. This method applies to any yeast species, although it has been generally used for Saccharomyces cerevisiae cells. First, the yeast growth in specific conditions is tested. Then, cells are washed and resuspended in deionized water with a 1:1 ratio (w/v). Cells are then placed in an oximeter chamber with constant stirring, and a Clark electrode is used to quantify oxygen consumption. Some molecules are sequentially placed into the chamber and selected according to this effect on the electron transport chain or ATP synthesis. ATPase inhibitor oligomycin is first added to measure respiration coupled to ATP synthesis. Afterward, an uncoupler is used to measure the maximal respiratory capacity. Finally, a mix of electron transport chain inhibitors is added to discard oxygen consumption unrelated to mitochondrial respiration. Data are analyzed using a linear regression to obtain the slope, representing the oxygen consumption rate. The advantage of this method is that it is specific for yeast mitochondrial respiration, maintaining the cellular context. It is essential to highlight that inhibitors used in mitochondrial respiration quantification could vary between yeast species.

Mitochondrial respiration in yeast whole cells is a valuable indicator of cell bioenergetics. Here, we present a protocol to quantify this phenotype applicable to different yeast species.

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Mitochondrial Transformation in Baker's Yeast to Study Translation and Respiratory Complex Assembly

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