This work was carried out with the supports of Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ010953012018), Rural Development Administration, and the National Research Foundation (Korea) grant funded by the Ministry of Science and ICT (No. 2016R1E1A1A02922014), Republic of Korea.

1. Growth of Arabidopsis Plants
<ol>
	<li>Prepare 1 L Gamborg B5 (B5) medium by adding 3.2 g of B5 medium including vitamins, 20 g of sucrose, 0.5 g of 2-(N-morpholino) ethane sulfonic acid (MES) to approximately 800 mL of deionized water, and adjust the pH to 5.7 with potassium hydroxide (KOH). Add more deionized water bring the total volume to 1 L. Add 8 g of phytoagar and autoclave for 15 min at 121 &#176;C.</li>
	<li>Allow the medium to cool down to 55 &#176;C and pour 20 &#8211; 25 mL of the B5 medium into a Petri dish (9 cm in diameter, 1.5 cm in height) at a clean bench. Dry plates for 2 &#8211; 3 days, pack them with clean wrap, and keep them in a refrigerator until use.</li>
	<li>Sterilize Arabidopsis thaliana seeds with 1 mL of 70% (v/v) ethanol in a centrifuge tube (e-tube) by continuously shaking for 2 &#8211; 3 min. Remove the supernatant, add 1 mL of 1% (v/v) sodium hypochlorite solution, and shake continuously for 2 &#8211; 3 min. Prepare 100 &#8211; 150 seeds per Petri dish.
	<ol>
		<li>Remove the supernatants and wash the seeds with 1 mL of distilled water. Repeat this step 4x. Place the sterilized seeds at 4 &#176;C for 3 days to synchronize seed germination.</li>
	</ol>
	</li>
	<li>Sow 100 &#8211; 150 seeds onto B5 plates using a micropipette and seal the plate with surgical tape.</li>
	<li>Grow plants in a growth room with a 16 h/8 h light/dark cycle at 100 &#181;mol&#183;m-2&#183;s-1 light intensity, 70% relative humidity and 20 &#176;C temperature for 2 weeks.</li>
</ol>
2. Preparation of the Plasmid
NOTE: High-purity plasmids should be used for transformation; the use of a commercial plasmid purification kit is recommended.
<ol>
	<li>Purify the plasmid (RbcS-nt:GFP) using a DNA isolation kit. To concentrate DNA, perform ethanol precipitation in a 1.5 mL tube and dissolve the DNA pellet in 100 &#181;L of distilled water. Determine the concentration of the plasmid using a spectrophotometer at 260 nm. Dilute the plasmid to a final concentration of ~ 2 &#181;g/&#181;L. Keep the plasmid at -20 &#176;C until use.</li>
</ol>
3. Isolation of Protoplasts
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>Prepare the enzyme solution to contain 0.25% (w/v) macerozyme, 1.0% (w/v) cellulase, 400 mM D-mannitol, 8 mM calcium chloride (CaCl2), 0.1% (w/v) bovine serum albumin (BSA), and 5 mM MES. Adjust the pH to 5.6 with KOH.
		<ol>
			<li>Filter the enzyme solution using a cellulose acetate filter unit with a 0.45 &#956;m pore size. Aliquot 25 mL of the enzyme solution into 50 mL conical tubes and keep at -20 &#176;C. Thaw the enzyme solution at room temperature (RT) and mix well just before use.</li>
		</ol>
		</li>
		<li>Prepare a 21% (w/v) sucrose solution by dissolving 21 g of sucrose in 100 mL of deionized water and autoclave the solution.</li>
		<li>Prepare W5 solution to contain 154 mM sodium chloride (NaCl), 125 mM CaCl2, 5 mM potassium chloride (KCl), 5 mM glucose, and 1.5 mM MES. Adjust the pH to 5.6 with KOH and autoclave the solution.</li>
		<li>Prepare MaMg solution to contain 400 mM D-mannitol, 15 mM magnesium chloride (MgCl2), and 5 mM MES. Adjust the pH to 5.6 with KOH and autoclave the solution.</li>
		<li>Prepare 1 M D-mannitol and 1 M calcium nitrate stock solutions to make the PEG solution. Autoclave these solutions.</li>
	</ol>
	</li>
	<li>Harvest intact leaf tissues from 2 weeks-old plants from ~ 1 &#8211; 2 B5 plates using a surgical knife and place the harvested leaves into a 50 mL conical tube containing 25 mL of enzyme solution. Place the conical tube containing the enzyme solution and leaf tissues horizontally on a rotary shaker with gentle agitation in the dark. It takes 8 &#8211; 12 h for full digestion of leaf tissues.</li>
	<li>At 8 &#8211; 12 h after incubation, pour the enzyme solution (containing protoplasts released from leaf tissues) into a fresh Petri dish through a mesh with 140 &#181;m pore size. Carefully layer the protoplast-containing enzyme solution on top of 15 mL of 21% (w/v) sucrose solution and centrifuge it at 98 x g for 10 min with the lowest acceleration and deceleration settings in a swinging-bucket rotor.</li>
	<li>Using a Pasteur pipette, carefully transfer the protoplasts from the upper-most layer (enzyme solution) and at the interface between the enzyme solution and sucrose solution to a 50 mL conical tube containing 30 mL of W5 solution. Centrifuge this tube at 51 x g for 6 min. At this stage, protoplasts will be in the pellet at the bottom of the conical tube.</li>
	<li>Discard the supernatant carefully using a pipette without disturbing the protoplasts pellet. Add 25 mL of W5 solution, and gently resuspend the protoplasts. Incubate the protoplast solution in a 4 &#176;C refrigerator for at least 1 h for stabilization.</li>
</ol>
4. Protoplast Transformation using Polyethylene Glycol
<ol>
	<li>Prepare a 40% PEG solution by adding 4 g of PEG 8000, 4 mL of 1 M mannitol solution, 1 mL of 1 M Ca(NO3)2, and 1.8 mL of distilled water into a 50 mL conical tube and mix well. Dissolve PEG by heating in a microwave oven for 20 &#8211; 30 s. Place the 40% PEG solution at RT for cooling down. 
	NOTE: The 40% PEG solution should be prepared freshly every time. If protoplasts are not pelleted completely during the 4 &#176;C incubation in the refrigerator, centrifuge the material at 46 x g for 2 min.</li>
	<li>Remove the supernatant carefully but completely and add MaMg solution to the protoplast pellet to yield the concentration of 5 x 106/mL. 
	NOTE: The number of protoplast can be determined by a hemocytometer viewed under a microscope.</li>
	<li>Place 10 &#181;g of the plasmid DNA each empty 13 mL round-bottom tube and add 300 &#181;L of protoplast solution using a pipette. 
	NOTE: The end of the pipette tip should be cut off. Whenever protoplasts are sampled, the protoplast-containing solution should be resuspended right before pipetting so that the same number of protoplasts is added to each tube.</li>
	<li>Mix the plasmid DNA with protoplasts by gently rotating the tubes and immediately add 300 &#181;L of 40% PEG solution using a pipette. Mix gently but completely by rotating tubes and incubate for 30 min at room temperature; tilt the tube almost horizontally and rotate it several times.</li>
	<li>Add 1 mL of W5 solution and mix gently but completely by rotating the tube by hand in a similar manner. Incubate the sample for 10 min at room temperature.</li>
	<li>Repeat this step two times more using 1.5 mL and 2 mL of W5 solution, respectively. Incubate for 30 min after the final addition of W5 solution.</li>
	<li>Centrifuge at 46 x g for 4 min. Discard the supernatant and add 2 mL of W5 solution. Mix gently but completely. Incubate at 22 &#176;C in a dark chamber.</li>
</ol>
5. Analysis of the Protein Import into Chloroplasts
NOTE: After PEG-mediated transformation of protoplasts, incubation time ranges from 8 to 24 h.
<ol>
	<li>Fluorescence Microscopy
	<ol>
		<li>Place 10 &#181;L of the protoplast solution on a slide glass using a pipette with a trimmed tip and carefully cover with a coverslip to avoid damaging the protoplasts.</li>
		<li>Place the slide on the stage of a fluorescence microscope equipped with excitation/emission filter sets for observing green fluorescent protein (GFP) and chlorophyll auto-fluorescence.</li>
		<li>Capture images with a cooled charge-coupled device (CCD) camera and process images using an image-editing software to produce pseudo-color images.</li>
	</ol>
	</li>
	<li>Total Protein Extraction and Immunoblotting
	<ol>
		<li>Prepare denaturation buffer containing 2.5% (w/v) sodium dodecylsulfate (SDS), and 2% (v/v) 2-mercaptoethanol. 
		NOTE: Protein import into chloroplasts can be quantified by measuring the degree of transit peptide processing via immunoblot analysis using anti-GFP antibody.</li>
		<li>Transfer protoplasts into a centrifuge tube and centrifuge at 46 x g for 4 min.</li>
		<li>Remove the supernatant and add 80 &#181;L of denaturation buffer. Vigorously vortex for 5 s and add 5x SDS sample buffer (250 mM Tris-Cl (pH 6.8), 0.5 M DTT, 10% (w/v) SDS, 0.05% (w/v) Bromophenol blue, and 50% (v/v) glycerol). Mix well and boil for 10 min.</li>
		<li>Subject this protein sample to standard SDS-PAGE and immunoblotting with anti-GFP antibody.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Jarvis, P., Lopez-Juez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+homeostasis+of+chloroplasts+and+other+plastids.">Biogenesis and homeostasis of chloroplasts and other plastids.</a> Nature Reviews Molecular Cell Biology. 14 (12), 787-802 (2013).</li><li>Neuhaus, H. E., Emes, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonphotosynthetic+Metabolism+in+Plastids.">Nonphotosynthetic Metabolism in Plastids.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 51, 111-140 (2000).</li><li>Rolland, N., 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+biosynthetic+capacities+of+the+plastids+and+integration+between+cytoplasmic+and+chloroplast+processes.">The biosynthetic capacities of the plastids and integration between cytoplasmic and chloroplast processes.</a> Annual Review of Genetics. 46, 233-264 (2012).</li><li>Jarvis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+nucleus-encoded+proteins+to+chloroplasts+in+plants.">Targeting of nucleus-encoded proteins to chloroplasts in plants.</a> New Phytologist. 179 (2), 257-285 (2008).</li><li>Li, H. 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Energy-dependent uptake of precursors by isolated mitochondria.</a> Journal of Biological Chemistry. 257 (21), 13034-13041 (1982).</li><li>Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., Weisbeek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+transit+peptide+in+the+routing+of+precursors+toward+different+chloroplast+compartments.">The role of the transit peptide in the routing of precursors toward different chloroplast compartments.</a> Cell. 46 (3), 365-375 (1986).</li><li>Lee, D. W., 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=Arabidopsis+nuclear-encoded+plastid+transit+peptides+contain+multiple+sequence+subgroups+with+distinctive+chloroplast-targeting+sequence+motifs.">Arabidopsis nuclear-encoded plastid transit peptides contain multiple sequence subgroups with distinctive chloroplast-targeting sequence motifs.</a> Plant Cell. 20 (6), 1603-1622 (2008).</li><li>Lee, 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=Mitochondrial+targeting+of+the+Arabidopsis+F1-ATPase+gamma-subunit+via+multiple+compensatory+and+synergistic+presequence+motifs.">Mitochondrial targeting of the Arabidopsis F1-ATPase gamma-subunit via multiple compensatory and synergistic presequence motifs.</a> Plant Cell. 24 (12), 5037-5057 (2012).</li><li>Jin, J. B., 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+new+dynamin-like+protein,+ADL6,+is+involved+in+trafficking+from+the+trans-Golgi+network+to+the+central+vacuole+in+Arabidopsis.">A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis.</a> Plant Cell. 13 (7), 1511-1526 (2001).</li><li>Lee, K. H., Kim, D. H., Lee, S. W., Kim, Z. H., Hwang, I. <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+import+experiments+in+protoplasts+reveal+the+importance+of+the+overall+context+but+not+specific+amino+acid+residues+of+the+transit+peptide+during+import+into+chloroplasts.">In vivo import experiments in protoplasts reveal the importance of the overall context but not specific amino acid residues of the transit peptide during import into chloroplasts.</a> Molecules and Cells. 14 (3), 388-397 (2002).</li><li>Lee, D. W., Lee, S., Oh, Y. J., Hwang, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sequence+motifs+in+the+rubisco+small+subunit+transit+peptide+independently+contribute+to+Toc159-dependent+import+of+proteins+into+chloroplasts.">Multiple sequence motifs in the rubisco small subunit transit peptide independently contribute to Toc159-dependent import of proteins into chloroplasts.</a> Plant Physiology. 151 (1), 129-141 (2009).</li><li>Lee, D. W., Woo, S., Geem, K. R., Hwang, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+motifs+in+transit+peptides+act+as+independent+functional+units+and+can+be+transferred+to+new+sequence+contexts.">Sequence motifs in transit peptides act as independent functional units and can be transferred to new sequence contexts.</a> Plant Physiology. 169 (1), 471-484 (2015).</li><li>Lee, J., 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=Both+the+hydrophobicity+and+a+positively+charged+region+flanking+the+C-terminal+region+of+the+transmembrane+domain+of+signal-anchored+proteins+play+critical+roles+in+determining+their+targeting+specificity+to+the+endoplasmic+reticulum+or+endosymbiotic+organelles+in+Arabidopsis+cells.">Both the hydrophobicity and a positively charged region flanking the C-terminal region of the transmembrane domain of signal-anchored proteins play critical roles in determining their targeting specificity to the endoplasmic reticulum or endosymbiotic organelles in Arabidopsis cells.</a> Plant Cell. 23 (4), 1588-1607 (2011).</li><li>Cleary, S. P., 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=Isolated+plant+mitochondria+import+chloroplast+precursor+proteins+in+vitro+with+the+same+efficiency+as+chloroplasts.">Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.</a> Journal of Biological Chemistry. 277 (7), 5562-5569 (2002).</li></ol>

The authors have nothing to disclose.

We provided a detailed protocol for the use of protoplasts of Arabidopsis to study protein import into chloroplasts. This method is powerful for investigating the protein import process. This simple, versatile technique is useful for examining the targeting of the intended cargo proteins to chloroplasts. Using this method, protoplasts are prepared from leaf tissues of Arabidopsis11,12 which can be obtained from plants at many different growth st...

The chloroplast is one of the most important organelles in plants. One of the main functions of chloroplasts is to carry out photosynthesis1. Chloroplasts also carry out many other biochemical reactions for the production of fatty acids, amino acids, nucleotides, and numerous secondary metabolites1,2. For all of these reactions, chloroplasts require a large number of different types of proteins. However, the chloroplast genome contains only approximately 100 genes3,4. Therefore, chloroplasts must import the majority of their proteins from the cytosol. In fact, most chloroplast proteins were shown to be imported from the cytosol after translation4,5,6. Plant cells require specific mechanisms to import proteins from the cytosol to chloroplasts. However, although these protein import mechanisms have been investigated for the past several decades, we still do not fully understand them at the molecular level. Here, we provide a detailed method for preparing protoplasts and exogenously expressing genes in protoplasts. This method could be valuable for elucidating the molecular mechanisms underlying protein import into chloroplasts in detail.
Protein import can be studied using many different approaches. One of these methods involves the use of an in vitro protein import system7,8. Using this approach, in vitro-translated protein precursors are incubated with purified chloroplasts in vitro, and protein import is analyzed by SDS-PAGE followed by western blot analysis. The advantage of this approach is that each step of protein import into chloroplasts can be studied in detail. Thus, this method has been widely used to define the components of the protein import molecular machinery and to dissect sequence information for transit peptides. More recently, another approach involving the use of protoplasts from leaf tissues was developed and it has become widely used to study protein import into chloroplasts9,10. The advantage of this approach is that protoplasts provide a cellular environment that is closer to that of intact cells than the in vitro system. Thus, the protoplast system allows us to address many additional aspects of this process, such as the associated cytosolic events and how the specificity of targeting signals is determined. Here, we present a detailed protocol for the use of protoplasts to study protein import into chloroplasts.

<table>
	<tbody>
		<tr>
			<td>GAMBORG B5 MEDIUM INCLUDING VITAMINS</td>
			<td>Duchefa Biochemie</td>
			<td>G0210.0050</td>
			<td></td>
		</tr>
		<tr>
			<td>SUCROSE</td>
			<td>Duchefa Biochemie</td>
			<td>S0809.5000</td>
			<td></td>
		</tr>
		<tr>
			<td>MES MONOHYDRATE</td>
			<td>Duchefa Biochemie</td>
			<td>M1503.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, powder</td>
			<td>JUNSEI</td>
			<td>24440S1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore Surgical tape</td>
			<td>3M</td>
			<td>1530-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade stainless No.10</td>
			<td>FEATHER</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tube, 50ml</td>
			<td>SPL LIFE SCIENCES</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Macerozyme R-10</td>
			<td>YAKULT PHARMACEUTICAL IND.</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase ONOZUKA R-10</td>
			<td>YAKULT PHARMACEUTICAL IND.</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>ALBUMIN, BOVINE (BSA)</td>
			<td>VWR</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>SIGMA</td>
			<td>M1902-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CALCIUM CHLORIDE, DIHYDRATE</td>
			<td>MP BIOMEDICALS</td>
			<td>0219463505-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Twister</td>
			<td>VISION SCIENTIFIC</td>
			<td>VS-96TW</td>
			<td></td>
		</tr>
		<tr>
			<td>Screen cup for CD-1</td>
			<td>SIGMA</td>
			<td>S1145</td>
			<td></td>
		</tr>
		<tr>
			<td>Screens for CD-1</td>
			<td>SIGMA</td>
			<td>S3895</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>SPL LIFE SCIENCES</td>
			<td>10090</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>HILGENBERG</td>
			<td>3150102</td>
			<td></td>
		</tr>
		<tr>
			<td>LABORATORY CENTRIFUGE / BENCH-TOP</td>
			<td>VISION SCIENTIFIC</td>
			<td>VS-5500N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>JUNSEI</td>
			<td>19015S0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>SIGMA</td>
			<td>P3911-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-GLUCOSE, ANHYDROUS</td>
			<td>BIO BASIC</td>
			<td>GB0219</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>DUKSAN</td>
			<td>40</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium nitrate tetrahydrate</td>
			<td>SIGMA</td>
			<td>C2786-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>SIGMA</td>
			<td>P2139-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>SIGMA</td>
			<td>M2393-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube 13ml, 100x16mm, PP</td>
			<td>SARSTEDT</td>
			<td>55.515</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>MARIENFELD</td>
			<td>1000412</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses</td>
			<td>MARIENFELD</td>
			<td>101030</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Chamber</td>
			<td>MARIENFELD</td>
			<td>650030</td>
			<td></td>
		</tr>
		<tr>
			<td>Axioplan 2 Imaging Microscope</td>
			<td>Carl Zeiss</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tube 1.5ml</td>
			<td>SARSTEDT</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>SIGMA</td>
			<td>M3148-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS), Proteomics Grade</td>
			<td>VWR</td>
			<td>M107-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS, Ultra Pure Grade</td>
			<td>VWR</td>
			<td>0497-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (DL-Dithiothreitol), Biotechnology Grade</td>
			<td>VWR</td>
			<td>0281-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue sodium salt ACS</td>
			<td>VWR</td>
			<td>0312-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>JUNSEI</td>
			<td>27210S0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Colors A.v. Monoclonal Antibody (JL-8)</td>
			<td>TAKARA</td>
			<td>632381</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying protein import into chloroplasts using protoplasts

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.

The import of proteins into chloroplasts can be examined using two approaches: fluorescence microscopy and immunoblot analysis after SDS-PAGE-mediated separation. Here, we used RbcS-nt:GFP, a fusion construct encoding the 79 N-terminal amino acid residues of RbcS containing the transit peptide fused to GFP. When proteins are imported into chloroplasts, green fluorescence signals from the target protein RbcS-nt:GFP should merge with the red fluorescent signals from chlorophyll auto-fluores...

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Studying Protein Import into Chloroplasts Using Protoplasts

Here we describe a protocol to express proteins into protoplasts by using PEG-mediated transformation method. The method provides easy expression of proteins of interest, and efficient investigation of protein localization and the import process for various experimental conditions in vivo.

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

studying-protein-import-into-chloroplasts-using-protoplasts

Research

JoVE Journal

Biochemistry

9.6K Views.  Pohang University of Science and Technology. Here we describe a protocol to express proteins into protoplasts by using PEG-mediated transformation method. The method provides easy expression of proteins of interest, and efficient investigation of protein localization and the import process for various experimental conditions in vivo.

Video: Studying Protein Import into Chloroplasts Using Protoplasts

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...