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/pubmed?term=((Biogenesis+and+homeostasis+of+chloroplasts+and+other+plastids%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/15012188">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/pubmed?term=((The+biosynthetic+capacities+of+the+plastids+and+integration+between+cytoplasmic+and+chloroplast+processes%5BTitle%5D)+AND+%22Annual+Review+of+Genetics%22%5BJournal%5D)">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/pubmed?term=((Targeting+of+nucleus-encoded+proteins+to+chloroplasts+in+plants%5BTitle%5D)+AND+%22New+Phytologist%22%5BJournal%5D)">Targeting of nucleus-encoded proteins to chloroplasts in plants.</a> New Phytologist. 179 (2), 257-285 (2008).</li>
<li>Li, H. M., Chiu, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+Transport+into+Chloroplasts%5BTitle%5D)+AND+%22Annual+Review+of+Plant+Biology%22%5BJournal%5D)">Protein Transport into Chloroplasts.</a> Annual Review of Plant Biology. 61, 157-180 (2010).</li>
<li>Keegstra, K., Cline, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+import+and+routing+systems+of+chloroplasts%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Protein import and routing systems of chloroplasts.</a> Plant Cell. 11 (4), 557-570 (1999).</li>
<li>Gasser, S. M., Daum, G., Schatz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Import+of+proteins+into+mitochondria.+Energy-dependent+uptake+of+precursors+by+isolated+mitochondria%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Import of proteins into mitochondria. 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/pubmed?term=((The+role+of+the+transit+peptide+in+the+routing+of+precursors+toward+different+chloroplast+compartments%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Arabidopsis+nuclear-encoded+plastid+transit+peptides+contain+multiple+sequence+subgroups+with+distinctive+chloroplast-targeting+sequence+motifs%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((Mitochondrial+targeting+of+the+Arabidopsis+F1-ATPase+gamma-subunit+via+multiple+compensatory+and+synergistic+presequence+motifs%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((A+new+dynamin-like+protein%2C+ADL6%2C+is+involved+in+trafficking+from+the+trans-Golgi+network+to+the+central+vacuole+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Molecules+and+Cells%22%5BJournal%5D)">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/pubmed?term=((Multiple+sequence+motifs+in+the+rubisco+small+subunit+transit+peptide+independently+contribute+to+Toc159-dependent+import+of+proteins+into+chloroplasts%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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/pubmed?term=((Sequence+motifs+in+transit+peptides+act+as+independent+functional+units+and+can+be+transferred+to+new+sequence+contexts%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((Isolated+plant+mitochondria+import+chloroplast+precursor+proteins+in+vitro+with+the+same+efficiency+as+chloroplasts%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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 stages ranging from very early to fully mature leaves. However, care must be taken when growing plants used for protoplast preparation. One should use very healthy plants, as protoplasts prepared from healthy plants can withstand the many steps involved in PEG-mediated transformation. Another important precaution is to use fresh solutions. Slight changes in the concentrations of the solutions can greatly damage the protoplasts, since they are fragile and very sensitive to osmotic pressure.
We have been using protoplasts to study protein import into chloroplasts9,13,14. Based on these studies, we were able to dissect the sequence motifs in various transit peptides. In addition, we used protoplasts to identify the targeting signals (the positively charged region flanking the C-terminus of the transmembrane domain) of proteins targeted to the outer envelope membrane of the chloroplast15. Similarly, we used protoplasts to investigate protein import into the mitochondria10. Again, we were able to identify many critical sequence motifs in the presequences of mitochondrial proteins. In addition, the outer membrane proteins of the mitochondria also contain a positively charged region flanking the transmembrane domain as their targeting signal15. These targeting signals share a high degree of similarity in amino acid composition. Indeed, chloroplast proteins were mistargeted to mitochondria during in vitro import experiments16. However, chloroplast and mitochondrial proteins are specifically imported into their target organelles in vivo. Thus, protoplasts can be used to elucidate the mechanisms underlying how targeting specificity is determined between chloroplasts and mitochondria.
Protoplasts represent an ideal system for analyzing the import of proteins into chloroplasts in vivo. However, one caveat is that protoplasts may be under strong stress conditions such as wounding stress. Thus, we cannot rule out the possibility that such stress may affect the import process. Thus, in certain cases, the results should be interpreted with caution when protoplasts are used for protein import experiments.

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-fluorescence, as examined by fluorescence microscopy (Figure 1). The close overlap of the two signals indicates protein import into chloroplasts. Often the GFP signals are spread throughout the chloroplasts or are concentrated at the center of chloroplasts, with weakly diffuse signals throughout the chloroplasts, depending on the individual protein. The import of proteins can be confirmed by immunoblot analysis using GFP antibody. Total proteins are prepared from protoplasts and separated by SDS-PAGE followed by Western blot analysis (Figure 2). In most cases, two protein bands should be observed in the immunoblot if a protein was properly imported into chloroplasts: the upper band corresponds to the full-length precursor and the lower band to the processed form after import into chloroplasts. The amount of the processed form of the protein should increase in a time-dependent manner. Such results would imply that the protein RbcS-nt:GFP is imported into chloroplasts in Arabidopsis protoplasts. Moreover, the ratio of the processed form to the total amount of expressed proteins (the processed form plus precursor) can be used as a measure of import efficiency. If necessary, the chloroplasts can be purified from gently lysed protoplasts, and proteins from the chloroplast fraction can be analyzed by western blotting to further confirm the import of proteins into chloroplasts.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58441/58441fig1.jpg" /> 
Figure 1. In vivo localization of GFP fused to the RbcS transit peptide to chloroplasts. 
Images were taken 18 h after transformation under a fluorescence microscope. Green (a), red (b), merged (c), and bright (d) labels indicate GFP image, chlorophyll image, a merged image of the two signals, and bright field image, respectively. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58441/58441fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58441/58441fig2.jpg" /> 
Figure 2. Western blot analysis of RbcS-nt:GFP to investigate protein import into chloroplasts. 
Total protein extracts were prepared from protoplasts and separated by 10% (w/v) SDS-PAGE, followed by western blot analysis using anti-GFP antibody. <a href="https://www.jove.com/files/ftp_upload/58441/58441fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying Protein Import into Chloroplasts Using Protoplasts at JoVE.com

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

Nanyang Technological University

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

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Biology

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with 35S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

Protein Transport to the Thylakoids

Thylakoids are membrane-bound sac-like structures within the chloroplast that serve as sites for photosynthesis. Thylakoid lumen contains many electron transport proteins and is enclosed by a thylakoid membrane rich in the light-harvesting complex. Proteins targeted to the thylakoids are transported as precursors and are sorted by the general TOC/TIC import pathway. Once the precursor reaches the stroma, stromal processing peptidases remove their transit signal and expose thylakoid signal sequences. The processed precursors use distinct mechanisms to reach the thylakoid lumen or membrane.
The first mechanism is the chloroplast signal recognition particle or cpSRP-dependent pathway that transports chlorophyll-binding proteins to the thylakoid membrane using energy from GTP hydrolysis. cpSRP proteins and FtsY receptors bind the processed precursor and stimulate GTP hydrolysis. The energy derived facilitates precursor entry through the Alb3 translocase and their subsequent insertion into the thylakoid membranes. In a second mechanism, small thylakoid membrane proteins form soluble stromal intermediates with short hydrophobic signal-like peptides that allow these proteins to fold and insert into the membrane. Once inserted, the signal-like peptides are cleaved by thylakoid processing peptidases to form the mature transmembrane protein. A third pathway employs Oxa-like integrases for inserting thylakoid membrane protein spontaneously without any energy input or targeting factors.
Some proteins targeted to the lumen employ the chloroplast secretory or cpSec pathway, which transports unfolded proteins across the thylakoid membrane. Processed precursors carrying a twin-arginine or RR motif in the N terminal domain often get folded by the stromal chaperonin Cpn60. Folded peptides are then translocated to the thylakoid lumen by the chloroplast twin-arginine or cpTat pathway. cpTat peptidases possess a basic amino acid in their C-terminal domain that helps distinguish and avoid cpSec precursors and cleaves cpTat precursors only.

Thylakoids are membrane-bound sac-like structures within the chloroplast that serve as sites for photosynthesis. Thylakoid lumen contains many electron ...

Education

JoVE Core

Cell Biology

Unit 1

Intracellular Compartments and Protein Sorting

KEY TERMS AND CONCEPTS

Protein Transport to the Stroma

Chloroplasts are triple membrane structures with an outer membrane, an inner membrane, and a thylakoid membrane, each containing distinct metabolite transporters, membrane translocons, and enzymes. Appropriate sorting and translocating these proteins to their correct membrane systems is essential for chloroplast function.
Protein complexes called the translocon of the outer chloroplast membrane or TOC complex, and the translocon of the inner chloroplast membrane or TIC complex mediate the correct sorting of chloroplast proteins. Newly synthesized precursors targeted to the stroma bind cytosolic chaperones Hsp90 and Hsp70-Hsp90 Organizing Protein (HOP). Hsp90 and HOP use energy from ATP hydrolysis to keep the precursor in an unfolded state. A cleavable 13 to 146 amino acids long transit peptide sequence at the N-terminal and a non-cleavable internal signal sequence help precursors translocate to the correct chloroplast subcompartment. The transit peptide is recognized by two homologous GTPases, TOC159 and TOC34. TOC159 is the initial receptor for the transit peptide, while TOC34 facilitates GTP hydrolysis to help the precursor enter through the TOC channel.
The precursor is then transported across the inner membrane through the TIC complex. Sufficient ATP concentration coupled with proper temperature facilitates the translocation of the precursor across the outer and inner chloroplast membrane into the stroma. TIC40 also stimulates Hsp93 to utilize the energy of ATP hydrolysis and pull the precursor completely into the stroma. Stromal processing peptidases cleave the transit signal as the processed precursor is released into the stroma and folded by Cpn60 to form the active protein. Hsp93 undergoes ADP to ATP exchange and helps in consecutive cycles of import.

Chloroplasts are triple membrane structures with an outer membrane, an inner membrane, and a thylakoid membrane, each containing distinct metabolite ...

Protein Transport to the Outer Chloroplast Membrane

Chloroplast outer membrane proteins encoded by the nucleus are synthesized in the cytosol. Soon after synthesis, they bind cytosolic factors such as 14-3-3 protein and the Hsp70 chaperones that keep these precursors in an unfolded state until their translocation.
Two models describe the mechanism of precursor recognition and entry across the outer membrane through the TOC complex. Model 1 suggests the newly synthesized precursor binds to the TOC receptor 159 and forms a complex. TOC159-precursor complex docks onto the outer chloroplast membrane via another TOC receptor, TOC34, stimulating GTP hydrolysis of&#160; TOC34 and TOC159. This allows TOC159 to associate with TOC central channel and facilitate precursor translocation across the outer membrane.
Model 2 suggests that the transit signal of the precursor protein is phosphorylated at its C-terminal by an unknown kinase. TOC34 receptor functions as the initial receptor and binds to the phosphorylated transit signal, undergoing GTP hydrolysis. TOC34 dephosphorylates the transit peptide itself before transferring it to the TOC159 GTPase. GTP hydrolysis by TOC159 further promotes peptide translocation through the TOC complex into the intermembrane space.
As the precursors reach the TIC complex on the inner membrane, a stretch of polyglycine residues close to the transit signal, blocks further translocation. As a result, the precursor gets stalled across the TIC complex and is not translocated into the stroma. Plastid type 1 peptidases present in the intermembrane space cleave the polyglycine signal. The processed precursor is transferred to an insertion protein called outer envelope protein (OEP) or outer membrane protein (OMP) that folds and inserts the processed proteins into the outer membrane of the chloroplast.

Chloroplast outer membrane proteins encoded by the nucleus are synthesized in the cytosol. Soon after synthesis, they bind cytosolic factors such as ...

Studying Protein Import into Chloroplasts Using Protoplasts

Summary

Explore More Videos

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Scalable Transfection of Maize Mesophyll Protoplasts

Protein Transport to the Thylakoids

Protein Transport to the Stroma

Protein Transport to the Outer Chloroplast Membrane