Name: Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins (Video) | JoVE Journal
Uploaded: 2019-08-15T14:30:00.000+00:00
Duration: 2 min 45 s
Description: Learn chloroplast protein import and localization methods using Arabidopsis thaliana. This collection covers in vitro and in vivo assays, subfractionation, and complex analysis techniques for plant cell biology research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Research in the author&#8217;s laboratory is funded by the Biotechnology and Biological Sciences Research Council (BBSRC; grant references BB/N006372/1, BB/R005591/1, BB/R009333/1, BB/R016984/1).

<ol><li>Jarvis, P., López-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>Ling, Q., Jarvis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+protein+import+into+chloroplasts+isolated+from+stressed+plants%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Analysis of protein import into chloroplasts isolated from stressed plants.</a> Journal of Visualized Experiments. (117), (2016).</li>
<li>Aronsson, H., Jarvis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+method+for+isolating+import-competent+Arabidopsis+chloroplasts%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">A simple method for isolating import-competent Arabidopsis chloroplasts.</a> FEBS Letters. 529 (2-3), 215-220 (2002).</li>
<li>Fitzpatrick, L. M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+isolating+a+high+yield+of+Arabidopsis+chloroplasts+capable+of+efficient+import+of+precursor+proteins%5BTitle%5D)+AND+%22Plant+Journal%22%5BJournal%5D)">A method for isolating a high yield of Arabidopsis chloroplasts capable of efficient import of precursor proteins.</a> Plant Journal. 27 (1), 59-65 (2001).</li>
<li>Ling, Q., Jarvis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+chloroplast+protein+import+by+the+ubiquitin+E3+ligase+SP1+is+important+for+stress+tolerance+in+plants%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 is important for stress tolerance in plants.</a> Current Biology. 25 (19), 2527-2534 (2015).</li>
<li>Lee, J., Kang, H., Hwang, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+protein+import+into+chloroplasts+using+protoplasts%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Studying protein import into chloroplasts using protoplasts.</a> Journal of Visualized Experiments. (142), (2018).</li>
<li>Asher, A., Ganesan, I., Klasek, L., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+physiologically+active+thylakoids+and+their+use+in+energy-dependent+protein+transport+assays%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Isolation of physiologically active thylakoids and their use in energy-dependent protein transport assays.</a> Journal of Visualized Experiments. (139), (2018).</li>
<li>Bouchnak, I., Moyet, L., Salvi, D., Kuntz, M., Rolland, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+chloroplast+sub-compartments+from+Arabidopsis+for+the+analysis+of+protein+Localization+by+immunoblotting+or+proteomics%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Preparation of chloroplast sub-compartments from Arabidopsis for the analysis of protein Localization by immunoblotting or proteomics.</a> Journal of Visualized Experiments. (140), (2018).</li>
<li>Salvi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AT_CHLORO%3A+the+first+step+when+looking+for+information+about+subplastidial+localization+of+proteins%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">AT_CHLORO: the first step when looking for information about subplastidial localization of proteins.</a> Methods in Molecular Biology. 1829, 395-406 (2018).</li>
<li>Shanmugabalaji, V., Douet, V., Agne, B., Kessler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Affinity+purification+of+chloroplast+translocon+protein+complexes+using+the+TAP+tag%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Affinity purification of chloroplast translocon protein complexes using the TAP tag.</a> Journal of Visualized Experiments. (141), (2018).</li>
<li>Kikuchi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Ycf2-FtsHi+heteromeric+AAA-ATPase+complex+is+required+for+chloroplast+protein+import%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">A Ycf2-FtsHi heteromeric AAA-ATPase complex is required for chloroplast protein import.</a> Plant Cell. 30 (11), 2677-2703 (2018).</li>
<li>Rantala, M., Paakkarinen, V., Aro, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+thylakoid+membrane+protein+complexes+by+blue+native+gel+electrophoresis%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Analysis of thylakoid membrane protein complexes by blue native gel electrophoresis.</a> Journal of Visualized Experiments. (139), (2018).</li>
<li>Kikuchi, S., Hirohashi, T., Nakai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+preprotein+translocon+at+the+outer+envelope+membrane+of+chloroplasts+by+blue+native+PAGE%5BTitle%5D)+AND+%22Plant+and+Cell+Physiology%22%5BJournal%5D)">Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE.</a> Plant and Cell Physiology. 47 (3), 363-371 (2006).</li>
<li>Chen, L. J., Li, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stable+megadalton+TOC-TIC+supercomplexes+as+major+mediators+of+protein+import+into+chloroplasts%5BTitle%5D)+AND+%22Plant+Journal%22%5BJournal%5D)">Stable megadalton TOC-TIC supercomplexes as major mediators of protein import into chloroplasts.</a> Plant Journal. 92 (2), 178-188 (2017).</li>
<li>Jarvis, R. P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJarvis%2C+RP+%22Methods+in+Molecular+Biology%22">Methods in Molecular Biology</a>. JM, W. alker 775, Humana Press. New York City. 432(2011).</li>
<li>Jarvis, R. P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJarvis%2C+RP+%22Methods+in+Molecular+Biology%22">Methods in Molecular Biology</a>. JM, W. alker 774, Humana Press. New York City. 374(2011).</li>
</ol>

Commercial application of the author&#8217;s research is covered by patent applications GB1803833.1, GB1803834.9, GB1815206.6 and US 16/643507.

Chloroplasts are the organelles that define plants1. Along with many other metabolic, developmental and signaling functions, chloroplasts are responsible for photosynthesis &#8211; the process whereby sunlight energy is harnessed to power the cellular activities of life. Consequently, chloroplasts are essential, not only for plants but also for the myriad of ecosystems that depend on plants, and for agriculture. Chloroplasts are composed of thousands of different proteins, most of which are nucleus-encoded and imported from the cytosol before being routed internally to one of a number of clearly-distinct intraorganellar compartments1. To achieve a more complete understanding of chloroplast development and functions, and to enable biotechnological strategies involving chloroplast manipulation that address global challenges linked to food security or bioenergy, it will be essential to determine the targeting, localization and interactions of important chloroplast proteins. This method collection describes a set of critically important and complementary techniques that may be used to accomplish these goals. The collection mostly focuses on the widely-used model plant Arabidopsis thaliana (thale cress), but the methods may also be adapted and applied to other organisms.
The collection includes descriptions of two different techniques for analyzing the import of nucleus-encoded proteins into chloroplasts across the double-membrane envelope. The article by Ling and Jarvis2 describes an in vitro method in which isolated chloroplasts are incubated with a radioactively-labelled precursor protein. The extent to which the chloroplasts take up the precursor protein is determined by monitoring the protein size change that occurs as a result of transit peptide (targeting leader sequence) cleavage, using SDS-PAGE and phosphor-imaging. The presented method is a development of an approach that has been used to study chloroplast protein import in vitro for several decades3,4, and incorporates additional steps that enable assessment of the responsiveness of the import machinery to stress conditions experienced by the plant5. On the other hand, the article by Lee et al.6 describes an in vivo method based on the transient expression of a chimeric precursor protein, carrying a fluorescent protein domain, in intact cells (protoplasts). In this assay, the extent of chloroplast protein import can be followed in two different ways: by monitoring the localization and intensity of the fluorescence signal using fluorescence microscopy; and, by analyzing the protein size change that occurs as a result of transit peptide cleavage using immunoblotting. These two methods are highly complementary, and can deliver compelling results when used together in parallel5.
Once a protein has been imported across the envelope into the chloroplast, and its transit peptide has been removed, it may either take on its final conformation in the stroma (the main internal aqueous compartment of the organelle), or engage one of a number of internal sorting pathways1. As the site of the highly-abundant photosynthetic complexes, the thylakoid membranes are a major destination for such internal sorting; in fact, thylakoid protein targeting involves multiple, mechanistically-distinct pathways. The article by Asher et al.7 describes a range of in vitro methods that enable different thylakoid protein translocation pathways to be studied. These methods involve the incubation of isolated thylakoids with radioactively-labelled precursor protein and, in some cases, a concentrated stromal extract. The extent to which the protein is taken up by the thylakoids is monitored by assessing cleavage of the targeting signal, and protection of the protein from exogenously-applied thermolysin protease, using SDS-PAGE and phosphor-imaging. Of course, the thylakoids are not the only subcompartment of the chloroplast, and it is often desirable to have the ability to assess other compartments as well. In this regard, the article by Bouchnak et al.8 is particularly important, as it describes methods for the subfractionation of chloroplasts to produce highly-pure samples corresponding to the envelope membranes, the stroma, and the thylakoids. Once prepared, these fractions may be analyzed by immunoblotting and/or mass spectrometry, in order to provide a wealth of information on the suborganellar localization of chloroplast proteins9.
With regard to the analysis of protein-protein interactions and multiprotein complex assemblies, two different methodologies are described in the collection. The article by Shanmugabalaji et al.10 presents a method for the affinity purification of chloroplast multiprotein complexes. This technique involves the analysis of transgenic plants expressing a component of the complex of interest that has been engineered to carry an affinity tag (the so-called tandem affinity purification tag, or TAP tag). The ability of this tag to bind strongly to an otherwise inert matrix is exploited as part of the purification strategy. The presented method focuses specifically on purification of the chloroplast protein import machinery (this comprises multiprotein complexes called TOC and TIC embedded in the envelope membranes1), but in principle it can be adapted to study any of the other multiprotein assemblies that exist in chloroplasts11. The article by Rantala et al.12 describes a complementary approach to complex characterization based on electrophoresis under native conditions. The technique, as presented here, involves the liberation of photosynthetic complexes from purified thylakoids using mild, non-ionic detergent, followed by their separation using blue native (BN)-PAGE. Primary resolution of the complexes may be followed by a second dimension of electrophoresis under denaturing conditions, and this enables visualization of the individual components of each complex identified in the first dimension. As with the TAP method, this native PAGE approach can be successfully adapted to study other protein complexes within the organelle13,14. With either approach, the purified complexes may be analyzed in a variety of ways, including by immunoblotting and mass spectrometry.
Together, the articles included in this methods collection present a powerful set of complementary techniques that, in conjunction with existing resources15,16, may be employed to greatly improve our understanding of diverse aspects of chloroplast biogenesis and function, particularly those closely linked to the organellar proteome. Because chloroplasts are responsible for the bulk of terrestrial photosynthetic primary production, and furthermore have vital roles in the responses of plants to the environment (including both biotic and abiotic stresses), these remarkable organelles will inevitably remain a major focus of fundamental and applied research across the globe for years to come.&#160;

chloroplast research methods: probing the targeting, localization and interactions of chloroplast proteins

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins

Chloroplasts are the organelles that define plants1. Along with many other metabolic, developmental and signaling functions, chloroplasts are responsible ...

Welcome to the JoVE Methods Collections on Chloroplast Research Methods curated by our guest editor, Paul Jarvis. This collection covers a wide array of in vitro and in vivo techniques that characterize protein interactions inside chloroplasts. Researchers at the University of Oxford describe an in vitro method using isolated chloroplasts, which are incubated with radioactively labeled proteins to monitor their uptake.This method is a development of previous approaches used to study chloroplast protein import in vitro. Next, in a complimentary method, chloroplast protein import is examined in vivo. This method uses transient expression of chimeric proteins and protoplasts to examine protein size changes and localization via immunoblotting and fluorescence microscopy.In this method video, the authors examined the thylakoid membrane system present in chloroplasts. By isolating the membranes, protein translocation pathways are elucidated in chloroplasts by SDS-PAGE and phosphor imaging. Researchers in France apply different centrifugation methods to isolate the sub-compartments found in chloroplasts.These isolated cellular fractions are then analyzed by immunoblotting and proteomics to provide information on the sub-organellar localization of chloroplast proteins. Next, Swiss researchers used affinity purification of chloroplast multi-protein complexes to examine the molecular machinery involved in protein import. Specifically the talk and tick multi-protein complexes embedded in envelope membranes.In a complimentary approach, scientists characterize the plant thylakoid protein complex in Arabidopsis using Blue Native PAGE, or BN-PAGE, which allows for the separation of protein complexes in their native and functional form. The complexes are further analyzed via 2D SDS-PAGE. You've just had a glimpse of the various methods used to study chloroplast proteins in our JoVE Methods Collection.You can view the articles in depth at the Chloroplast Research Methods Collection webpage shown at the link on screen.

chloroplast-research-methods-probing-targeting-localization

Research

JoVE Journal

Biochemistry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

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.

Analysis of Protein Import into Chloroplasts Isolated from Stressed 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.

mRNA Interactome Capture from Plant Protoplasts

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.

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

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.

Studying Protein Import into Chloroplasts Using Protoplasts

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.

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

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.

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

Many plant species change the positioning of chloroplasts to optimize light absorption. This protocol describes how to use a straightforward, home-built instrument to investigate chloroplast movement in Arabidopsis thaliana leaves using changes in the transmission of light through a leaf as a proxy.

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Learn chloroplast protein import and localization methods using Arabidopsis thaliana. This collection covers in vitro and in vivo assays, subfractionation, and complex analysis techniques for plant cell biology research.

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins

Explore More Videos