Studying Protein Import into Chloroplasts Using Protoplasts | Protocol

Dec, 10th 2018, 20:00 by

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 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.
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.
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 µm. Please click here to view a larger version of this figure.
Total protein extracts were prepared from protoplasts and separated by 10% (w/v) SDS-PAGE, followed by western blot analysis using anti-GFP antibody. Please click here to view a larger version of this figure.
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.

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