UPDATED BOTH IMAGES: Please help me answering ONLY THE \"e\" PART of the questio
ID: 224597 • Letter: U
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UPDATED BOTH IMAGES:
Please help me answering ONLY THE "e" PART of the question.
Chloroplasts contain six compartments – outer membrane, intermembrane space, inner membrane, stroma, thylakoid membrane, and thylakoid lumen (Figure 1) –each of which is populated by specific sets of proteins. To investigate the import of nucleusencoded proteins into chloroplasts, you have chosen to study ferredoxin (FD), which is located in the stroma, and plastocyanin (PC), which is located in the thylakoid lumen, as well as two hybrid genes: ferredoxin with plastocyanin signal peptide (pcFD) and plastocyanin with ferredoxin signalpeptide (fdPC). You translate mRNAs from these four genes in vitro, mix the translation products with isolated chloroplasts for a few minutes, reisolate the chloroplasts after protease treatment, and fractionate them to find which compartments the proteins have entered (Figure 2A). The status of the normaland hybrid proteins at each stage of the experiment is shown in Figure 2B: eachlane in the gel corresponds to a stage of the experiment as indicated alongsidethe experimental protocol in Figure 2A.
a. How efficient is chloroplast uptake of ferredoxin and plastocyanin in your in vitro system? How can you tell?
b. Are ferredoxin and plastocyanin localized to their appropriate chloroplast compartments in these experiments? How can you tell?
c. Are the hybrid proteins imported as you would expect if the Nterminal signal peptides determined their final location? Comment on any significant differences.
d. Why are there three bands in experiments with plastocyanin and pcFD but only two bands in experiments with ferredoxin and fdPC? To the extentyou can, identify the molecular species in the bands and their relationship to one another.
e. Based on your experiments, propose a model for the import of proteins into the stroma and thylakoid lumen.
Figure 1. The six compartments of a chloroplast.
Figure 2. Import of ferredoxin and plastocyanin into chloroplast compartments. (A)Experimental protocol. (B) Gel analysis. Samples from each stage in the experimental protocol were analyzed by gel electrophoresis. Each lane in (B) corresponds to a particular experimental treatment in (A). Ferredoxin gene segments are black; plastocyanin genesegments are white. The signal peptides in all genes are located at the left (Nterminal) end. The ferredoxin signal peptide in the hybrid gene is shown as fd; the plastocyanin signal peptide in the hybrid is shown as pc.
outer nne membrane membrane stroma intermembrane thylakoid thylakoid space membraneExplanation / Answer
The TIC-TOC model is ideal for the import of proteins into the stroma and thylakoid lumen. About 90% of the 3000 different proteins present in the mature chloroplasts are encoded by nuclear DNA and are translated in the cytosol. These proteins are synthesized in their precursor form's comprising an amino-terminal targeting signal or a transit peptide and are imported into the organelle by an active, post-translational targeting process. This process is mediated by molecular machines in the outer and inner envelope membranes. Upon arrival in the stroma, the transit peptide is removed first and the protein either takes on its final conformation or is sorted to one of its several internal compartments in a separate targeting process. Most nucleus-encoded plastid proteins are imported by the TOC (translocation at the outer envelope membrane of chloroplast) and TIC (translocation at the inner envelope membrane of chloroplasts) complexes that are located in the outer envelope membrane (OEM) and inner envelope membrane (IEM).
Proteins destined for the stroma contain a stroma targeting domain and a thylakoid transfering domain at their N-terminus. The stromal protein remain in the stroma a
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