Deletion Mutants of Chlorophyll a/b Binding Proteins Are Efficiently Imported into Chloroplasts but Do Not Integrate into Thylakoid Membranes.
Chlorophyll a/b binding polypeptides (CABp) are integral thylakoid membrane proteins containing three membrane-spanning helices. We have created a series of mutations in tomato CABp to test whether individual membrane helices with hydrophilic flanking sequences, when fused to a transit peptide, can be imported into chloroplasts and correctly targeted to thylakoid membranes. All of the mutated precursors, including those with large C-terminal and internal deletions, were imported successfully, showing that these regions of the mature CABp are not required for import into chloroplasts. All mutants tested, containing either one or two membrane helices, were found primarily in the stroma and not in the thylakoids. The small amount of protein found associated with the thylakoids was largely resistant to alkali extraction but was sensitive to protease, unlike wild-type protein, which is resistant to both treatments. When incubated with thylakoids in the absence of stroma and/or ATP, a significant amount of wild-type protein assumes a form that is resistant to alkali extraction but is protease sensitive, like the imported deletion proteins. This form of the wild-type protein is not chased into a protease-resistant form by adding stroma and/or ATP. These results suggest that CABp can spontaneously associate with membranes as an aberrant species that is not an intermediate in the process of integration. The inability of the deletion forms of CABp to assume a protease-resistant conformation suggests that correct integration is afforded by elements within the entire protein that collectively contribute to the proper conformation of the protein. The ability of deletion mutants to associate with thylakoids in a nonphysiological way suggests that the study of such mutants may not be useful in elucidating thylakoid-targeting signals.
Huang
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Carnegie Institution of Washington, Stanford, California 94305.
Adam
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Hoffman
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