Functional Characterizations of Sequence Motifs in the Transit Peptide of Pisum sativum Tic40
Date Issued
2011
Date
2011
Author(s)
CHAN, PO-TING
Abstract
Most chloroplast proteins are encoded in the nucleus, translated in the cytosol, and then transported into chloroplasts through the Toc (Translocon at the outer envelope membrane of chloroplasts) and Tic (Translocon at the inner envelope membrane of chloroplasts) complexes. Nucleus-encoded chloroplast proteins are usually synthesized as higher molecular weight precursors with N-terminal targeting signals called the transit peptides. Even though transit peptides are necessary and sufficient for chloroplast precursor proteins import, what constitute the critical sequence features for receptor recognition in the transit peptides is still poorly understood. Previous research has shown that precursors can be classified into three groups based on their preference to be imported into chloroplasts of different ages and the age-selective signal is located within the transit peptide of each precursor. In this study, I tried to identify the transit peptide features that confer preference for older chloroplasts. I divided the transit peptide of prPsTic40 (precursor of Tic40 from pea Pisum sativum), one of the precursors that prefer to be imported into older chloroplasts, into eight blocks and generated serial alanine scanning mutants. When block 4 (residues 28-36) was mutated, import efficiency into older chloroplasts was significantly decreased, while import efficiency into younger chloroplasts was not affected. Further mutations within block 4 through site-directed mutagenesis revealed that two positive charges, arginine and lysine of residues 29 and 30, are necessary and sufficient to confer preference for older chloroplasts. Moreover, two positive charges in the transit peptide of prAtL11 (precursor of L11 from Arabidopsis thaliana), another precursor that also prefers to be imported into older chloroplasts, are also responsible for prAtL11 preference for older chloroplasts. These results indicate that two consecutive positive charges in the transit peptide are the critical feature constituting the age-selective signal for older chloroplasts preference.
Subjects
chloroplast
transit peptide
age selectivity
Type
thesis
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