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Chloroplast proteome analysis: new insights into intracellular protein trafficking

English title Chloroplast proteome analysis: new insights into intracellular protein trafficking
Applicant Baginsky Sacha
Number 112565
Funding scheme Project funding (Div. I-III)
Research institution Departement Umweltsystemwissenschaften ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Botany
Start/End 01.04.2006 - 30.11.2009
Approved amount 169'474.00
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All Disciplines (2)

Discipline
Botany
Biochemistry

Keywords (4)

plastid development; proteomics; protein targeting; mechanisms of protein translocation

Lay Summary (English)

Lead
Lay summary
Chloroplasts are typical plant cell organelles that play numerous unique roles in processes of global significance, e.g. photosynthesis and amino acid biosynthesis. Chloroplasts are of cyanobacterial origin, but during evolution they lost their autonomy and transferred most of their genes to the nucleus. For proper functioning, chloroplasts must therefore import a substantial number of nuclear encoded proteins from the cytosol. Some of the chloroplast-targeted proteins can be recognized by an N-terminal extension (transit peptide), that directs their import into chloroplast.Recent analyses of the chloroplast proteome however suggested, that a substantial number of the chloroplast-localized proteins do not possess a transit sequence that can be recognized by currently existing tools. Thus the question, which proteins are imported into chloroplasts is still central for plant cell biology.

The research project is designed to provide answers to the above question.To this end, we will follow two different experimental strategies: first we plan to extend the list of known chloroplast-localized proteins by a proteomics approach and second we plan to elucidate the targeting mechanism of those proteins without a predicted transit sequence in greater detail. To extend existing proteomics efforts, we will employ newly developed protein and peptide fractionation strategies prior to high throughput mass spectrometric protein identification from isolated chloroplasts. These analyses will provide a more comprehensive list of chloroplast proteins and allow for a global analysis of plastid function in the plant cell. The other strategy is designed to validate the hypothesis that some proteins are imported into the plastid despite the lack of a predictable N-terminal transit peptide. Unusual or missing transit sequences could potentially indicate a novel, to date unknown import route that uses components different from the established TIC/TOC translocation system. To address this question in greater detail, we start out by fusing selected proteins without a transit peptide to GFP and assess the subcellular localization of the fusion proteins by confocal laser scanning microscopy. To check for GFP position effects on subcellular localization, we will fuse GFP C-, N-terminal or internally to the protein of interest. In the next step, we plan to expand the targeting analyses e.g. by analyzing protein targeting in known chloroplast protein import mutants and by introducing mutations in the amino acid sequences of some selected chloroplast-localized proteins to find the region that carries the targeting information.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Associated projects

Number Title Start Funding scheme
127202 Completion of the plastid proteome map for genome-scale constraint-based modeling by functional characterization of protein import and targeted proteomics 01.02.2010 Project funding (Div. I-III)

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