Research Projects Hsp93 / Chloroplast Import
The chloroplast, the characteristic organelle of plants and green algae, is responsible for various essential functions, including photosynthesis, lipid metabolism, starch and amino acid biosynthesis. Although each chloroplast carries its own genome, the vast majority of its proteins are encoded in the cell nucleus. Following their synthesis in the cytosol, these proteins must be transported through the chloroplast envelope, a double lipid bilayer, to reach their final destination. Polypeptide translocation is carried out by a translocase
, an assembly of proteins embedded in the outer and inner chloroplast membrane (or associated with it). The components of the translocase are called Toc or Tic proteins, reflecting their location (outer or inner membrane) and molecular weight. Cargo proteins destined for chloroplasts carry an N-terminal transit peptide
that is recognized by Toc159 and/or Toc34, two GTPases associated with the outer membrane. From there, the protein is transferred to Toc75, which forms a channel through the outer membrane. The pore of the inner membrane presumably consists of Tic20/21. The energy for the translocation process is provided by ATP hydrolysis on the stromal side.
Simplified model of protein translocation into the chloroplast.
In other organelles featuring protein translocation, such as mitochondria and the endoplasmic reticulum, Hsp70 is believed to serve as the import motor. In the case of chloroplasts, however, the role of stromal Hsp70 chaperones is less clear. Various studies indicate that it may not be the central player in protein import, suggesting that other chaperones provide the driving force required to pull polypeptides across the membrane.
Hsp93 - A Putative Protein Import Motor
One potential candidate is Hsp93
, a member of the AAA+ protein superfamily, which encompasses several ATP-driven molecular motors. Hsp93 is localized in the stroma, but a fraction of it appears to be associated with the inner envelope membrane, presumably by interacting with the TIC complex.
contains two Hsp93 homologues termed Hsp93-V and Hsp93-III according to their chromosomal location. They are ~88% identical and contain two nucleotide binding domains. Attempts to generate Hsp93 double knockouts have failed so far, suggesting that plant cells need at least one version of Hsp93 for viability. Hsp93-V knockout plants display impaired protein import into chloroplasts, resulting in smaller and paler plants in comparison to wild-type strains.
Our long-term goal
is to determine whether Hsp93 is a motor for chloroplast import. The lethality of the Hsp93 double knockout makes a genetic approach very difficult. We therefore address this question by studying Hsp93 in vitro and correlate its properties with a possible role as an import motor. Features that we investigate include ATP hydrolysis, its ability to interact with and translocate chloroplast precursor proteins through its central channel, and its ability to interact with components of the TIC complex.
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