Development and Evolution of Research Questions in the Inoue Lab
version 1 (CAUTION!! ..references are not complete..)
Some of the ongoing projects address general questions that have "evolved" from rather specific questions about a protein called Toc75. This page will develop to describes these "evolutionary processes".
Fig. 1. Evolution of questions with Toc75
<Background>
Chloroplast Biogenesis
√ Chloroplasts have evolved from an ancient form of cyanobacteria.
√ The successful conversion of the cyanobacterial endosymbiont to the chloroplasts depends on gene transfer, which requires at least three steps: gene duplication, gene expression, and gene loss (from the endosymbiont genome).
√ For "gene loss", the pre-organelle endosymbiont needs to establish a system to uptake proteins encoded in the host nucleus and synthesized in the cytoplasm.
√ Consequently, a chloroplast depend nucleus for >95% of its protein constituents.
√ Thus, protein import is essential for i) successful endosymbiosis; and ii) maintenance and development of the organelle.
Toc75
√ Toc75 is one of the major proteins in the chloroplast outer envelope.
√ Based on its primary structure, Toc75 is postulated to form a beta-barrel structure in the lipid bilayers. Integral beta-barrel proteins are found only in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts.
√ Toc75 belongs to a group of proteins called Omp85/BamA family, which includes proteins in the outer membranes of Gram-negative bacteria (including cyanobacteria) and mitochondria (Knowles et al. 2009).
√ Thus, Toc75 has most probably derived from a protein in the outer membrane of the cyanobacterial endosymbiont (Bölter et al. 1998; Reumann et al. 1999).
√ Various biochemical studies have shown that Toc75 is involved in the translocation of precursor proteins that are synthesized in the cytoplasm.
√ Based on the way it interacts with the incoming precursor, how it looks like, and how it behaves when reconstituted in liposomes, Toc75 is believed to form the protein conducting channel in the outer envelope.
√ Toc75 is also encoded in the nuclear genome. Hence, it has to be sorted to the outer envelope posttranslationally.
√ Unlike other proteins found in the outer membranes of chloroplasts and mitochondria, Toc75 is synthesized as a large precursor with an N-terminal extension (tp75), which is cleaved by two steps (Tranel et al. 1995).
√ The first portion of tp75 (called tpn75) acts as a general stromal targeting sequence, whereas the second segment of tp75 (tpc75) appears to function as a stop-transfer to prevent the mature Toc75 from traversing the inner envelope (Tranel and Keegstra 1996).
<Evolution of Questions>
Original questions about Toc75 (see Fig. 1)
Q. How does Toc75 function as a protein conducting channel? (mechanism of protein translocation)
Q1. How has Toc75 evolved from the ancestral protein in the endosymbiont? (evolution of membrane proteins)
Q2. How does tpc75 direct Toc75 to the outer envelope? (protein targeting to the envelope)
Q3. How is tpc75 processed? (protein maturation)
How have these Questions been evolving?
Q1 => Identification of a paralogous protein OEP80/Toc75-V (Eckart et al. 2002; Inoue and Potter 2004), which belongs to Omp85 family and is essential for plant viability from the embryonic stage (Patel et al. 2008; Hsu et al. 2008).
Q2 => Discovery of polyglycine-mediated envelope targeting pathway; Inoue and Keegstra 2003; Baldwin and Inoue 2006). This pathway appears to be utilized by a subset of proteins located in the chloroplast envelope (unpublished).
Q3 => Discovery of Plsp1 (Plastidic type I signal peptidase 1), which plays multiple roles and is necessary for proper development of thylakoids (the internal membrane structures of chloroplasts) (Inoue et al. 2005; Shipman and Inoue 2009).
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Last updated on July 28th, 2009
Kentaro Inoue(C) 2009
