Reconstitution of protein targeting to the inner envelope membrane of chloroplasts

Abstract

The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), we investigated the targeting of the nucleus-encoded integral IM protein, atTic40. We found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. We also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. Collectively, our experiments are consistent with a “postimport” mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.

Requirements for a conservative protein translocation pathway in chloroplasts

“…Association of Tic110 with the inner envelope independent of an exogenous energy source was observed, which was also resistant to the extraction at pH11. The two different systems described here and in the manuscript by Li and Schnell [12] are difficult to compare, but the latter study used an extremely different ratio of preproteins to inner envelope than in our in-organellar system, therefore it could be out of the biochemically defined linear range for the reaction partner inner envelope resulting in non-selective adhesion or insertion. On the other side, resistance to extraction with Na 2 CO 3 does not necessarily mean productive transmembrane insertion, but could also be caused by strong hydrophobic interaction of the transmembrane helices with the surface of the lipid bilayer.…”

“…Our results clearly promote our understanding on the molecular basis of the re-export pathway, which was previously thought to be independent on any proteinaceous factors and to occur spontaneously into the inner envelope. In these studies [12] isolated inner envelope vesicles were incubated with in vitro translated Tic110 in a wheat germ lysate. Association of Tic110 with the inner envelope independent of an exogenous energy source was observed, which was also resistant to the extraction at pH11.…”

“…In chloroplasts very little is known about the molecular characteristics of those pathways, though both seem to exist. While some inner envelope preproteins seem to import without a soluble stromal intermediate, like the phosphate-triose-phosphate-translocator [11], others have been shown to accumulate in the stroma, like Tic110 or Tic40, before being inserted into the inner envelope [12,13].…”

Plastids Contain a Second Sec Translocase System with Essential Functions

“…Several nucleus-encoded envelope proteins are candidate SCY2 substrates. TIC40 and TIC110 are transiently present in the stroma as soluble intermediates and integrated into the inner envelope membrane, possibly by a conservative sorting pathway of unknown identity (Li and Schnell, 2006;Tripp et al, 2007). Although soluble intermediates of TIC21 and TIC110 accumulate in null Tic40 mutants, TIC40 is thought to play an accessory, rather than a central, role, because translocation is slowed but not blocked (Chiu and Li, 2008).…”

“…The signals and systems involved in targeting to the thylakoid membranes and lumen are relatively well studied and show clear homologies with bacterial transport systems (for review, see Schü nemann, 2007;Cline and DabneySmith, 2008). Although it has been clearly established that certain inner membrane proteins, most notably TIC21, TIC40, and TIC110, also have soluble stromal intermediates (Li and Schnell, 2006;Tripp et al, 2007;Vojta et al, 2007;Chiu and Li, 2008) and therefore require a postimport pathway for integration, a translocase that mediates insertion into the inner membrane or translocation to the intramembrane space has not been identified (Tripp et al, 2007).…”