A post-import pathway for protein targeting to chloroplast inner membrane
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), I investigated the targeting of the nucleus-encoded integral IM protein, atTic40. I 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. I also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. These in vitro studies are further supported by in vivo evidence showing pre-atTic40 directly engineered into the chloroplast genome and expressed within chloroplasts can efficiently target to the inner envelope membrane. Taken together, my experiments are consistent with a "post import" mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.