Import pathways of chloroplast interior proteins and the outer-membrane protein OEP14 converge at Toc75.
Most chloroplast outer-membrane proteins are synthesized at their mature size without cleavable targeting signals. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. It has therefore been assumed that insertion of outer-membrane proteins proceeds through a different pathway from import into the interior of chloroplasts, which requires a thermolysin-sensitive translocon complex and ATP. Here, we show that a model outer-membrane protein, OEP14, competed with the import of a chloroplast interior protein, indicating that the two import pathways partially overlapped. Cross-linking studies showed that, during insertion, OEP14 was associated with Toc75, a thermolysin-resistant component of the outer-membrane protein-conducting channel that mediates the import of interior-targeted precursor proteins. Whereas almost no OEP14 inserted into protein-free liposomes, OEP14 inserted into proteoliposomes containing reconstituted Toc75 with a high efficiency. Taken together, our data indicate that Toc75 mediates OEP14 insertion, and therefore plays a dual role in the targeting of proteins to the outer envelope membrane and interior of chloroplasts.
Tu
Shih-Long
SL
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan.
Chen
Lih-Jen
LJ
Smith
Matthew D
MD
Su
Yi-Shin
YS
Schnell
Danny J
DJ
Li
Hsou-Min
HM
D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000502:physiology
D029681:Arabidopsis Proteins; D008081:Liposomes; D008565:Membrane Proteins; C103933:OEP75 protein precursor, plant; D010940:Plant Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; C491244:TOC75 protein, Arabidopsis