Insertion of PsaK into the thylakoid membrane in a "Horseshoe" conformation occurs in the absence of signal recognition particle, nucleoside triphosphates, or functional albino3.
The photosystem I subunit PsaK spans the thylakoid membrane twice, with the N and C termini both located in the lumen. The insertion mechanism of a thylakoid membrane protein adopting this type of topology has not been studied before, and we have used in vitro assays to determine the requirements for PsaK insertion into thylakoids. PsaK inserts with high efficiency and we show that one transmembrane span (the C-terminal region) can insert independently of the other, indicating that a "hairpin"-type mechanism is not essential. Insertion of PsaK does not require stromal extract, indicating that signal recognition particle (SRP) is not involved. Removal of nucleoside triphosphates inhibits insertion only slightly, both in the presence and absence of stroma, suggesting a mild stimulatory effect of a factor in the translation system and again ruling out an involvement of SRP or its partner protein, FtsY. We, furthermore, find no evidence for the involvement of known membrane-bound translocation apparatus; proteolysis of thylakoids destroys the Sec and Tat translocons but does not block PsaK insertion, and antibodies against the Oxa1/YidC homolog, Alb3, block the SRP-dependent insertion of Lhcb1 but again have no effect on PsaK insertion. Because YidC is required for the efficient insertion of every membrane protein tested in Escherichia coli (whether SRP-dependent or -independent), PsaK is the first protein identified as being independent of YidC/Alb3-type factors in either thylakoids or bacteria. The data raise the possibility of a wholly spontaneous insertion pathway.
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Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Woolhead
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Moore
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Henry
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Robinson
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D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins; D002462:Cell Membrane; D002467:Cell Nucleus / Q000378:metabolism; D002734:Chlorophyll / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000737:chemistry; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D045331:Photosystem I Protein Complex; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D018271:Signal Recognition Particle / Q000378:metabolism*; D020524:Thylakoids / Q000737:chemistry*; D014158:Transcription, Genetic
C106404:ALBINO 3 protein, Arabidopsis; D029681:Arabidopsis Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; C438010:PsaK protein, barley; D018271:Signal Recognition Particle; D002734:Chlorophyll; D004247:DNA