Characterization of the early steps of OE17 precursor transport by the thylakoid DeltapH/Tat machinery.
In order to probe the structure and protein translocation function of the thylakoid Tat machinery, a 25-residue C-terminal extension containing a 13-residue in vivo biotinylation tag and a 6x His tag was added to a mutant precursor of the 17-kDa subunit of the oxygen-evolving complex to form pOE17(C)-BioHis. When avidin was attached to biotinylated precursor in situ, the precursor-avidin complex was neither imported nor did it form a membrane-spanning translocation intermediate. It did, however, competitively inhibit the translocation of unbiotinylated precursor with an apparent KI unaffected by avidin. It is shown that the precursor protein achieves a stable folded structure upon dilution from urea, suggesting that the avidin-induced inhibition of transport results from a folding-induced proximity of N-terminal and C-terminal domains. It is further demonstrated that the majority of precursor rapidly binds to the thylakoid membrane, remaining import competent and yet undissociable by high salt or high pH treatment at ice temperature. The membrane binding event is unaffected by avidin. Import kinetics reveal that nonproton motive force-driven transport steps make up a major fraction of the transport time. These observations suggest that the N-terminal presequence on the avidin-bound precursor is available for membrane binding and initial recognition by the transport machinery, but the attached avidin signals the machinery that the precursor is an incorrectly configured substrate and thus import is aborted. Consequently, the DeltapH/Tat machinery's proofreading mechanism must operate after precursor recognition but before the committed step in transport.
Musser
S M
SM
Section of Plant Biology, University of California, Davis, USA.
Theg
S M
SM
D000595:Amino Acid Sequence; D001360:Avidin / Q000494:pharmacology; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D020524:Thylakoids / Q000187:drug effects / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D011498:Protein Precursors; C408585:twin-arginine translocase complex, E coli; D001360:Avidin