Interactions between Sec complex and prepro-alpha-factor during posttranslational protein transport into the endoplasmic reticulum.
Posttranslational translocation of prepro-alpha-factor (ppalphaF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex. We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step. We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules. To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppalphaF was generated with photoreactive probes incorporated into the mature portion of the polypeptide. Cross-linking to multiple regions of Sec61p was observed. In contrast to the signal sequence, neighboring positions of the mature portion of ppalphaF had similar interactions with Sec61p. These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.
Plath
Kathrin
K
Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Wilkinson
Barrie M
BM
Stirling
Colin J
CJ
Rapoport
Tom A
TA
D001692:Biological Transport; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum; D007425:Intracellular Membranes / Q000378:metabolism; D000072235:Mating Factor; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary / Q000502:physiology; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; C074213:SEC62 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor