Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane.
The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.
Pilon
M
M
Howard Hughes Medical Institute and Department of Molecular Cell Biology, University of California at Berkeley, Berkeley California 94720, USA.
Römisch
K
K
Quach
D
D
Schekman
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001693:Biological Transport, Active; D017931:DNA Primers / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006360:Heat-Shock Proteins; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D017931:DNA Primers; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; C074215:SEC63 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate