Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex.
The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH(2) and COOH terminus of Sec61alpha was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61alpha. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.
Raden
D
D
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0103, USA.
Song
W
W
Gilmore
R
R
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport; D003593:Cytoplasm / Q000378:metabolism*; D004285:Dogs; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D010179:Pancreas; D010442:Peptide Chain Initiation, Translational / Q000502:physiology; D010447:Peptide Hydrolases / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000378:metabolism*; D000069816:SEC Translocation Channels; D012639:Seeds; D018271:Signal Recognition Particle / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism / Q000494:pharmacology; D014908:Triticum
D046911:Macromolecular Substances; D008565:Membrane Proteins; D000069816:SEC Translocation Channels; D018271:Signal Recognition Particle; D010447:Peptide Hydrolases; D013820:Thermolysin