Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex
Graduate School of Biomedical Sciences; Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology
Life Sciences | Medicine and Health Sciences
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.
J Cell Biol. 2000 Jul 10;150(1):53-64.
The Journal of cell biology
Raden, David; Song, Weiqun; and Gilmore, Reid, "Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex" (2000). GSBS Student Publications. 1007.