|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/2000//53 $5.00
The Journal of Cell Biology, Volume 150, Number 1,
, 2000 53-64
Original Article |
Role of the Cytoplasmic Segments of Sec61
in the Ribosome-Binding and Translocation-Promoting Activities of the Sec61 Complex
reid.gilmore{at}umassmed.edu
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 NH2 and COOH terminus of Sec61
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 Sec61. 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.
Key Words: endoplasmic reticulum • protein targeting • protein translocation • translocon structure • protein topology
© 2000 The Rockefeller University Press
Abbreviations used in this paper: CX-PK-RM, chymotrypsin-digested PK-RM; ECL, enhanced chemiluminescence; NAC, nascent chain–associated complex; OST, oligosaccharyltransferase; PIC, protease inhibitor cocktail; PK-RM, puromycin high salt–washed RM; RM, rough microsomes; RNC, ribosome nascent chain complex; SR, signal recognition particle receptor; SRP, signal recognition particle; TEA, triethanolamine acetate, pH 7.5; TM, transmembrane; TX-PK-RM, trypsin-digested PK-RM; ThX-PK-RM, thermolysin-digested PK-RM; VX-PK-RM, endoproteinase Glu-C–digested PK-RM.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
|
|
