Ribosome-independent regulation of translocon composition and Sec61alpha conformation.
In this study, the contributions of membrane-bound ribosomes to the regulation of endoplasmic reticulum translocon composition and Sec61alpha conformation were examined. Following solubilization of rough microsomes (RM) with digitonin, ribosomes co-sedimented in complexes containing the translocon proteins Sec61alpha, ribophorin I, and TRAPalpha, and endoplasmic reticulum phospholipids. Complexes of similar composition were identified in digitonin extracts of ribosome-free membranes, indicating that the ribosome does not define the composition of the digitonin-soluble translocon. Whereas in digitonin solution a highly electrostatic ribosome-translocon junction is observed, no stable interactions between ribosomes and Sec61alpha, ribophorin I, or TRAPalpha were observed following solubilization of RM with lipid-derived detergents at physiological salt concentrations. Sec61alpha was found to exist in at least two conformational states, as defined by mild proteolysis. A protease-resistant form was observed in RM and detergent-solubilized RM. Removal of peripheral proteins and ribosomes markedly enhanced the sensitivity of Sec61alpha to proteolysis, yet the readdition of inactive ribosomes to salt-washed membranes yielded only modest reductions in protease sensitivity. Addition of sublytic concentrations of detergents to salt-washed RM markedly decreased the protease sensitivity of Sec61alpha, indicating that a protease-resistant conformation of Sec61alpha can be conferred in a ribosome-independent manner.
Potter
M D
MD
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Nicchitta
C V
CV
D000818:Animals; D003902:Detergents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007202:Indicators and Reagents / Q000494:pharmacology; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry*; D008861:Microsomes / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D020033:Protein Isoforms / Q000737:chemistry; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism*; D000069816:SEC Translocation Channels; D013997:Time Factors
D003902:Detergents; D007202:Indicators and Reagents; D008244:Lysophosphatidylcholines; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D020033:Protein Isoforms; D000069816:SEC Translocation Channels; C038470:1,2-heptanoylphosphatidylcholine; D004072:Digitonin