Reconstitution of glycopeptide export in mixed detergent-solubilised and resealed microsomes depleted of lumenal components.
Export of macromolecules from the endoplasmic reticulum (ER) lumen into the cytosol is a major aspect of the quality control systems operating within the early secretory system. Glycopeptides are exported from the ER by an ATP- and GTP-dependent pathway, which shares many similarities to the protein export system. Significantly, for glycopeptides, there is no requirement for cytosolic factors, biochemically distinguishing the glycopeptide and protein paths and probably reflecting the lower conformational complexity of the former substrate. Genetic studies in yeast, and biochemical data from higher eukaryotes, indicate that glycopeptides utilise the Sec61 translocon. Here, we report a new system allowing access to lumenal ER components, facilitating assessment of their importance in glycopeptide retrotranslocation and potentially other processes. Saponin, in combination with CHAPS, but not saponin alone, facilitated removal of >95% of lumenal protein disulphide isomerase (PDI) and BiP. Upon resealing, these microsomes retained glycopeptide export competence. These data suggest that the majority of lumenal components of the ER are most likely nonessential for glycopeptide export. In addition, export competence was highly sensitive to the addition of external protease, indicating a role for protein factors with cytoplasmically exposed determinants.
Ali
Bassam R S
BR
Department of Biological Sciences, Imperial College, London, SW7 2AY, UK.
Edwards
Laura C
LC
Field
Mark C
MC
D000818:Animals; D002478:Cells, Cultured; D002793:Cholic Acids / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012503:Saponins / Q000494:pharmacology*
D002793:Cholic Acids; D012503:Saponins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate