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...

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Bibliographic Details
Published in:Journal of biochemical and biophysical methods 2005-01, Vol.62 (1), p.1-12
Main Authors: Ali, Bassam R.S., Edwards, Laura C., Field, Mark C.
Format: Article
Language:English
Subjects:
Animals
Cells, Cultured
Cholic Acids - pharmacology
Dose-Response Relationship, Drug
Endoplasmic reticulum
Endoplasmic Reticulum - drug effects
Endoplasmic Reticulum - metabolism
Glycopeptide
Microsomes, Liver - drug effects
Microsomes, Liver - secretion
Quality control
Rats
Reconstitution
Retrograde transport
Saponins - pharmacology
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Summary: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.
ISSN:0165-022X
1872-857X
DOI:10.1016/j.jbbm.2004.01.013