Studying endoplasmic reticulum function in vitro using siRNA.
In eukaryotic cells, N-glycosylation is typically the most common protein modification that occurs in the endoplasmic reticulum (ER) lumen. N-glycosylation is facilitated by a large heterologous protein complex called the oligosaccharyltransferase (OST) that allows the attachment of a high mannose oligosaccharide from a dolichol pyrophosphate donor en bloc onto suitable asparagine residues of newly synthesized nascent chains during translocation into the ER lumen (1). While the complexity of the OST is highly conserved in eukaryotes, the role of its different subunits is poorly defined. We have investigated the function of three OST subunits, the ER translocon-associated component ribophorin I, and two isoforms of the presumptive catalytic subunit, STT3. We use a combination of siRNA-mediated knockdown of individual proteins combined with a semi-permeabilized mammalian cell system to provide a robust read out for OST subunit function during N-glycosylation of model substrates in vitro. This approach is equally applicable to the study of other cellular components.
Wilson
Cornelia M
CM
Faculty of Medicine, University of Limoges, Limoges, France.
High
Stephen
S
D000818:Animals; D015153:Blotting, Western; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006367:HeLa Cells; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D034622:RNA Interference; D034741:RNA, Small Interfering
D008565:Membrane Proteins; D034741:RNA, Small Interfering; C034241:ribophorin; D006602:Hexosyltransferases; C099573:STT3A protein, human; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase