Abstract
The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N(cyt)-C(lum) orientation.
Highlights
Mammalian cells express two homologues of Stt3p (STT3-A and STT3-B) that form a complex with other oligosaccharyl transferase (OT) subunits [4]
A photoreactive probe incorporated into a nascent polypeptide chain carrying a cryptic glycosylation sequon (Gln-X-Thr) can be cross-linked to STT3-A in dog pancreatic microsomes [5], lending further support to the idea that STT3 is the catalytic subunit of OT
Glycosylation of the invertase fragment indicates a luminal location of the corresponding loop; in the second approach, glycosylation of the reporter again suggests a luminal orientation, whereas the ability of transformed his4Ϫ cells to grow on media lacking histidine but including histidinol suggests a cytosolic location of the reporter [19]
Summary
Mammalian cells express two homologues of Stt3p (STT3-A and STT3-B) that form a complex with other OT subunits (ribophorins I and II, OST48, and DAD1) [4]. We present in vivo and in vitro topology mapping studies of S. cerevisiae Stt3p and mouse STT3-A, using Cterminal reporter fusions and insertion of glycosylation sites in the loops between the putative transmembrane segments.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
