Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex.
In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61alpha and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST. No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.
Nilsson
IngMarie
I
Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843, USA.
Kelleher
Daniel J
DJ
Miao
Yiwei
Y
Shao
Yuanlong
Y
Kreibich
Gert
G
Gilmore
Reid
R
von Heijne
Gunnar
G
Johnson
Arthur E
AE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000528:radiation effects; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006602:Hexosyltransferases; D008027:Light; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding / Q000528:radiation effects; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism*
D008565:Membrane Proteins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D006602:Hexosyltransferases; C096554:STT3 protein, S cerevisiae; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase