ABSTRACT
Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytoplasm and degraded by the proteasome. Processing intermediates of N-linked oligosaccharides on incompletely folded glycoproteins have an important role in their folding/refolding, and also in their targeting to proteolytic degradation. In Saccharomyces cerevisiae, we have identified a gene coding for a non-essential protein that is homologous to mannosidase I (HTM1) and that is required for degradation of glycoproteins. Deletion of the HTM1 gene does not affect oligosaccharide trimming. However, deletion of HTM1 does reduce the rate of degradation of the mutant glycoproteins such as carboxypeptidase Y, ABC-transporter Pdr5-26p and oligosaccharyltransferase subunit Stt3-7p, but not of mutant Sec61-2p, a non-glycoprotein. Our results indicate that although Htm1p is not involved in processing of N-linked oligosaccharides, it is required for their proteolytic degradation. We propose that this mannosidase homolog is a lectin that recognizes Man8GlcNAc2 oligosaccharides that serve as signals in the degradation pathway.
Subject(s)
Fungal Proteins/metabolism , Glycoproteins/metabolism , Protein Folding , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Animals , Carbohydrate Conformation , Carboxypeptidases/genetics , Carboxypeptidases/metabolism , Cathepsin A , Cysteine Endopeptidases/metabolism , Fungal Proteins/genetics , Gene Deletion , Genes, Fungal , Glycoproteins/chemistry , Glycoproteins/genetics , Hexosyltransferases , Humans , Immunoblotting , Mannosidases/chemistry , Mannosidases/genetics , Mannosidases/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins , Molecular Sequence Data , Multienzyme Complexes/metabolism , Oligosaccharides/metabolism , Proteasome Endopeptidase Complex , SEC Translocation Channels , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Temperature , Ubiquitins/metabolismABSTRACT
N-linked protein glycosylation is an essential process in eukaryotic cells. In the central reaction, the oligosaccharyltransferase (OTase) catalyzes the transfer of the oligosaccharide Glc3Man9GlcNac2 from dolicholpyrophosphate onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum. The product of the essential gene STT3 is required for OTase activity in vivo, but is not present in highly purified OTase preparations. Using affinity purification of a tagged Stt3 protein, we now demonstrate that other components of the OTase complex, namely Ost1p, Wbp1p and Swp1p, specifically co-purify with the Stt3 protein. In addition, different conditional stt3 alleles can be suppressed by overexpression of either OST3 and OST4, which encode small components of the OTase complex. These genetic and biochemical data show that the highly conserved Stt3p is a component of the oligosaccharyltransferase complex.
