PMT1, the gene for a key enzyme of protein O-glycosylation in Saccharomyces cerevisiae.
The integral endoplasmic reticulum membrane protein catalyzing the initial reaction of protein O-glycosylation in Saccharomyces cerevisiae has been purified to homogeneity. The 92-kDa N-glycosylated protein transfers mannose residues from dolichyl phosphate-D-mannose to specific serine/threonine residues of proteins entering the secretory pathway. This type of mannosyl transfer reaction has so far been observed only in fungal cells. Oligonucleotides derived from peptide sequences of the transferase were used to screen a genomic yeast library. A clone was isolated which contains an open reading frame of 2451 bp corresponding to an mRNA transcript of 3 kb. The predicted protein consists of 817 amino acids including three potential N-glycosylation sites. The hydropathy plot indicates a tripartite structure of the protein: an amino-terminal third and a carboxyl-terminal third, both with multiple potential transmembrane helices, and a central hydrophilic part. Expression of the clone in Escherichia coli resulted in mannosyltransferase activity. Gene disruption led to a complete loss of in vitro mannosyltransferase activity from dolichyl phosphate-D-mannose to a peptide used as acceptor in the enzymatic assay. In vivo it was observed, however, that protein O-mannosylation in the disruptant had decreased only to about 40-50%, indicating the existence of an additional transferase which had not been measured by the in vitro enzyme assay.
Strahl-Bolsinger
S
S
Lehrstühle für Zellbiologie, Universität Regensburg, Germany.
Immervoll
T
T
Deutzmann
R
R
Tanner
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation; D008364:Mannosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase