Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment

The difference between the expected (51.5 kD) and observed molecular mass of Kre2p ( Fig. 1 B) is likely to be at Kre2p is oriented as a type II membrane-anchored protein, a topology characteristic of all isolated glycosyltransferases (Shaper and Shaper, 1992; Kleene and Berger, 1993; Gleeson et al., 1994) . Kre2p consists of a short amino-terminal cytoplasmic domain, a hydrophobic transmembrane domain, and a large carboxy-terminal luminal catalytic domain. The catalytic domain is linked to the transmembrane domain by a polypeptidic "stem" region. The stem region is generally thought to be devoid of secondary structure. (B) Immunological detection of Kre2p. Yeast total-protein extracts from kre2::TRP1 in SEY6210 and from the same strain expressing KRE2 from the multicopy plasmid YEp352 (Hill et al., 1986) , were immunoblotted with affinity-purified anti-Kre2p polyclonal antibodies (see Materials and Methods). The molecular mass standards are shown in kilodaltons. (C) Kre2p is membrane associated. SEY6210 cells overexpressing KRE2 were lysed, and cell debris was removed by centrifugation. The resulting crude homogenate was aliquoted, and fractions were rendered 0.1 M with Na2CO 3 or 1.6 M with urea. After 30 min, each fraction was centrifuged at 100,000 g for 1 h and after SDS-PAGE, Kre2p was detected by immunoblotting with the anti-Kre2p Ab in pellet (P) and supernatant (S) fractions. least partly due to N-glycosylation, since Kre2p is expected to act in the secretory pathway and the protein possesses a single site for N-glycosyl attachment in its predicted luminal domain (Asna97-Gln-Thr) (see Fig. 6 ). To test for Kre2p N-glycosylation, yeast cells were [35S]methionine labeled in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Immunoprecipitation and S D S -P A G E analysis of labeled cell lysates revealed that Kre2p was N-glycosylated (see Fig. 3 , lanes 1 and 2), with the position of the sole N-glycosylation attachment site at A s n 197 being consistent with a type II topology for Kre2p. The molecular mass of Kre2p in the presence of tunicamycin is about 54 kD, still 2.5 kD larger than its pre-dicted molecular mass. The membrane nature of Kre2p or other posttranslational modifications could explain this discrepancy.