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

The product encoded by the KRE2 gene is a predicted type II membrane protein of 442 amino acid residues with a short cytoplasmic NH2 terminus, a hydrophobic transmembrane region, and a large luminal enzymatic domain containing one potential N-glycosylation site ( Fig. 1 A) . To identify and subsequently analyze Kre2p, a specific rabbit antiserum was raised, affinity purified, and used to detect antigen-antibody complexes by Western blotting of total-cell protein extracts of yeast strains harboring a KRE2 disruption or containing a KRE2 multicopy plasmid to facilitate immunological detection. The affinity-purified Ab detected Kre2p as a 59-60 kD protein in the KRE2overexpressing strain that was absent from the kre2::TRP1 strain ( Fig. 1 B) , and not detected by preimmune antiserum (data not shown). The in vivo-produced Kre2p is ~8.0 kD larger than the 51.5-kD molecular mass predicted from the D N A sequence. The possible integral membrane protein nature of Kre2p was examined by using reagents extracting cytoplasmic, vesicle-enclosed, and peripheral membrane proteins but leaving intact tightly associated membrane proteins. Ceils expressing KRE2 at high levels were lysed with glass beads and treated with sodium carbonate or urea. After high-speed centrifugation of the treated cell lysates, the distribution of Kre2p in the pellet and supernatant fractions was assessed by Western immunoblotting. As can be seen in Fig. 1 C, Kre2p was found only in membrane pellet fractions.