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

Wild-type, mnnl, and mnn9 mutant yeast strains carrying KRE2 were [35S]methionine labeled for 10 rain and chased for 45 min, and the extent of glycosylation of immunoprecipitated Kre2p was examined (Fig. 3) . The size of Kre2p produced in a wild-type strain after a 10-min radiolabeling is ~57 kD (major band, lane 2). After an additional 45-min chase, an apparent increase of molecular mass from 57 to ~59-60 kD was seen (Fig. 3 , lanes 2 and 5). This apparent 2-3 kD increase in mass suggests that Kre2p may undergo a post-ER modification not involving extensive outer chain elaboration. The molecular mass of Kre2p produced in an mnn9 strain after a 10-min pulse (Fig. 3, lanes 2 and 4) and a 45 min chase (lanes 5 and 7) was identical to the mass of Kre2p from a wild-type cell, indicating that Kre2p does not receive an outer chain oligosaccharide. We tested whether the time-dependent additional carbohydrate modification required the MNN1encoded Golgi al,3-mannosyltransferase, an enzyme that acts on both core and outer chains ( Fig. 2 ; Ballou et al., 1990; Graham and Emr, 1991; Graham et al., 1994; Yip et al., 1994) . After a short pulse, Kre2p synthesized in a mnnl strain is approximately of wild-type size (Fig. 3, lane 3) . After a 45-min chase, however, the mnnl-produced Kre2p was ~1-2 kD smaller than the wild-type protein (lanes 5-7) indicating that Kre2p is normally exposed to a Golgi compartment where the Mnnlp cxl,3-mannosyltransferase adds at least three mannose residues to the Kre2p N-glycosyl core. Mnnlp therefore contributes to most of the observed Kre2p post-ER modifications (Fig. 2) . GlcNAc2 carbohydrate structure. In other cases, core structures may be extended by an outer chain of variable size (up to 200 mannose residues) that is composed of a backbone of al,6-mannosyl residues to which are attached branched al,2-and cd,3-mannosyl side chains. Recent evidence suggests that the initiating Ochlp al,6-mannosyltransferase (Nakayama et al., 1992 ) defines a very early Golgi compartment that appears to be distinct from a subsequent early Golgi compartment responsible for cxl,6-mannosyl side chains elongation (Gaynor et al., 1994) . The ~1,2-and al,3mannosyl side chain modifications are predicted to occur in the medial-Golgi. The possible S. cerevisiae N-linked oligosaccharide structures are shown (adapted from Ballou, 1990 and Ballou et al., 1990) . Arrows depict [31,4, al,6, cd,2, and cd,3 linkages between mannoses of the core and outer chain. X = 10 on average. The mannose units not present in the mnn9 mutation are indicated. The Mnnlp-terminal al,3-mannosyltransferase is responsible for the addition of all al,3-mannosyl residues in a medial-Golgi compartment (Ballou, 1990; Graham et al., 1994; Yip et al., 1994) . [] represents mannose residues that are thought to be added to the Kre2p core oligosaccharide in a MNNl-dependent manner (see Fig. 3 ). Fully glycosylated proteins are then transported to a late Golgi compartment which includes the proteolytic enzymes (Kex2p, Kexlp, and DPAP A) responsible for maturation of secreted protein precursors. Glycoproteins that are not retained in the Golgi complex can be subsequently directed to the vacuole through an endosomal compartment or to the cell surface where they can be (a) incorporated into the plasma membrane, (b) secreted and retained in the periplasmic space/cell wall, or (c) secreted extracellularly.