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

The fact that Kre2p requires more than one topological segment to achieve proper Golgi localization, emphasizes that there may be distinct, but not necessarily mutually exclusive, mechanisms of retention. Golgi proteins that possess a cytoplasmic targeting signal have been proposed to be sorted via a receptor-mediated mechanism (Wilsbach and Payne, 1993; Gleeson et al., 1994; Low and Hong, 1994) . Overexpression of the late Golgi yeast proteins Kexlp, Kex2p, and DPAP A leads to some vacuolar mislocalization, indicating saturation of the capacity of a receptor-based sorting process (Cooper and Bussey, 1992; Wilcox et al., 1992; Nothwehr et al., 1993; Wilsbach and Payne, 1993) . Kre2p overexpression also results in some mistargeting to the vacuole, and could be similarly explained. When wild-type Kre2p is expressed from a 21xbased multicopy vector in a background with mutations in the major vacuolar hydrolases, all positive cells display a punctiform pattern of fluorescence with 15 % of the stained cells also showing vacuolar fluorescence. This small percentage of doubly stained cells are likely expressing high levels of Kre2p as determined by the intensity of the fluorescence. In contrast, overexpression of animal glycosyltransferases does not bring about saturation of the retention mechanism (Munro, 1991; Aoki et al., 1992; Burke et al., 1992; Colley et al., 1992; Teasdale et al., 1992; Gleeson et al., 1994) . Interpreting our data in the simplest way, the Kre2p segment encompassing the NH 2 terminus along with the TMD and stem region could interact directly with a medial-Golgi localized receptor spanning both sides of the Golgi lipid bilayer. For Kre2p to be retained in the Golgi, an interaction between the putative receptor and the NH2-terminal domain is essential. Part of the presumed Kre2p receptor complex could be part of the Golgi extracisternal space matrix which in mammals was recently shown to bind medial-Golgi enzymes presumably through their cytoplasmic tails . Interestingly, examination of the cytoplasmic NHz-terminal domains of the six members of the KRE2 mannosyltransferase family (Lussier et al., 1993; Mallet et al., 1994) reveals that the Kre2p (MALFLSKRLLR) sequence resembles only that of Ktr4p (MRFLSKRILK; Mallet et al., 1994) where the sequence FLSKR(I/L)L(K/R) is conserved in both enzymes. This may imply a common recep-tor for the two proteins. Interaction of the presumed receptor protein with a chimeric construct lacking the Kre2p stem domain but including a reporter luminal domain (KKP) may be partial, and insufficient to retain the fusion protein in the Golgi complex. To achieve full Golgi retention of a reporter luminal protein, interaction of the postulated receptor with the three nonenzymatic domains would be required.