Title: Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment Document date: 1995_11_2
ID: q1jx0n0l_38
Snippet: The capacity of chimeric proteins to function in vivo in Golgi-localized mannosylation was assessed by using a K1 killer toxin sensitivity assay (Fig. 9 ). K1 killer yeast strains secrete a small pore-forming toxin that requires a cell-wall receptor for function (Bussey, 1991) . KRE2 null mutations lead to shorter mannose chains on cell-wall mannoproreins disrupting the toxin receptor and leading to resistance (Hill et al., 1992; H/~usler et al.,.....
Document: The capacity of chimeric proteins to function in vivo in Golgi-localized mannosylation was assessed by using a K1 killer toxin sensitivity assay (Fig. 9 ). K1 killer yeast strains secrete a small pore-forming toxin that requires a cell-wall receptor for function (Bussey, 1991) . KRE2 null mutations lead to shorter mannose chains on cell-wall mannoproreins disrupting the toxin receptor and leading to resistance (Hill et al., 1992; H/~usler et al., 1992) . Yeast cells containing different chimeric proteins were assayed for killer toxin sensitivity and comparisons were made between a sensitive wild-type strain, a resistant kre2 null strain, and the null strain containing plasmids bearing wild-type KRE2 or different chimeric constructions. As can be seen in Fig. 9 , the kre2 null mutant is toxin resistant, showing no killing zone, but the same strain containing the KRE2 gene, or expressing KDKK or KPKK displays a large clear killing zone of ,-~20 mm similar to wild-type cells consistent with correct Golgi localization. Chimeric protein KD-K has also been shown to be localized to the Golgi complex but has a reduced zone size (15 mm) likely a consequence of its reduced mannosyltransferase activity. The reduced killing zone (10 mm) of cells expressing DKKK which has wild-type enzymatic activity in vitro indicates that it is not correctly retained in the Kre2p Golgi subcompartment. MR/KKK has also been shown to be localized to the vacuole, but displays an almost wild-type zone size (17.5 mm). Its catalytic domain appears to have a normal conformation as it possesses wild-type mannosyltransferase activity, but it is partially retained in the ER (see above). Thus, MR/KKK while slowly passing through the secretory pathway en route to the vacuole, is likely able to correctly mannosylate cell-wall proteins.
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