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

Data obtained with chimeric proteins KDKK, KPKK, KD-K, and KKP clearly show that the Kre2p TMD is dispensable for Golgi retention and not sufficient to retain a reporter protein in the yeast Golgi complex. Our results are, however, at variance with conclusions reached in a recent study of Chapman and Munro (1994) and we address this below. In a first set of experiments, Chapman and Munro (1994) tested the role of the Kre2p TMD with a chimeric protein consisting of a full-length fusion of Kre2p/Mntlp linked to the SUC2-encoded invertase by a small region containing a Kex2p-dependent cleavage site (chimeric protein MMMI). The mislocalization of this protein was expected to result in production of soluble invertase when passing through the Kex2p-containing late Golgi compartment. This fusion protein was inferred to be in the Golgi complex as no invertase was secreted from the cell. When the TMD of Kre2p was replaced with that of Pho8p (MPMI) in this protein, it appeared to be mislocalized to some extent, as some invertase was secreted from the cell. This result was interpreted to indicate a role for the Kre2p TMD in Golgi retention. However, the intracellular localization of this chimeric protein was not visualized by immunofluorescence. We replaced the Kre2p TMD in the native protein with two different nonrelated TMD's, and showed that both proteins (KDKK and KPKK) were Golgi localized by using indirect immunofluorescence microscopy. In addition, we tested that the proteins had the right conformation by measuring their glycosylation in vivo, and their mannosyltransferase activity in vitro and in vivo. These proteins (KDKK and KPKK) behaved phenotypically as wild-type Kre2p in our tests with no indication that the Kre2p TMD in the context of a native protein is necessary for proper retention. A possible explanation of the Chapman and Munro (1994) results is that the Kre2p TMD is somehow necessary for Golgi retention in the context of those large heterologous proteins perhaps because some retention property of the luminal domain of Kre2p has been disrupted in the invertase fusion. Unfortunately, the ER retention of chimeric protein KPKP did not permit resolution of this question.