The cytoplasmic region of -1,6-mannosyltransferase Mnn9p is crucial for retrograde transport from the Golgi to the endoplasmic reticulum in Saccharomyces cerevisiae

Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan; Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan

^* To whom correspondence should be addressed. Email: t.yoko-o{at}aist.go.jp.

	Abstract

In Saccharomyces cerevisiae, Och1p and Mnn9p mannosyltransferases are localized in the cis-Golgi. Attempts to live image Och1p and Mnn9p tagged with green fluorescent protein or red fluorescent protein, respectively, using a high-performance confocal laser scanning microscope system, resulted in simultaneous visualization of the native proteins in a living cell. Our observations revealed that Och1p and Mnn9p are not always co-localized to the same cisternae. The difference in the dynamics of these mannosyltransferases may reflect differences in the mechanism for their retention in the cis-Golgi, since it has been reported that Mnn9p cycles between the endoplasmic reticulum and the cis-Golgi, whereas Och1p does not (Todorow et al., 2000. Proc. Natl. Acad. Sci. USA 97:13643-13648). We investigated the localization of chimeric proteins of Mnn9p and Och1p in sec12 and erd1 mutant cells. A chimeric protein, M16/O16, which consists of the N-terminal cytoplasmic region of Mnn9p and the transmembrane and luminal region of Och1p, behaved like Mnn9p, suggesting that the N-terminal cytoplasmic region is important for the intracellular dynamics of Mnn9p. This observation is supported by results from subcellular fractionation experiments. Mutational analysis revealed that two arginine residues in the N-terminal region of Mnn9p are important for the chimeric protein to cycle between the endoplasmic reticulum and the Golgi.