Open AccessThe Cytoplasmic Region of α-1,6-Mannosyltransferase Mnn9p Is Crucial for Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum in Saccharomyce s cerevisiae
Author(s) -
Michiyo Okamoto,
Takehiko Yoko-o,
Tokichi Miyakawa,
Yoshifumi Jigami
Publication year - 2008
Publication title -
eukaryotic cell
Language(s) - English
Resource type - Journals
eISSN - 1535-9778
pISSN - 1535-9786
DOI - 10.1128/ec.00333-07
Subject(s) - golgi apparatus , endoplasmic reticulum , biology , cytoplasm , secretory pathway , microbiology and biotechnology , subcellular localization , copi , saccharomyces cerevisiae , transport protein , calnexin , fusion protein , fluorescence recovery after photobleaching , green fluorescent protein , biochemistry , yeast , gene , calreticulin , membrane , recombinant dna
InSaccharomyces cerevisiae , Och1p and Mnn9p mannosyltransferases are localized in thecis -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 colocalized to the same cisternae. The difference in the dynamics of these mannosyltransferases may reflect differences in the mechanisms for their retention in thecis -Golgi, since it has been reported that Mnn9p cycles between the endoplasmic reticulum and thecis -Golgi whereas Och1p does not (Z. Todorow, A. Spang, E. Carmack, J. Yates, and R. Schekman, Proc. Natl. Acad. Sci. USA97: 13643-13648, 2000). We investigated the localization of chimeric proteins of Mnn9p and Och1p insec12 anderd1 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 apparatus.