Ohashi Takao, Hegi Seijirou, Fukunaga Takamasa, Hosomi Akira, Takegawa Kaoru
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
ASPEX Division, Research Center, Asahi Glass Co., Ltd., Yokohama, Kanagawa, 221-8755, Japan.
Appl Microbiol Biotechnol. 2020 Oct;104(20):8897-8909. doi: 10.1007/s00253-020-10881-9. Epub 2020 Sep 12.
The majority of Golgi glycosyltransferases are type II membrane proteins with a small cytosolic tail at their N-terminus. Several mechanisms for localizing these glycosyltransferases to the Golgi have been proposed. In Saccharomyces cerevisiae, the phosphatidylinositol-4-phosphate-binding protein ScVps74p interacts with the cytosolic tail of a Golgi glycosyltransferase and contributes to its localization. In this study, we investigated whether a similar mechanism functions in the fission yeast Schizosaccharomyces pombe. First, we identified gpp74 (GPP34 domain-containing Vps74 homolog protein), a gene encoding the S. pombe homolog of S. cerevisiae Vps74p. Deletion of the gpp74 gene resulted in the missorting of three Golgi glycosyltransferases, SpOch1p, SpMnn9p, and SpOmh1p, to vacuoles, but not SpAnp1p, indicating Gpp74p is required for targeting some glycosyltransferases to the Golgi apparatus. Gpp74p with an N-terminal GFP-tag localized to both the Golgi apparatus and the cytosol. Golgi localization of Gpp74p was dependent on the phosphatidylinositol 4-kinase SpPik1p. Site-directed mutagenesis of hydrophobic and basic amino acids in the cytosolic tails of SpOch1p and SpMnn9p resulted in their missorting to vacuoles, indicating these cytosolic N-terminal residues are important for localization in the Golgi. Unexpectedly, no prominent alternations in protein glycosylation were observed in S. pombe gpp74Δ cells, probably due to the residual Golgi localization of some SpOch1p and SpMnn9p in these cells. Collectively, these results demonstrate that both Gpp74p-dependent and Gpp74p-independent mechanisms are responsible for the Golgi localization of glycosyltransferases to the Golgi in S. pombe. KEY POINTS: • Gpp74p is involved in the localization of glycosyltransferases to the Golgi. • The cytosolic tails of glycosyltransferases are important for Golgi localization. • Gpp74p localizes to the Golgi in a SpPik1p-dependent manner.
大多数高尔基体糖基转移酶是II型膜蛋白，其N端有一个小的胞质尾巴。已经提出了几种将这些糖基转移酶定位到高尔基体的机制。在酿酒酵母中，磷脂酰肌醇-4-磷酸结合蛋白ScVps74p与高尔基体糖基转移酶的胞质尾巴相互作用，并有助于其定位。在本研究中，我们调查了类似的机制是否在裂殖酵母粟酒裂殖酵母中起作用。首先，我们鉴定了gpp74（含GPP34结构域的Vps74同源蛋白），一个编码粟酒裂殖酵母中酿酒酵母Vps74p同源物的基因。gpp74基因的缺失导致三种高尔基体糖基转移酶SpOch1p、SpMnn9p和SpOmh1p错误分选到液泡中，但SpAnp1p没有，这表明Gpp74p是将一些糖基转移酶靶向高尔基体所必需的。带有N端GFP标签的Gpp74p定位于高尔基体和胞质溶胶。Gpp74p的高尔基体定位依赖于磷脂酰肌醇4-激酶SpPik1p。对SpOch1p和SpMnn9p胞质尾巴中的疏水和碱性氨基酸进行定点诱变导致它们错误分选到液泡中，表明这些胞质N端残基对于在高尔基体中的定位很重要。出乎意料的是，在粟酒裂殖酵母gpp74Δ细胞中未观察到蛋白质糖基化的明显变化，这可能是由于这些细胞中一些SpOch1p和SpMnn9p仍定位于高尔基体。总的来说，这些结果表明，Gpp74p依赖性和Gpp74p非依赖性机制都负责粟酒裂殖酵母中糖基转移酶向高尔基体的定位。要点：• Gpp74p参与糖基转移酶向高尔基体的定位。• 糖基转移酶的胞质尾巴对于高尔基体定位很重要。• Gpp74p以SpPik1p依赖性方式定位于高尔基体。
