Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
PLoS One. 2013 Sep 3;8(9):e74715. doi: 10.1371/journal.pone.0074715. eCollection 2013.
Vps74p is a member of the PtdIns(4)P-binding protein family. Vps74p interacts with Golgi-resident glycosyltransferases and the coat protein COPI complex to modulate Golgi retention of glycosyltransferases and with the PtdIns(4)P phosphatase Sac1p to modulate PtdIns(4)P homeostasis at the Golgi. Genetic analysis has shown that Vps74p is required for the formation of abnormal elongated buds in cdc34-2 cells. The C-terminal region of Vps74p is required for Vps74p multimerization, Golgi localization, and glycosyltransferase interactions; however, the functional significance of the N-terminal region and three putative phosphorylation sites of Vps74p have not been well characterized. In this study, we demonstrate that Vps74p executes multiple cellular functions using different domains. We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing. Deletion of the N-terminal 90 amino acids, but not the 66 amino acids, of Vps74p impaired its ability to restore the elongated bud phenotype in cdc34-2/vps74Δ cells. Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells. Furthermore, we found that three N-terminal phosphorylation sites contribute to rapamycin hypersensitivity, although these phosphorylation residues are not involved in Vps74p localization, ability to modulate glycosyltransferase retention, or elongated bud formation in cdc34-2 cells. Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions.
Vps74p 是 PtdIns(4)P 结合蛋白家族的成员。Vps74p 与驻留在高尔基体中的糖基转移酶和 COPI 衣壳蛋白复合物相互作用，以调节糖基转移酶在高尔基体中的保留，与 PtdIns(4)P 磷酸酶 Sac1p 相互作用，以调节高尔基体中 PtdIns(4)P 的稳态。遗传分析表明，Vps74p 是 cdc34-2 细胞中异常伸长芽形成所必需的。Vps74p 的 C 端区域是 Vps74p 多聚化、高尔基体定位和糖基转移酶相互作用所必需的；然而，Vps74p 的 N 端区域和三个假定的磷酸化位点的功能意义尚未得到很好的表征。在这项研究中，我们证明了 Vps74p 使用不同的结构域执行多种细胞功能。我们发现，Vps74p 的 N 端 66 个氨基酸对于其高尔基体定位和细胞壁完整性的调节是可有可无的，但对于糖基转移酶的保留和糖蛋白的加工是必需的。Vps74p 的 N 端 90 个氨基酸的缺失，但不是 66 个氨基酸的缺失，削弱了它在 cdc34-2/vps74Δ 细胞中恢复伸长芽表型的能力。Sac1p 和 Arf1p 的缺失也特异性地减少了 cdc34-2 细胞中的异常伸长芽表型。此外，我们发现三个 N 端磷酸化位点导致雷帕霉素的超敏反应，尽管这些磷酸化残基不参与 Vps74p 的定位、调节糖基转移酶保留的能力或 cdc34-2 细胞中伸长芽的形成。因此，我们提出 Vps74p 可能使用不同的结构域与特定的效应物相互作用，从而不同地调节各种细胞功能。
