Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095.
Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095
Proc Natl Acad Sci U S A. 2017 Mar 28;114(13):3433-3438. doi: 10.1073/pnas.1615163114. Epub 2017 Mar 13.
Phosphoinositides serve as key membrane determinants for assembly of clathrin coat proteins that drive formation of clathrin-coated vesicles. At the -Golgi network (TGN), phosphatidylinositol 4-phosphate (PtdIns4P) plays important roles in recruitment of two major clathrin adaptors, Gga (Golgi-localized, gamma-adaptin ear homology, Arf-binding) proteins and the AP-1 (assembly protein-1) complex. The molecular mechanisms that mediate localization of phosphatidylinositol kinases responsible for synthesis of PtdIns4P at the TGN are not well characterized. We identify two motifs in the yeast phosphatidylinositol 4-kinase, Pik1, which are required for binding to the VHS domain of Gga2. Mutations in these motifs that inhibit Gga2-VHS binding resulted in reduced Pik1 localization and delayed accumulation of PtdIns4P and recruitment of AP-1 to the TGN. The Pik1 homolog in mammals, PI4KIIIβ, interacted preferentially with the VHS domain of GGA2 compared with VHS domains of GGA1 and GGA3. Depletion of GGA2, but not GGA1 or GGA3, specifically affected PI4KIIIβ localization. These results reveal a conserved role for Gga proteins in regulating phosphatidylinositol 4-kinase function at the TGN.
磷酯酰肌醇作为包被蛋白组装的关键膜决定因素，驱动网格蛋白包被小泡的形成。在高尔基网络（TGN），磷酸肌醇 4-磷酸（PtdIns4P）在募集两种主要的网格蛋白衔接蛋白（Gga 蛋白（高尔基体定位，γ 衔接蛋白耳同源，Arf 结合）和 AP-1（组装蛋白-1）复合物）中发挥重要作用。介导负责 TGN 中 PtdIns4P 合成的磷酯酰肌醇激酶定位的分子机制尚未得到很好的描述。我们在酵母磷酸肌醇 4-激酶 Pik1 中鉴定出两个基序，它们与 Gga2 的 VHS 结构域结合所必需的。这些基序的突变抑制了 Gga2-VHS 结合，导致 Pik1 定位减少、PtdIns4P 积累延迟以及 AP-1 募集到 TGN。哺乳动物的 Pik1 同源物 PI4KIIIβ 与 GGA2 的 VHS 结构域优先相互作用，而不是与 GGA1 和 GGA3 的 VHS 结构域相互作用。GGA2 的耗竭，而不是 GGA1 或 GGA3，特异性地影响 PI4KIIIβ 的定位。这些结果揭示了 Gga 蛋白在调节 TGN 中磷酯酰肌醇 4-激酶功能方面的保守作用。
