Yao R, Maeda T, Takada S, Noda T
Department of Cell Biology, The Cancer Institute, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-Ku, Tokyo 170-8455, Japan.
Biochem Biophys Res Commun. 2001 Aug 31;286(4):771-8. doi: 10.1006/bbrc.2001.5430.
The frizzled gene is evolutionally conserved in a wide variety of organisms including mammals, and in Drosophila, frizzled is implicated in the development of planar polarity. We describe here the isolation and characterization of a Golgi protein, GOPC, as a frizzled interacting protein. GOPC comprises one PDZ domain, two coiled-coil motifs and two evolutionally conserved regions. Immunofluorescence studies indicated that a significant fraction of GOPC protein was localized in the Golgi apparatus. Using a series of deletion mutants, we show that both coiled-coil motifs and a C-terminal conserved region were required for its Golgi localization. Interestingly, deletion mutants that lack a N-terminal conserved region or coiled-coil motifs formed aggresome-like perinuclear structure. Interaction of GOPC and frizzled was observed both in vivo and in vitro, and the PDZ domain of GOPC and the C-terminal Ser/Thr-X-Val motif of frizzled were required for their interaction. Immunofluorescence studies indicated that, although frizzled was a membrane protein, it was localized at the Golgi apparatus as well, and colocalization of GOPC and frizzled at the Golgi apparatus was observed. Furthermore, when GOPC was coexpressed with frizzled, translocation of GOPC to the plasma membrane was observed. Importantly, brefeldin A interrupted not only the localization of GOPC to the Golgi apparatus but also the translocation of frizzled to the plasma membrane, indicating that the Golgi structure was required for the proper subcellular localization of frizzled. Taken together, these results indicate that GOPC may play a role in the vesicle transport of frizzled from the Golgi apparatus to the plasma membrane.
卷曲蛋白基因在包括哺乳动物在内的多种生物中具有进化保守性，在果蝇中，卷曲蛋白与平面极性的发育有关。我们在此描述了一种高尔基体蛋白GOPC作为卷曲蛋白相互作用蛋白的分离和特性。GOPC包含一个PDZ结构域、两个卷曲螺旋基序和两个进化保守区域。免疫荧光研究表明，相当一部分GOPC蛋白定位于高尔基体。使用一系列缺失突变体，我们表明卷曲螺旋基序和C末端保守区域对于其高尔基体定位都是必需的。有趣的是，缺乏N末端保守区域或卷曲螺旋基序的缺失突变体形成了类似聚集体的核周结构。在体内和体外均观察到GOPC与卷曲蛋白的相互作用，GOPC的PDZ结构域和卷曲蛋白的C末端丝氨酸/苏氨酸-X-缬氨酸基序是它们相互作用所必需的。免疫荧光研究表明，尽管卷曲蛋白是一种膜蛋白，但它也定位于高尔基体，并且观察到GOPC和卷曲蛋白在高尔基体中共定位。此外，当GOPC与卷曲蛋白共表达时，观察到GOPC向质膜的转位。重要的是，布雷菲德菌素A不仅中断了GOPC向高尔基体的定位，也中断了卷曲蛋白向质膜的转位，表明高尔基体结构对于卷曲蛋白正确的亚细胞定位是必需的。综上所述，这些结果表明GOPC可能在卷曲蛋白从高尔基体到质膜的囊泡运输中起作用。
