Pan X, Roberts P, Chen Y, Kvam E, Shulga N, Huang K, Lemmon S, Goldfarb D S
Department of Biology, University of Rochester, Rochester NY, 14627, USA.
Mol Biol Cell. 2000 Jul;11(7):2445-57. doi: 10.1091/mbc.11.7.2445.
Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including beta-catenin and importin alpha, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus-vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40-60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in "orphan" rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Delta cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Delta and vac8-Delta cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.
Vac8p是一种液泡膜蛋白，对于液泡的有效遗传与融合、胞质到液泡的靶向运输以及孢子形成来说必不可少。通过与其他犰狳结构域蛋白（包括β-连环蛋白和输入蛋白α）进行类比，我们推测Vac8p会在液泡膜上对接各种因子。双杂交和共纯化分析表明，Vac8p确实会与多个结合伴侣形成复合物，包括Apg13p、Vab2p和Nvj1p。在此，我们描述了Vac8p-Nvj1p复合物在核-液泡（NV）连接形成过程中出人意料的作用。Nvj1p是核膜的一种整合膜蛋白，通过其C端的40-60个氨基酸（aa）在胞质溶胶中与Vac8p相互作用。Nvj1p绿色荧光蛋白（GFP）集中在细胞核与一个或多个液泡紧密接触部位的小斑块或筏状结构中。此前，我们发现Vac8p-GFP集中在液泡间筏状结构中，可能在其中促进液泡-液泡融合，还集中在液泡簇边缘的“孤立”筏状结构中。Vac8p红色荧光标记的绿色荧光蛋白（YFP）的孤立筏状结构与Nvj1p蓝色荧光标记的绿色荧光蛋白（CFP）在NV连接部位共定位。绿色荧光蛋白标记的核孔复合体（NPC）被排除在NV连接之外。在vac8缺失细胞中，Nvj1p-GFP通常无法集中到筏状结构中，而是环绕细胞核分布。nvj1缺失细胞和vac8缺失细胞中均不存在NV连接。Nvj1p的过表达导致NV连接大量增殖。我们得出结论，Vac8p和Nvj1p是一种新型细胞器间连接装置的必要组成部分。
