Sorting And Transport Of Yeast Membrane Proteins

描述：（申请人提供） 本提案所述工作的总体目标是制定一项 对TGN和液泡膜蛋白分选的机制的理解， 在简单的真核生物酿酒酵母中组装。酵母已被证明 是一个很好的模型系统，既用于识别调节蛋白质， 真核细胞的膜运输和研究分子 这些蛋白质的功能。本提案的重点是 探讨TGN滞留和V-ATPase的分子机制 生物合成、组装、运输和靶向。突变分析已经 显示DPAP A的TGN保留涉及静态保留和 检索信号，Grdl 9 p已被发现直接结合到 DPAP A的胞质结构域。Grd 19 p复合物将被表征， 将分析其结合的DPAP A尾区。表征 的grd突变体揭示，Na+/H+交换Nhx 1 p是必需的， 运输出晚期内体/前液泡区室。Nhxlp的角色 离子转运将被调查，以了解如何内涵体膜的感觉 内腔离子环境 在酵母中存在两种V-ATP酶同工型，并且高尔基体/内体 由于靶向信号，同种型保留在液泡外。 Stvlp的胞质结构域，两个100 kDa整合膜亚基之一 同种型。这些信号将通过缺失和缺失来进一步表征。 定点突变以及具有保留Stv 1 p功能的蛋白质 将通过遗传和双杂交分析鉴定液泡外的基因。 先前的遗传分析已经确定了三种编码蛋白质的基因， 在液泡质子转运ATP酶（V-ATP酶）组装中的作用 在酵母内质网（ER）中。其中一种蛋白质，Vma 21 p， 一旦V-ATP酶被组装，就护送V-ATP酶复合物离开ER。一 将采用遗传学和生物化学相结合的方法来研究 Vma 21 p作为装配分子伴侣，囊泡包装蛋白， 和一个护送V-ATP酶复合体的人另外两个组装因子Vmal 2 p 和Vma 22 p，也将被调查的作用，在组装的复杂， 用于将V-ATP酶包装到从ER出芽的囊泡中。

DESCRIPTION: (provided by applicant) The overall goal of the work described in this proposal is to develop a mechanistic understanding of TGN and vacuole membrane protein sorting and assembly in the simple eukaryote Saccharomyces cerevisiae. Yeast has proved to be an excellent model system both for identifying the proteins regulating membrane traffic in eukaryotic cells and for investigating the molecular mechanisms by which these proteins function. The focus of this proposal to investigate the molecular mechanisms of TGN retention and V-ATPase biosynthesis, assembly, transport and targeting. Mutational analysis has revealed that TGN retention of DPAP A involves both static retention and retrieval signals, and the Grdl9p has been found to bind directly to the cytosolic domain of DPAP A. The Grd19p complex will be characterized, and the region of the DPAP A tail to which it binds will be analyzed. Characterization of the grd mutants has revealed that the Na+/H+ exchanger Nhx1p is required for transport out of the late endosomal/prevacuolar compartment. The role for Nhxlp ion transport will be investigated to learn how endosomal membranes sense the lumenal ion environment. There are two isoforms of the V-ATPase in yeast, and the Golgi/endosomal isoform is retained outside the vacuole due to targeting signals in the cytosolic domain of Stvlp, one of two 100 kDa integral membrane subunit isoforms. These signals will be further characterized by deletion and site-directed mutagenesis, and the proteins that function to retain Stv1p outside the vacuole will be identified by genetic and two-hybrid analyses. Previous genetic analysis has identified three genes that encode proteins that function in the assembly of the vacuolar proton-translocating ATPase (V-ATPase) in the yeast endoplasmic reticulum (ER). One of these proteins, Vma21p, appears to escort the V-ATPase complex out of the ER once the V-ATPase is assembled. A combined genetic and biochemical approach will be taken to investigate the potential role of Vma21p as an assembly chaperone, vesicle packaging protein, and an escort for the V-ATPase complex. The other two assembly factors, Vmal2p and Vma22p, will also be investigated for roles in assembly of the complex and for packaging of the V-ATPase into vesicles budding from the ER.