Sorting And Transport Of Yeast Membrane Proteins

描述：(申请人提供) 本提案中描述的工作的总体目标是开发一种 对TGN和液泡膜蛋白分选的机理认识 在简单的真核生物酿酒酵母中组装。酵母菌已经证明 是一种既能识别调节蛋白又能识别调节蛋白的优秀模型系统 真核细胞的膜运输及其分子研究 这些蛋白质发挥作用的机制。这项提案的重点是 探讨TGN滞留和V-ATPase的分子机制 生物合成、组装、运输和靶向。突变分析有 揭示了DPAP A的TGN保留包括静态保留和 检索信号，Grd19P已被发现直接与 DPAP A的胞液结构域将被表征，Grd19p复合体 DPAP的区域将分析它绑定到的尾巴。表征 已经揭示了Na/H交换器Nhx1p是 转运出晚期内体/泡前间隔。Nhxlp的角色 将对离子转运进行研究，以了解内膜如何感知 腔内离子环境。 酵母中的V-ATPase有两种亚型，一种是高尔基体，另一种是内体。 由于靶向信号位于液泡外，异构体被保留在液泡外。 Stvlp的胞液结构域，它是两个100 kDa的完整膜亚基之一 异构体。这些信号将进一步表现为删除和 定点突变和保留Stv1p的蛋白质 液泡外部将通过遗传和双杂交分析进行鉴定。 此前的遗传分析已经确定了三个编码蛋白质的基因 液泡质子转运ATPase(V-ATPase)组装中的功能 在酵母内质网(ER)中。其中一种蛋白质Vma21p出现了 一旦V-ATPase组装好，护送V-ATPase复合体离开ER。一个 将采取遗传和生化相结合的方法来研究 Vma21p作为组装伴侣、囊泡包装蛋白、 以及V-ATPase综合体的护送人员。另外两个装配因数VMal2p 和Vma22p，也将被调查在复合体和 用于将V-ATPase包装成从内质网萌发的囊泡。

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.