Membrane Dynamics and Its Regulation During Intracellular Vesicular Transport

酵母Sec 12 p是一种II型跨膜糖蛋白，是内质网（ER）形成转运囊泡所必需的。生化和形态学分析表明，Sec 12 p是本地化的ER通过两种机制：静态保留在ER和动态检索从高尔基体。我们分离的rer 1突变体将真实的Sec 12 p错误定位到高尔基体的后隔室。为了了解RER 1在Sec 12 p定位中的作用，我们克隆了该基因。RER 1编码一个188个氨基酸残基的疏水蛋白，具有四个膜跨度。rer 1无效突变体是可行的。即使在rer 1破坏的细胞中，Sec 12 p的免疫荧光染色ER，这意味着保留系统仍然在突变体中运行。为了确定Rer 1 p的亚细胞定位，通过免疫荧光显微镜观察HA表位标记的Rer 1 p。抗HA单克隆抗体以点状模式染色细胞，这是高尔基体蛋白的典型模式，与ER染色明显不同。亚细胞分级实验表明，Rer 1 p的行为像一个早期的高尔基体蛋白。从这些，我们得出结论，Rer 1 p的功能在高尔基体膜返回Sec 12 p已经逃脱了静态保留系统的ER。我们还进行了系统的分析，以确定ER定位信号在Sec 12 p对应于这两种机制。Sec 12 p的跨膜结构域（TMD）和N-末端结构域都显示出将蛋白定位于ER的能力，TMD的作用本身对于ER定位是有效和足够的，并且强烈依赖于RER 1。另一方面，N-末端结构域显示出适度的ER定位能力，其独立于RER 1。已测量甘露糖基修饰的速率以区分保留和回收。结果表明，TMD主要作为检索信号，N端结构域主要作为保留信号。

Yeast Sec12p, a type II transmembrane glycoprotein, is required for formation of transport vesicles from the endoplasmic reticulum (ER). Biochemical and morphological analyzes have suggested that Sec12p is localized to the ER by two mechanisms : static retention in the ER and dynamic retrieval from the Golgi apparatus. The rer1 mutant we isolated mislocalizes the authentic Sec12p to the later compartments of the Golgi. To understand the role of RER1 on Sec12p localization, we cloned the gene. RER1 encodes a hydrophobic protein of 188 amino acid residues with four membrane spans. The rer1 null mutant is viable. Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER,implying that the retention system is still operating in the mutant. To determine the subcellular localization of Rer1p, an HA-epitope-tagged Rer1p were observed by immunofluorescence microscopy. The anti-HA monoclonal antibody stains the cells in a punctate pattern which is typical for Golgi proteins and clearly distinct from the ER staining. Subcellular fractionation experiments indicated that the Rer1p behaves like an early Golgi protein. From these, we conclude that Rer1p functions in the Golgi membrane to return Sec12p that has escaped from the static retention system of the ER.We have also performed a systematic analysis to determine the ER localization signals in Sec12p corresponding to these two mechanisms. Both the transmembrane domain (TMD) and the N-terminal domain of Sec12p show the ability to localize the protein to the ER.The effect of the TMD is potent and sufficient by itself for the ER localization and is strongly dependent on RER1. On the other hand, the N-terminal domain shows a moderate ER-localization capability which is independent of RER1. The rate of mannosyl modification has been measured to distinguish between retention and retrieval. The results suggest that the TMD mainly acts as the retrieval signal and the N-terminal domain the retention signal.