Membrane Dynamics and Its Regulation During Intracellular Vesicular Transport

酵母Sec12p是一种II型跨膜糖蛋白，是从内质网(ER)形成运输小泡所必需的。生化和形态分析表明，Sec12p通过两种机制定位于内质网：静态保留在内质网和动态从高尔基体检索。我们分离到的rer1突变体将真正的Sec12p错误地定位到高尔基体的后面的隔室。为了了解RER1在Sec12p定位中的作用，我们克隆了该基因。RER1编码188个氨基酸残基、4个膜跨度的疏水蛋白。Rer1零突变体是可行的。即使在rer1被破坏的细胞中，Sec12p的免疫荧光也对ER染色，这意味着保留系统仍然在突变体中运行。为了确定Rer1p的亚细胞定位，用免疫荧光显微镜观察了HA表位标记的Rer1p。抗HA单抗将细胞染成点状，这是高尔基体蛋白的典型特征，与ER染色明显不同。亚细胞分离实验表明，Rer1p的行为类似于早期的高尔基体蛋白。由此，我们得出结论：Rer1p在高尔基体膜上起作用，使逃逸出内质网静态保留系统的Sec12p返回。我们还进行了系统的分析，确定了与这两种机制相对应的Sec12p中的ER定位信号。Sec12p的跨膜结构域(TMD)和N-末端结构域都显示出将蛋白质定位到内质网的能力，TMD本身对内质网定位是有效和充分的，并且强烈依赖于RER1。另一方面，N-末端结构域显示出中等的ER本地化能力，该能力独立于RER1。甘露醇修饰的速度已经被测量以区分保留和恢复。结果表明，TMD主要起恢复信号的作用，N端域主要起保留信号的作用。

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.