Rieder S E, Banta L M, Köhrer K, McCaffery J M, Emr S D
Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla 92093-0668, USA.
Mol Biol Cell. 1996 Jun;7(6):985-99. doi: 10.1091/mbc.7.6.985.
In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.
在酿酒酵母中，诸如羧肽酶Y等液泡蛋白通过类似内体的中间区室从高尔基体转运至类似溶酶体的液泡。液泡蛋白分选（vps）突变体vps28是“E类”vps突变体的成员之一，它会在异常的类似内体的E类区室中积累液泡、内吞和晚期高尔基体标记物。对VPS28的序列分析揭示了一个开放阅读框，预计可编码一种由242个氨基酸组成的亲水性蛋白质。与此相符的是，针对Vps28p产生的多克隆抗血清识别出一种28 kDa的细胞质蛋白。VPS28的破坏导致了去往液泡的生物合成运输和内吞运输均出现中度缺陷。在vps28缺失突变体细胞中，可溶性液泡水解酶向液泡转运受损（约40 - 50%的羧肽酶Y分选错误）。内吞标记物FM 4 - 64（一种活性亲脂染料）内化后，在δvps28细胞中一个与增大的液泡相邻的小细胞内区室产生强烈染色。此外，液泡H⁺ - ATP酶在δvps28细胞的液泡周围E类区室中积累，从质膜内化的a因子受体Ste3p也是如此。电子显微镜分析显示存在一种由弯曲膜囊泡堆叠组成的新型区室。免疫定位研究表明液泡H⁺ - ATP酶与这种杯状囊泡结构相关，表明它对应于荧光显微镜下观察到的E类区室。我们的数据表明，vps28突变体中前液泡区室顺行和逆行运输的动力学缺陷导致蛋白质和膜在一个过度多层的内体区室中积累。我们提出Vps28p以及其他E类Vps蛋白可能（可能作为包被蛋白）促进从前液泡内体高效运输所需的运输中间体的形成。
