Gleeson P A, Anderson T J, Stow J L, Griffiths G, Toh B H, Matheson F
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria, Australia.
J Cell Sci. 1996 Dec;109 ( Pt 12):2811-21. doi: 10.1242/jcs.109.12.2811.
Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTP gamma S or AlF4- resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (approximately 10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.
运输囊泡的形成需要胞质蛋白与膜结合。我们之前描述过一种对布雷菲德菌素A敏感的亲水性蛋白（p230），它含有非常高频率的七肽重复序列，存在于胞质溶胶中并与高尔基体膜相关联。我们在此表明，通过使用α2,6唾液酸转移酶作为区室特异性标记对HeLa细胞冷冻切片进行免疫金标记，p230定位于反式高尔基体网络。研究了G蛋白激活剂在p230与高尔基体膜结合以及囊泡生物发生中的作用。用GTPγS或AlF4-处理经链球菌溶血素-O通透处理的HeLa细胞，导致p230在高尔基体膜上积累。此外，用AlF4-处理分离的高尔基体膜以诱导囊泡积累后的免疫标记显示，p230主要定位于反式高尔基体网络衍生的出芽结构和小被膜囊泡的细胞质表面。p230标记的囊泡有一层薄的（约10nm）电子致密细胞质被膜，并且可以很容易地与网格蛋白包被囊泡区分开来。对穿孔细胞或处理过的高尔基体膜冷冻切片进行双重免疫金标记显示，p230和我们在此表明是不同分子的与反式高尔基体网络相关的p200，似乎定位于从反式高尔基体网络出芽的不同囊泡群体上。这些结果强烈表明存在源自反式高尔基体网络的不同群体的非网格蛋白包被囊泡。由于p230在胞质溶胶与TGN膜的芽/囊泡之间循环，这一过程受G蛋白调节，我们提出p230参与特定群体的非网格蛋白包被囊泡的生物发生。
