Darchen F, Senyshyn J, Brondyk W H, Taatjes D J, Holz R W, Henry J P, Denizot J P, Macara I G
CNRS URA 1112, Institut de Biologie Physico-Chimique, Paris, France.
J Cell Sci. 1995 Apr;108 ( Pt 4):1639-49. doi: 10.1242/jcs.108.4.1639.
Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise subcellular localization. It has been suggested that Rab3a, which is mainly expressed in neural and endocrine cells, might regulate exocytosis. Recently, direct experimental evidence supporting this hypothesis has been obtained. Consistent with such a role for Rab3a in regulated exocytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin cells. Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localization of this GTP-binding protein by using a combination of morphological techniques. Bovine chromaffin cells were labelled with an affinity-purified polyclonal anti-Rab3a antibody and analyzed by confocal microcopy. Rab3a was found to colocalize partially with dopamine beta-hydroxylase, a chromaffin granule marker. In agreement with this observation, immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicles were labelled. To eliminate the possibility that the staining was due to a Rab3a-related protein, we investigated by immunoelectron microscopy the localization of an epitope-tagged Rab3a expressed in rat PC12 cells. Secretory granules were specifically labelled, whereas clear microvesicles were not. These results provide further evidence supporting a specific association of the GTPase Rab3a with large dense core secretory vesicles.
Rab家族的小GTP酶控制着真核细胞内的囊泡运输。尽管Rab蛋白活性的分子机制尚未阐明，但已知这些蛋白的功能取决于其精确的亚细胞定位。有人提出，主要在神经和内分泌细胞中表达的Rab3a可能调节胞吐作用。最近，已获得支持这一假设的直接实验证据。与Rab3a在调节性胞吐作用中的这种作用一致的是，先前报道的Rab3a与突触小泡以及肾上腺嗜铬细胞中的分泌颗粒有特异性关联。由于基于亚细胞分级分离的后一结果存在争议，我们通过结合形态学技术重新研究了这种GTP结合蛋白的亚细胞定位。用亲和纯化的多克隆抗Rab3a抗体标记牛嗜铬细胞，并通过共聚焦显微镜进行分析。发现Rab3a与嗜铬颗粒标记物多巴胺β-羟化酶部分共定位。与该观察结果一致，免疫电子显微镜显示嗜铬颗粒有特异性染色。除了大的致密核心囊泡外，一些小囊泡也被标记。为了排除染色是由于Rab3a相关蛋白引起的可能性，我们通过免疫电子显微镜研究了在大鼠PC12细胞中表达的表位标记的Rab3a的定位。分泌颗粒被特异性标记，而清亮的微囊泡则未被标记。这些结果提供了进一步的证据，支持GTP酶Rab3a与大的致密核心分泌囊泡有特异性关联。
