Herman G A, Bonzelius F, Cieutat A M, Kelly R B
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0534.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12750-4. doi: 10.1073/pnas.91.26.12750.
Some cell types have cytoplasmic storage vesicles whose fusion with the cell surface is triggered by an extracellular signal. To explore the relationship between different classes of storage vesicles, we expressed, in the neuro-endocrine cell line PC12, the facilitative glucose transporter GLUT4, which is stored in small cytoplasmic vesicles in fat and muscle cells and mobilized to the cell surface when insulin is present. PC12 cells have two known types of storage vesicles, secretory granules and synaptic vesicles, but GLUT4 is targeted to neither. It is recovered, however, in a class of small vesicles that sediment approximately twice as fast as synaptic vesicles. Immunoelectron microscopy confirmed the presence of such small vesicles in transfected PC12 cells. By velocity sedimentation analysis, GLUT4 vesicles efficiently exclude the synaptic vesicle markers synaptophysin, SV2, and synaptobrevin; the transferrin receptor, a marker of conventional endocytosis; and the polymeric immunoglobulin receptor, a marker of transcytosis. The exclusion of synaptophysin and the transferrin receptor from most of the GLUT4-containing structures was confirmed by confocal immunofluorescence microscopy. Like synaptic vesicles, therefore, GLUT4 vesicles of PC12 cells appear to be a unique type of organelle. A GLUT4-containing organelle of identical sedimentation properties was found in transfected fibroblast cell lines and in rat adipocytes. On stimulation of the adipocytes with insulin, GLUT4 was translocated from the peak of small vesicles to faster sedimenting membranes. We propose that the class of vesicles described here is present in a wide range of cell types and is involved in transient modification of the cell surface.
某些细胞类型具有细胞质储存囊泡，其与细胞表面的融合由细胞外信号触发。为了探究不同类型储存囊泡之间的关系，我们在神经内分泌细胞系PC12中表达了易化型葡萄糖转运蛋白GLUT4，该蛋白在脂肪和肌肉细胞的小细胞质囊泡中储存，在有胰岛素存在时被转运到细胞表面。PC12细胞有两种已知类型的储存囊泡，即分泌颗粒和突触囊泡，但GLUT4并不靶向这两种囊泡。然而，它在一类小囊泡中被回收，这类小囊泡的沉降速度大约是突触囊泡的两倍。免疫电子显微镜证实了转染的PC12细胞中存在此类小囊泡。通过速度沉降分析，GLUT4囊泡能有效排除突触囊泡标记物突触素、SV2和突触结合蛋白；传统内吞作用的标记物转铁蛋白受体；以及转胞吞作用的标记物多聚免疫球蛋白受体。共聚焦免疫荧光显微镜证实了突触素和转铁蛋白受体被排除在大多数含GLUT4的结构之外。因此，与突触囊泡一样，PC12细胞的GLUT4囊泡似乎是一种独特的细胞器类型。在转染的成纤维细胞系和大鼠脂肪细胞中发现了具有相同沉降特性的含GLUT4细胞器。在用胰岛素刺激脂肪细胞时，GLUT4从小囊泡峰位转运至沉降更快的膜上。我们提出，这里描述的这类囊泡存在于多种细胞类型中，并参与细胞表面的瞬时修饰。
