Biogenesis of Synaptic Vesicle-like Structures in a Pheochromocytoma Cell Line PC-12

The presence of unique proteins in synaptic vesicles of neurons suggests selective targeting during vesicle formation. Endocrine, but not other cells, also express synaptic vesicle membrane proteins and target them selectively to small intracellular vesicles. We show that the rat pheochromocytoma cell line, PC12, has a population of small vesicles with sedimentation and density properties very similar to those of rat brain synaptic vesicles. When synaptophysin is expressed in nonneuronal cells, it is found in intracellular organelles that are not the size of synaptic vesicles. The major protein in the small vesicles isolated from PC12 cells is found to be synaptophysin, which is also the major protein in rat brain vesicles. At least two of the minor proteins in the small vesicles are also known synaptic vesicle membrane proteins. Synaptic vesicle-like structures in PC12 cells can be shown to take up an exogenous bulk phase marker, HRP. Their proteins, including synaptophysin, are labeled if the cells are surface labeled and subsequently warmed. Although the PC12 vesicles can arise by endocytosis, they seem to exclude the receptor-mediated endocytosis marker, transferrin. We conclude that PC12 cells contain synaptic vesicle-like structures that resemble authentic synaptic vesicles in physical properties, protein composition and endocytotic origin. S VNAPTIC vesicles were first identified when electron micrographs of nerve terminals revealed clusters of spherical vesicles with remarkably constant diameters. Generation of antibodies to purified synaptic vesicles showed that they contained unique proteins (Carlson and Kelly, 1980; Jones et al., 1981). To understand how synaptic vesicles are made therefore, we need to know how unique synaptic vesicle proteins are targeted selectively to the synaptic vesicle, and how uniform diameters are generated. A cell line containing synaptic vesicles would facilitate the study of synaptic vesicle biogenesis. Endocrine cell lines are good candidates since they express four of the known synaptic vesicle membrane proteins: p65 (Matthew et al., 1981), SV2 (Buckley and Kelly, 1985), synaptophysin or p38 (Jahn et al., 1985; Wiedenmann and Franke, 1985), and synaptobrevin (Baumert et al., 1989). Although some of these proteins can be detected in dense core secretory granules (Lowe et al., 1988; Obendorf et al., 1988), there is general consensus that in the pheochromocytoma cells line, PC12, the majority of the proteins are in small, electron-lucent vesicles of unknown function (Navone et al., 1986; Wiedenmann et al., 1988). The relationship of PC12 cell vesicles to authentic brain synaptic vesicles is not clear. Johnston et al. (1989) have recently suggested that the small PC12 vesicles are pleiomorphic, larger than synaptic vesicles, and are involved Anson W. Lowe's present address is Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305. in transferrin transport. On the other hand, synaptophysincontaining vesicles from PC12 cells cannot be distinguished from authentic rat brain synaptic vesicles on sizing columns (Wiedenmann et al., 1988). If the vesicles in PC12 cells are valid analogues of brain synaptic vesicles, and if they can be isolated, then analysis of membrane protein targeting to PC12 vesicles should clarify how synaptic vesicles are generated. We can identify in PCI2 cells, vesicles that are similar to authentic rat brain synaptic vesicles by several criteria. They have the same sedimentation velocity and buoyant density and contain at least two of the other synaptic vesicle membrane proteins, p65 and SV2. We can isolate them and show that their major protein appears to be synaptophysin, as is true for authentic synaptic vesicles. By two techniques we show that PC12 vesicles arise by endocytosis. The PC12 vesicles can therefore be considered valid analogues of brain synaptic vesicles by four criteria: size, density, protein composition, and endocytotic origin. They may not be completely analogous, however, because we have failed to demonstrate neurotransmitter retention. Since PC12 vesicles can be readily isolated, it is possible to compare targeting data generated by biochemical analysis to earlier morphological studies. The distribution of synaptophysin and endocytosed transferrin overlapped in immunofluorescence studies of both PC12 cells and also fibroblasts transfected with DNA encoding synaptophysin (Johnston et al., 1989). The conclusion was that a significant fraction of the synaptophysin is targeted to an organelle common to © The Rockefeller University Press, 0021-9525/90/05/1693/11 $2.00 The Journal of Cell Biology, Volume II0, May 199