Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.

Abstract

Abstract. We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles . Based on preliminary im munofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Siidhof . 1989. EMBO (Eur Mol . Biol. Organ.) J. 8:2863-2872) . In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke . 1989. Cell. 59 :433-446) . We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found T HE release of classical, nonpeptide neurotransmitters from nerve endings is mediated by synaptic vesicles (SVs),' a homogeneously sized vesicle population (Carlson et al., 1978;Hutmer et al., 1983) that is concentrated in nerve endings at the presynaptic side of synapses (Peters et al ., 1976 ;De Camilli and Jahn, 1990). Within nerve terminals, SVs undergo an exo-endocytic recycling where at each local cycle they can be reloaded with neurotransmitter content (Ceccarelli et al ., 1973; Heuser and Reese, 1973). The recycling of synaptic vesicles appears to involve, at least under certain conditions, an endosome-like compartment (Heuser and Reese, 1973) . Thus, an identification of the proteins that participate in SV recycling may pro- that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifu gation . The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation . Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a cons...

EEA1, a Tethering Protein of the Early Sorting Endosome, Shows a Polarized Distribution in Hippocampal Neurons, Epithelial Cells, and Fibroblasts

“…The two populations are also similar morphologically with multivesicular and tubular domains, although the tubular domain is far more extensive in the somatodendritic region (Parton et al, 1992). The demonstration of differences in sensitivity to BFA, however, suggests that their molecular compositions may be distinct (Cameron et al, 1991). We have now shown that EEA1 is associated exclusively with endosomes of the somatodendritic domains of mature rat hippocampal neurons and not with axonal endosomes marked by internalized antibodies to synaptotagmin.…”

Redistribution of Synapsin I and Synaptophysin in Response to Electrical Stimulation in the Rat Neurohypophysial Nerve Endings.

“…Indeed, one recent line of evidence suggests that synaptophysin-positive microvesicles perform exocytotic-endocytotic recycling. Synaptophysin is targeted into a pathway of recycling microvesicles in both neuroendocrine cell line PC12 and synaptophysin-transfected fibroblasts (17), and in PC12 cells synaptophysin is further sorted into specialized vesicle population similar to authentic synaptic vesicles (3,4,20). Synaptophysin-positive microvesicles in pancreatic /3-cell are associated with GABAsynthesizing enzyme and are suggested to store GABA (31).…”

Role of the Vesicular Chloride Transporter ClC-3 in Neuroendocrine Tissue

“…Glycerol velocity gradients were performed as described (Cameron et al, 1991). Briefly, INS-1E cell supernatants were prepared as described above for sucrose gradients [except that the homogenization buffer was (in mM) 150 NaCl, 10 HEPES, 1 EGTA, 0.1 MgCl 2 , and 1ϫ Complete protease inhibitor (Roche), pH 7.4] and loaded onto a 5-25% continuous glycerol gradient with a 50% sucrose cushion.…”