Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells.

Abstract

Synaptophysin, an integral membrane protein of small synaptic vesicles, was expressed by transfection in fibroblastic CHO-Kl cells. The properties and localization of synaptophysin were compared between transfected CHO-Kl cells and native neuroendocrine PC12 cells. Both cell types similarly glycosylate synaptophysin and sort it into indistinguishable microvesicles. These become labeled by endocytic markers and are primarily concentrated below the plasmalemma and at the area of the Golgi complex and the centrosomes. A small pool of synaptophysin is transiently found on the plasma membrane. In CHO-Kl cells synaptophysin co-localizes with transferrin that has been internalized by receptor-mediated endocytosis. These findings suggest that synaptophysin in transfected CHO-Kl cells and neuroendocrine PC12 cells is directed into a pathway of recycling microvesicles which, in CHO cells, is shown to coincide with that of the transferrin receptor. They further indicate that fibroblasts have the ability to sort a synaptic vesicle membrane protein. Our results suggest a pathway for the evolution of small synaptic vesicles from a constitutively recycling organelle which is normally present in all cells.

Newly synthesized synaptophysin is transported to synaptic-like microvesicles via constitutive secretory vesicles and the plasma membrane

“…Taken together, these results indicate that PC12 cell SLMVs, like rat brain SSVs, are included in the column upon CPG chromatography but are slightly larger than the SSVs, which is consistent with previous morphological observations (Navone et al, 1986). We investigated the behaviour of constitutive secretory vesicles and plasma membrane fragments upon CPG chromatography, using as a marker the [35S]sulfate-labeled hsPG chased for 7 min ( Figure 8F days (data not shown), a value similar to that reported for total cellular synaptophysin (Johnston et al, 1989;Wiedenmann and Huttner, 1989 (Orci et al, 1985;Tooze and Tooze, 1986), occurred with the same kinetics and efficiency under the present conditions as reported previously after…”

“…We conclude that newly synthesized synaptophysin cycles between the plasma membrane and early endosomes prior to its incorporation into SLMVs. This conclusion is consistent with the observations that at steady state, some of the synaptophysin in PC12 cells is present in endosomal structures (Johnston et al, 1989;Clift-O'Grady et al, 1990) which have very recently been identified as early endosomes and the vesicles shuttling between them and the plasma membrane (Cameron et al, 1991). In light of these reports, we find it likely that the larger membranes containing [35S]sulfate-labeled synaptophysin after prolonged chase times, which were pelleted with the P2 fraction upon differential centrifugation, included early endosomes.…”

“…Fourth, the inhibitory effect of hypertonic sucrose on receptor-mediated endocytosis has been shown to be completely reversed within 5 min in isotonic medium (Daukas and Zigmond, 1985 Johnston et al (1989) and Clift-O'Grady et al (1990). After synthesis in the rough endoplasmic reticulum and passage through the Golgi complex, integral membrane proteins destined to SLMVs first move from the TGN to the plasma membrane in constitutive secretory vesicles (1, CSV).…”

“…as a membrane patch, throughout the exocytotic -endocytotic cycles. The colocalization of membrane proteins characteristic of SSVs and SLMVs with fluid phase markers of endocytosis (Johnston et al, 1989;Clift-O'Grady et al, 1990) and membrane proteins known to cycle between the plasma membrane and early endosomes (Cameron et al, 1991) does not allow one to distinguish between these two possibilities, de novo assembly versus membrane patch maintenance. Thus, a key question conceming the biogenesis of SSVs/SLMVs is: from which compartment are newly synthesized SSV/SLMV membrane proteins first assembled into these organelles?…”

Actin in the Drosophila embryo: Is there a relationship to developmental cue localization?

“…While neither of these proteins has been described as being present in the centrosomal region of Drosophila embryos, their distribution correlates with that of actin during the last four cleavages of the syncytial blastoderm (17,26,27) . Furthermore, the centrosome acts as a way-station for the recycling of small vesicles containing integral membrane proteins (28) , as well as for the transport of membranous organelles from the Golgi apparatus (29) . Thus, it is conceivable that peripheral membrane proteins, such as spectrin, might piggy-back on such vesicles to arrive at the apical or lateral membrane.…”