Abstract
Three major rat brain proteins were recently found to bind the SH3 domains of Grb2: synapsin I, dynamin, and a novel 145-kDa protein (p145) (McPherson, P. S., Czernik, A. J., Chilcote, T. J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J., and De Camilli, P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6486-6490). We have now used antibodies raised against p145 which had been purified by Grb2 affinity chromatography and SDS-polyacrylamide gel electrophoresis to characterize this protein. p145 is neuron-specific and co-distributes with dynamin in subcellular fractions of rat brain. Both proteins are partially recovered in soluble and particulate fractions. By immunofluorescence, p145 is closely co-localized with dynamin, which we show here to be highly concentrated in nerve terminals. In contrast to synapsin I, which is highly enriched in a purified synaptic vesicle fraction, both p145 and dynamin are de-enriched in this fraction. However, both proteins are found on membranes which are immunoisolated with antibodies directed against the synaptic vesicle membrane protein synaptophysin, suggesting that dynamin and p145 are localized in part on organelles which represent intermediate stages in the reformation of synapatic vesicles during recycling. Finally, we have determined that p145, like dynamin, is a phosphoprotein which undergoes dephosphorylation in response to nerve terminal depolarization. These findings suggest that p145 participates with dynamin in synaptic vesicle endocytosis and recycling.
Highlights
Both proteins are Another protein which participates in the synaptic vesicle found on membranes which are immunoisolated with cycle is dynamin
These findings suggest larity with the product of the Drosophila shibire gene (Chen et al, 1991; van der Bliek and Meyerowitz, 1991).A temperaturesensitive mutation in thisgene leads toa block of neurotransmitter release, due t o a lack of endocytosis of synaptic vesicle membranes following their exocytosis (Kosaka andIkeda, 1983; Ramaswami et al, 1994)
Synaptic vesicle membranes are internalized and reufsoerdthe generation of new neurotransmitter-filled synaptic vesicles by a pathway which is thought to involve clathrin-coated pits, coated vesicles, andearly endosomal intermediates(Heuser clathrin-mediated endocytosis in non-neuronal cells, as indicated by the impairment of coated vesicle formation in mammalian cells transfected with adominant negative mutant form of neuronal dynamin.Analogous to synapsinI, all dynaminisoforms have aproline-richdomain in their C termini (Obar et al, 1990; Nakata et al, 1993; Sontag et al, 1994; Cook et al, 1994)
Summary
In contrast to similar in their N-terminal halves, butdiverge in their C tersynapsin I, which is highly enriched in a purified syn- mini where synapsin I contains an uniqueproline-rich domain aptic vesicle fraction, both p145 and dynamin are de- (Sudhof et al, 1989). Both proteins are Another protein which participates in the synaptic vesicle found on membranes which are immunoisolated with cycle is dynamin.
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