PubMed · 9188441
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J H Exton. 1997-06-20. New developments in phospholipase D.. https://doi.org/10.1074/jbc.272.25.15579
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In mature neurons, synaptic vesicles continuously recycle within the presynaptic nerve terminal. In developing axons which are free of contact with a postsynaptic target, constitutive membrane recycling is not localized to the nerve terminal; instead, plasma membrane components undergo cycles of exoendocytosis throughout the whole axonal surface (Matteoli et al., 1992; Kraszewski et al., 1995). Moreover, in growing Xenopus spinal cord neurons in culture, acetylcholine (ACh) is spontaneously secreted in the quantal fashion along the axonal shaft (Evers et al., 1989; Antonov et al., 1998). Here we demonstrate that in Xenopus neurons ACh secretion is mediated by vesicles which recycle locally within the axon. Similar to neurotransmitter release at the presynaptic nerve terminal, ACh secretion along the axon could be elicited by the action potential or by hypertonic solutions. We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar. These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion. Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface. Maturation of this machinery in the process of synaptic development would improve the fidelity of synaptic transmission during high-frequency stimulation of the presynaptic cell.
ADP-ribosylation factors (ARFs) are highly conserved, approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and have an important role in vesicular transport. Several cDNAs for ARF-like proteins (ARLs) have been cloned from human, Drosophila, rat, and yeast, although the biological function(s) of ARLs is unknown. We have identified a yeast gene (yARL3) encoding a protein that is structurally related (>43% identical) to the mammalian ARF-like protein ARP. Biochemical studies of purified recombinant yARL3 protein revealed properties similar to those of ARF and ARL proteins, including the ability to bind and hydrolyze GTP. Like other ARLs, recombinant yARL3 did not stimulate cholera toxin-catalyzed auto-ADP-ribosylation. Anti-yARL3 antibodies did not cross-react with yARFs or yARL1. yARL3 was not essential for cell viability, but disruption of yARL3 resulted in cold-sensitive cell growth. At the nonpermissive temperature, processing of alkaline phosphatase and carboxypeptidase Y in arl3 mutant was slowed. yARL3 might be required for protein transport from endoplasmic reticulum to Golgi or from Golgi to vacuole at nonpermissive temperatures. On subcellular fractionation, unlike its mammalian homologue ARP, yARL3 was detected in the soluble fraction but not in the plasma membrane. Indirect immunofluorescence analysis revealed that yARL3 when overexpressed was associated in part with the endoplasmic reticulum-nuclear envelope. Thus, the structural and functional characteristics of yARL3 indicate that it may have a unique role(s) in vesicular trafficking.
ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane.