Giantin is thought to form a complex with p115 and Golgi matrix protein 130, which is involved in the reassembly of Golgi cisternae and stacks at the end of mitosis. The complex is involved in the tethering of coat pr...
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Giantin is thought to form a complex with p115 and Golgi matrix protein 130, which is involved in the reassembly of Golgi cisternae and stacks at the end of mitosis. The complex is involved in the tethering of coat protomer I vesicles to Golgi membranes and the initial stacking of reforming cisternae, Here we show that the nH2-terminal 15% of Giantin suffices to bind p115 in vitro and in vivo and to block cell-free Golgi reassembly. Because Giantin is a long, rod-like protein anchored to the membrane by its extreme COOH terminus, these results support the idea of a long, flexible tether linking vesicles and cisternae.
Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with nSF cause...
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Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with nSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the nMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not nMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.
Yeast Sec18p and its mammalian orthologue n-ethylmaleimide-sensitive fusion protein (nSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SnAPs), Sec18p/nSF induces ...
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Yeast Sec18p and its mammalian orthologue n-ethylmaleimide-sensitive fusion protein (nSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SnAPs), Sec18p/nSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SnAP receptors), During the ATP hydrolysis cycle, the Sec18p/nSF homohexamer undergoes a large-scale conformational change involving repositioning of the most n terminal of the three domains of each protomer, a domain that is required for SnAP-mediated interaction with SnAP receptors, Whether an internal conformational change in the n-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the n-terminal domain from Sec18p by x-ray crystallography. The Sec18p n-terminal domain consists of two beta-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SnAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled.
The n-ethylmaleimide-sensitive fusion protein (nSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes annSF homolog, dnSF1. Our previous work with...
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The n-ethylmaleimide-sensitive fusion protein (nSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes annSF homolog, dnSF1. Our previous work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dnSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dnSF1 function and to broaden our analysis of synaptic transmission to other gene products, we have performed a genetic screen for mutations that interact with comt. Here we report the isolation and analysis of four mutations that modify TS paralysis in comt, including two intragenic modifiers tone enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dnSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel alpha 1-subunit gene, cacophony (cac). Analysis of synaptic function in these mutants alone and in combination will further define the in vivo functions and interactions of specific gene products in synaptic transmission.
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