Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling

Mai-Lei Chen; David Green; Lei Liu; Yung Carmen Lam; Leona Mukai; Sujata Rao; Shobha Ramagiri; K S Krishnan; Jeff E Engel; Jim J-C Lin; Chun-Fang Wu

doi:10.1002/neu.10101

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Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling

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Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling

Mai-Lei Chen, David Green, Lei Liu, Yung Carmen Lam, Leona Mukai, Sujata Rao, Shobha Ramagiri, K S Krishnan, Jeff E Engel, Jim J-C Lin, …

Journal of neurobiology, Vol.53(3), pp.319-329

11/15/2002

DOI: 10.1002/neu.10101

PMID: 12382260

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Abstract

Dynamin is a GTPase protein that is essential for clathrin-mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi(ts1) changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton-X extraction. Addition of Na(+) or K(+) can extract dynamin to the cytosolic (supernatant) fraction. The shi(ts1) mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature-sensitive alleles or wild type. Sensitivity to salt extraction in shi(ts1) was enhanced by GTP and nonhydrolyzable GTP-gammaS. The shi(ts1) mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature-sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi(ts1) flies recovered much more slowly from heat-induced paralysis than did other temperature-sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd(msf15)), which reduces the paralytic temperature threshold of other temperature-sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi(ts1). Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses.

Dynamins - metabolism

Synaptic Vesicles - metabolism

Drosophila

Dynamins - genetics

Behavior, Animal - physiology

Drosophila Proteins - chemistry

Drosophila Proteins - metabolism

Mutation - genetics

Subcellular Fractions - metabolism

Animals

Dynamins - chemistry

Protein Conformation

Drosophila Proteins - genetics

Synaptic Vesicles - genetics

Details

Title: Subtitle: Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling
Creators: Mai-Lei Chen - Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA
David Green
Lei Liu
Yung Carmen Lam
Leona Mukai
Sujata Rao
Shobha Ramagiri
K S Krishnan
Jeff E Engel
Jim J-C Lin
Chun-Fang Wu
Resource Type: Journal article
Publication Details: Journal of neurobiology, Vol.53(3), pp.319-329
DOI: 10.1002/neu.10101
PMID: 12382260
NLM abbreviation: J Neurobiol
ISSN: 0022-3034
eISSN: 1097-4695
Publisher: Wiley; United States
Grant note: NS 34889 / NINDS NIH HHS HD 18577 / NICHD NIH HHS
Language: English
Date published: 11/15/2002
Academic Unit: Iowa Neuroscience Institute; Biology
Record Identifier: 9984070128602771

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