Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling.
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.
Chen
Mai-Lei
ML
Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA.
Green
David
D
Liu
Lei
L
Lam
Yung Carmen
YC
Mukai
Leona
L
Rao
Sujata
S
Ramagiri
Shobha
S
Krishnan
K S
KS
Engel
Jeff E
JE
Lin
Jim J-C
JJ
Wu
Chun-Fang
CF
D000818:Animals; D001522:Behavior, Animal / Q000502:physiology*; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D034281:Dynamins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D013347:Subcellular Fractions / Q000378:metabolism; D013572:Synaptic Vesicles / Q000235:genetics* / Q000378:metabolism
D029721:Drosophila Proteins; D034281:Dynamins; C439628:shi protein, Drosophila