2 ISOFORMS OF DROSOPHILA DYNAMIN IN WILD-TYPE AND SHIBIRE(TS) NEURAL TISSUE - DIFFERENT SUBCELLULAR-LOCALIZATION AND ASSOCIATION MECHANISMS

The temperature-sensitive mutations of the shibire (shi) gene in Droso phila cause endocytic arrest, resulting in neurotransmission block and paralysis at high temperatures. However, underlying mechanism for the defects is not yet known. We examined the subcellular distribution of dynamin, a product of the shi gene, by immunoblotting and immunocytoc hemical assays. Two isoforms of dynamin with apparent M(r) of 92 kD an d 94 kD have been detected in wild-type and shi(ts) adult neural tissu e. The two isoforms were reproducibly associated with different subcel lular fractions of head homogenates. The 94 kD isoform is fractionated in the low speed (2,000 x g) pellet containing plasma membrane fragme nts, and the 92 kD isoform in the high speed (130,000 x g) pellet. In this procedure, very little dynamin remained in the high speed superna tant fraction. The 94 kD isoform represents the majority (65-75%) of t otal dynamin and appears to be a peripheral membrane protein. It can b e extracted from the low speed membrane pellet by high salt, Na2CO3 (p H 11) or Triton X-100 treatments. Extracted 94 kD dynamin from both wi ld-type and mutant homogenates is able to reassociate with artificial phospholipid vesicles at both permissive and restrictive temperatures. Binding of the 94 kD dynamin to liposomes appears to be pH-dependent, varying most significantly within the physiological pH range, which m ay be functionally important. The 92 kD isoform cannot be released by high salt or Na2CO3 treatments and only a small fraction is released b y Triton X-100, suggesting a different mechanism of association with c ell structures. The distribution of the two isoforms is not altered by the presence of stabilized microtubules in homogenates. No apparent d egradation or subcellular redistribution of mutant dynamin was detecte d in two shi(ts) alleles after heat shock or block of the dynamin GTPa se activity, suggesting that intracellular redistribution or degradati on of mutant dynamin are not involved in the endocytosis arrest in the se mutants. These observations resemble the effect of endocytosis' arr est by GTP-gamma-S in rat brain synaptosomes (Takei el al., 1995), in which dynamin is trapped at the neck of invaginated pits but is absent in the clathrin-coated distal end undergoing internalization. Our fin ding that endocytosis arrest by shi(ts) mutations and GTP-gamma-S do n ot lead to cumulation of dynamin in the low speed pellet fraction furt her suggests that the 94 kD isoform remains associated with the plasma membrane during coated vesicle pinch-off and that the two isoforms do not appear to correspond to different functional states of dynamin bu t are likely to be involved in separate cellular compartments within t he membrane cycling pathway (e.g., the plasma membrane, endosomes, and endoplasmic reticulum).