Phillips A M, Smith M, Ramaswami M, Kelly L E
Department of Genetics, The University of Melbourne, Victoria, Australia 3010.
J Neurosci. 2000 Nov 15;20(22):8254-61. doi: 10.1523/JNEUROSCI.20-22-08254.2000.
The stoned locus of Drosophila melanogaster encodes two novel proteins, stonedA (STNA) and stonedB (STNB), both of which are expressed in the nervous system. Flies with defects at the stoned locus have abnormal behavior and altered synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicated a presynaptic function for stoned and suggested an involvement in vesicle cycling. Immunological studies revealed colocalization of the stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein synaptotagmin (SYT). We show here that stoned interacts genetically with synaptotagmin to produce a lethal phenotype. The STNB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the micro2-homology domain of STNB and the C2B domain of the SYTI isoform. The STNA protein is also found in association with vesicles, and it too exhibits an in vitro association with SYTI. However, we find that the bulk of STNA is in a nonmembranous fraction. By using the shibire mutant to block endocytosis, STNB is shown to be present on some synaptic vesicles before exocytosis. However, STNB is not associated with all synaptic vesicles. We hypothesize that STNB specifies a subset of synaptic vesicles with a role in the synaptic vesicle cycle that is yet to be determined.
果蝇的stoned基因座编码两种新蛋白，即stonedA（STNA）和stonedB（STNB），它们都在神经系统中表达。stoned基因座存在缺陷的果蝇具有异常行为和改变的突触传递。遗传相互作用，特别是与shibire（发动蛋白）突变的相互作用，表明stoned具有突触前功能，并提示其参与囊泡循环。免疫学研究揭示了stoned蛋白在神经肌肉接头处与整合突触囊泡蛋白突触结合蛋白（SYT）共定位。我们在此表明，stoned与突触结合蛋白发生遗传相互作用，产生致死表型。通过免疫共沉淀发现STNB蛋白与突触囊泡相关，谷胱甘肽S-转移酶下拉实验证明STNB的micro2-同源结构域与SYTI亚型的C2B结构域之间存在体外相互作用。还发现STNA蛋白与囊泡相关，并且它在体外也与SYTI存在关联。然而，我们发现大部分STNA存在于非膜部分。通过使用shibire突变体阻断内吞作用，显示STNB在胞吐作用之前存在于一些突触囊泡上。然而，STNB并非与所有突触囊泡都相关。我们假设STNB指定了一部分在突触囊泡循环中具有尚未确定作用的突触囊泡。
