Chen Shirui, Gendelman Hannah K, Roche John P, Alsharif Peter, Graf Ethan R
Department of Biology, Amherst College, Amherst, Massachusetts, United States of America.
PLoS One. 2015 Aug 28;10(8):e0136938. doi: 10.1371/journal.pone.0136938. eCollection 2015.
At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.
在突触处，神经递质的释放由聚集在称为活性区的特殊突触前释放位点的分子机制调控。每个位点的活性区蛋白组成是释放效率的决定因素，并且可以被重塑以改变突触功能。小GTP酶Rab3先前被确定在果蝇神经肌肉接头处控制活性区蛋白向单个释放位点的分布中发挥新作用。Rab3在突触小泡循环中的作用已得到广泛研究；然而，Rab3控制活性区发育的机制仍然未知。为了探究这一机制，我们进行了突变分析，以确定Rab3在果蝇突触处功能的分子和结构要求。我们发现Rab3转运到突触并在释放位点间分布活性区成分需要GTP结合。相反，Rab3的水解活性对于此功能并非必需。通过结构-功能分析，我们确定了效应器结合开关区域内Rab3功能所需的确切残基，并确定膜附着至关重要。我们的研究结果表明，Rab3通过与标准Rab蛋白功能一致的囊泡对接机制控制活性区成分的分布。
