Anggono Victor, Robinson Phillip J
Cell Signalling Unit, Children's Medical Research Institute, Wentworthville, New South Wales, Australia.
J Neurochem. 2007 Aug;102(3):931-43. doi: 10.1111/j.1471-4159.2007.04574.x. Epub 2007 Apr 16.
Dynamin I mediates vesicle fission during synaptic vesicle endocytosis (SVE). Its proline-rich domain (PRD) binds the Src-homology 3 (SH3) domain of a subset of proteins that can deform membranes. Syndapin I, amphiphysin I, and endophilin I are its major partners implicated in SVE. Syndapin binding is controlled by phosphorylation at Ser-774 and Ser-778 in the dynamin phospho-box. We now define syndapin and endophilin-binding sites by peptide competition and site-directed mutagenesis. Both bound the same region of the dynamin PRD and both exhibited unusual bidirectional binding modes around core PxxP motifs, unlike amphiphysin which employed a class II binding mode. Endophilin binds to tandem PxxP motifs in the sequence (778)SPTPQRRAPAVPPARPGSR(796) in dynamin, with SPTPQ being an overhang sequence. In contrast, syndapin binding involves two components in the region (772)RRSPTSSPTPQRRAPAVPPARPGSR(796). It required a single PxxP core and a non-PxxP N-terminally anchored extension which bridges the phospho-box and may contribute to binding specificity and affinity. Syndapin binding is exquisitely sensitive to the introduction of negative charges almost anywhere along this region, explaining why it is a highly tuned phospho-sensor. Over-expression of dynamin point mutants that fail to bind syndapin or endophilin inhibit SVE in cultured neurons. Due to overlapping binding sites the interactions between dynamin and syndapin or endophilin were mutually exclusive. Because syndapin acts as a phospho-sensor, this supports its role in depolarization-induced SVE at the synapse, which involves dynamin dephosphorylation. We propose syndapin and endophilin function either at different stages during SVE or in mechanistically distinct types of SVE.
发动蛋白I在突触小泡内吞作用（SVE）过程中介导小泡裂变。其富含脯氨酸的结构域（PRD）与一组能够使膜变形的蛋白质的Src同源结构域3（SH3）结合。Syndapin I、发动蛋白I结合蛋白I和内吞蛋白I是其参与SVE的主要伙伴。Syndapin的结合受发动蛋白磷酸化盒中Ser-774和Ser-778位点磷酸化的控制。我们现在通过肽竞争和定点诱变来确定Syndapin和内吞蛋白的结合位点。两者都结合发动蛋白PRD的同一区域，并且在核心PxxP基序周围都表现出不同寻常的双向结合模式，这与采用II类结合模式的发动蛋白结合蛋白不同。内吞蛋白与发动蛋白序列（778）SPTPQRRAPAVPPARPGSR（796）中的串联PxxP基序结合，其中SPTPQ是一个突出序列。相比之下，Syndapin的结合涉及区域（772）RRSPTSSPTPQRRAPAVPPARPGSR（796）中的两个成分。它需要一个单一的PxxP核心和一个非PxxP的N端锚定延伸，该延伸连接磷酸化盒，可能有助于结合特异性和亲和力。Syndapin的结合对该区域几乎任何位置引入负电荷都极其敏感，这解释了它为何是一个高度灵敏的磷酸化传感器。未能结合Syndapin或内吞蛋白的发动蛋白点突变体的过表达会抑制培养神经元中的SVE。由于结合位点重叠，发动蛋白与Syndapin或内吞蛋白之间的相互作用是相互排斥的。因为Syndapin作为一个磷酸化传感器，这支持了它在突触处去极化诱导的SVE中的作用，这涉及发动蛋白的去磷酸化。我们提出Syndapin和内吞蛋白要么在SVE的不同阶段发挥作用，要么在机制上不同类型的SVE中发挥作用。
