Rim1 and Rabphilin-3 Bind Rab3-GTP by Composite Determinants Partially Related through N-terminal α-Helix Motifs*
Rim1 and Rabphilin-3 Bind Rab3-GTP by Composite Determinants Partially Related through N-terminal α-Helix Motifs*
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Rim1 和 Rabphilin-3 通过 N 端 α-螺旋基序部分相关的复合决定因素结合 Rab3-GTP*
DOI：
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发表时间：
2001
影响因子：
4.8
通讯作者：
M. Kilimann
中科院分区：
文献类型：
--
作者：
Xiaolu Wang;Bin Hu;B. Zimmermann;M. Kilimann
Rim1是突触前活跃区的一种蛋白质，该区域是质膜上专门用于神经递质胞分泌的区域，并与Rab3相互作用，Rab3是一种参与神经递质囊泡动力学的小GTPase。在这里，我们研究了负责Rab3结合的Rim1的分子决定因素，采用表面等离子体共振和重组，细菌表达Rab3和Rim1蛋白。结合GTP-但不饱和gdp - Rab3的位点定位在Rim1 N端附近的短α-螺旋序列（氨基酸19-55）。Rab3异构体A、C和D结合的亲和度相似（K D = 1 ~ 2 μm）。Rab6A-GTP低亲和力结合（K d = 16 μm），而Rab1B、-5、-7、-8和-11A不结合。邻近序列直至387个氨基酸，包括Rim1的差异剪接序列、锌指模块和SGAWFF基序，对Rab3结合的强度或特异性没有显著贡献，而螺旋内的点突变（R33G）则消除了Rab3的结合。Rim1的Rab3结合位点与rabphilin-3 Rab3结合区的n端α-螺旋结构相似，并且我们确实观察到Rab3A与rabphilin-3的低亲和力特异性结合（K d在10-100 μm量级），而Rab3A与rabphilin-3的高亲和力结合（K d = 10-20 nm）需要额外的序列，最高可达178个氨基酸。相比之下，aczonin的n端α-螺旋基序与Rim1的rab3结合位点序列相似，不结合Rab3A、-C、-D或其他几种rabb蛋白。这些结果在Triton X-100脑裂解物的天然，戊酰化Rab3的下拉实验中得到定性证实。Munc13与Rim1的锌指结构域结合，但不与rabphilin-3或aczonin锌指结合。在有胆酸作为清洁剂的情况下，对脑裂解液进行下拉实验，检测到syntaxin和Rab3的下游Rim1序列在56和387氨基酸之间的结合。然而，后者受到GTP的抑制而不是刺激。
Rim1 is a protein of the presynaptic active zone, the area of the plasma membrane specialized for neurotransmitter exocytosis, and interacts with Rab3, a small GTPase implicated in neurotransmitter vesicle dynamics. Here, we have studied the molecular determinants of Rim1 that are responsible for Rab3 binding, employing surface plasmon resonance and recombinant, bacterially expressed Rab3 and Rim1 proteins. A site that binds GTP- but not GDP-saturated Rab3 was localized to a short α-helical sequence near the Rim1 N terminus (amino acids 19–55). Rab3 isoforms A, C, and D were bound with similar affinities (K d = 1–2 μm). Low affinity binding of Rab6A-GTP was also observed (K d = 16 μm), whereas Rab1B, -5, -7, -8, or -11A did not bind. Adjacent sequences up to amino acid 387, encompassing differentially spliced sequences, the zinc finger module, and the SGAWFF motif of Rim1, did not significantly contribute to the strength or the specificity of Rab3 binding, whereas a point mutation within the helix (R33G) abolished binding. This Rab3 binding site of Rim1 is reminiscent of the N-terminal α-helix that is part of the Rab3-binding region of rabphilin-3, and indeed we observed low affinity, specific binding of Rab3A (K d on the order of magnitude of 10–100 μm) to this region of rabphilin-3 alone (amino acids 40–88), whereas additional sequences up to amino acid 178 are needed for high affinity Rab3A binding to rabphilin-3 (K d = 10–20 nm). In contrast, an N-terminal α-helix motif in aczonin, with sequence similarity to the Rab3-binding site of Rim1, did not bind Rab3A, -C, or -D or several other Rab proteins. These results were qualitatively confirmed in pull-down experiments with native, prenylated Rab3 from brain lysate in Triton X-100. Munc13 bound to the zinc finger domain of Rim1 but not to the rabphilin-3 or aczonin zinc fingers. Pull-down experiments from brain lysate in the presence of cholate as detergent detected binding to downstream Rim1 sequences, between amino acids 56 and 387, of syntaxin and of Rab3. The latter, however, was inhibited rather than stimulated by GTP.
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小 GTP 结合蛋白 Rab3A 对 GTP 的裂解与 α-蛇毒素诱导的突触小泡的胞吐作用有关。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Stahl,B;vonMollard,GF;Walch-Solimena,C;Jahn,R
通讯作者：
Jahn,R
Rab3C 是一种突触小泡蛋白，在刺激胞吐作用后从突触小泡解离。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
FischervonMollard,G;Stahl,B;Khokhlatchev,A;Südhof,TC;Jahn,R
通讯作者：
Jahn,R
高亲和力 Rab3 结合对于 Rabphilin 依赖性刺激分泌的增强是可有可无的。
影响因子：
4
作者：
Joberty,G;Stabila,PF;Coppola,T;Macara,IG;Regazzi,R
通讯作者：
Regazzi,R
Rab3 通过类似于 ras 募集 raf 的机制可逆地将 rabphilin 募集到突触小泡。
DOI：
--
发表时间：
1996
期刊：
The EMBO journal
影响因子：
--
作者：
Stahl,B;Chou,JH;Li,C;Südhof,TC;Jahn,R
通讯作者：
Jahn,R
rabphilin 3a 对胰岛素分泌细胞调节胞吐作用的刺激作用不需要与 Rab 3 介导的膜的缔合-解离循环。
影响因子：
6.6
作者：
Arribas,M;Regazzi,R;Garcia,E;Wollheim,CB;DeCamilli,P
通讯作者：
DeCamilli,P