Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro.
Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro.
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带有 GAIP 的网格蛋白包被的囊泡在体外具有 GTP 酶激活蛋白活性。
DOI：
10.1073/pnas.96.12.6722
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发表时间：
1999
影响因子：
11.1
通讯作者：
Farquhar,MG
中科院分区：
文献类型：
--
作者：
Fischer,T;Elenko,E;McCaffery,JM;DeVries,L;Farquhar,MG
Gα-相互作用蛋白（GAIP）是Gα亚基的gtpase激活蛋白gap （gtpase激活蛋白）家族成员之一。先前，我们证明GAIP定位于网格蛋白包被囊泡（ccv）上。在这里，我们测试了富含gap的囊泡是否可以加速g αi蛋白的GTPase活性。定量免疫印迹法对含有囊泡载体（CV2）的大鼠肝脏部分进行了4.5倍的GAIP富集，免疫电镜下在CV2部分的部分囊泡上检测到GAIP。在重组g αi3中加入肝脏部分，检测GAP活性，只有CV2部分具有GAP活性。增加CV2的量可以增加GAP的活性，而使用针对GAIP C端抗体的CV2片段的免疫消耗则会降低GAP的活性。从大鼠肝脏中制备CCV组分，采用保持网格蛋白外壳的方案。GAIP在这些组分中富集，并通过免疫金标记在ccv上检测到。在重组g αi3蛋白中添加越来越多的CCV可提高GTPase的活性。我们得出结论，ccv具有Gαi3的GAP活性，并且膜相关的GAIP能够与Gαi3相互作用。异三聚体G蛋白与ccv上的GAP之间相互作用的重建为一个模型提供了生化证据，即G蛋白及其GAP在不同的膜上被区隔，并在囊泡融合时接触。或者，它们可能位于同一膜上，并在囊泡出芽时分离。
Gα-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for Gα subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIP-enriched vesicles could accelerate the GTPase activity of Gαiproteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5×) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant Gαi3and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant Gαi3protein increased the GTPase activity. We conclude that CCVs possess GAP activity for Gαi3and that membrane-associated GAIP is capable of interacting with Gαi3. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.