Characterization of RIN3 as a Guanine Nucleotide Exchange Factor for the Rab5 Subfamily GTPase Rab31
Characterization of RIN3 as a Guanine Nucleotide Exchange Factor for the Rab5 Subfamily GTPase Rab31
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DOI：
10.1074/jbc.m110.172445
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发表时间：
2011-07-08
影响因子：
4.8
通讯作者：
Katada, Toshiaki
中科院分区：
文献类型：
--
作者：
Kajiho, Hiroaki;Sakurai, Kyoko;Katada, Toshiaki
小的GTPase Rab5在gtp结合的无活性形式和gtp结合的活性形式之间循环，在早期内吞噬途径的膜出芽和运输中起重要作用。Rab5被多种液泡蛋白分选9 （VPS9）结构域的鸟嘌呤核苷酸交换因子激活。Rab21、Rab22和Rab31 （Rab5亚家族的成员）也参与早期核内体的转运。控制Rab5亚家族成员激活的机制尚不清楚。RIN （Ras和Rab相互作用因子）是一个多功能蛋白家族，除了Src同源性2 （SH2）和Ras关联结构域外，还具有VPS9结构域。我们研究了RIN家族成员是否在生化和细胞形态学水平上作为Rab5亚家族的鸟嘌呤核苷酸交换因子（gef）。在细胞游离和细胞GEF活性实验中，RIN3刺激gtp结合Rab31的形成。在HeLa细胞中，RIN3也形成增大的囊泡和管状结构，与Rab31共定位。相反，RIN3对Rab21没有明显的影响。我们还发现，在RIN3的SH2和RIN家族同源结构域之间的序列中，丝氨酸到丙氨酸的替换特异性地消除了它对Rab31的GEF作用，而不是Rab5。我们研究了RIN3是否影响阳离子依赖性甘露糖6-磷酸受体（CD-MPR）的定位，CD-MPR在反式高尔基网络和内吞区室之间运输。我们发现，RIN3部分地将CD-MPR从反式高尔基网络转运到外周囊泡，这取决于它的Rab31-GEF活性。这些结果表明，RIN3特异性地作为Rab31的GEF。
The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.