Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane
Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane
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DOI：
10.1091/mbc.e03-10-0733
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发表时间：
2004-05-01
影响因子：
3.3
通讯作者：
Caplan, S
中科院分区：
文献类型：
--
作者：
Naslavsky, N;Boehm, M;Caplan, S
EHD 1与内化蛋白质向质膜的再循环有关。然而，EHD 1介导再循环的机制及其与Rab-family-controlled事件的关系尚未建立。为了进一步研究EHD 1的作用模式，我们试图确定新的相互作用伙伴。GST-EHD 1被用作诱饵，从牛和小鼠脑胞质溶胶中分离出类似于120-kDa的物质，其通过质谱被鉴定为二价Rab 4/Rab 5效应物Rabenosyn-5。我们将相互作用的位点映射到EHD 1的EH结构域和Rabenosyn-5的五个NPF基序中的前两个。免疫荧光显微镜研究表明，EHD 1和Rabenosyn-5部分共定位于囊泡和管状结构在体内。为了解决EHD 1和Rabenosyn-5的功能作用，我们首先证明RNA干扰（RNAi）可以显着降低每种蛋白质单独或组合的表达水平。EHD 1或Rabenosyn-5的耗竭延迟了转铁蛋白和I类主要组织相容性复合物向质膜的再循环。然而，EHD 1的耗竭导致内化货物在紧凑的细胞核隔室中积累，而Rabenosyn-5-RNAi导致其保留在分散的外周隔室中。两种蛋白质的同时RNAi消耗导致与单独Rabenosyn-5-RNAi观察到的表型相似的表型，表明Rabenosyn-5在EHD 1之前调节内吞再循环。我们的研究表明，Rabenosyn-5和EHD 1依次作用于蛋白质从早期内体转运到内体再循环区室并返回质膜。
EHD1 has been implicated in the recycling of internalized proteins to the plasma membrane. However, the mechanism by which EHD1 mediates recycling and its relationship to Rab-family-controlled events has yet to be established. To investigate further the mode of EHD1 action, we sought to identify novel interacting partners. GST-EHD1 was used as bait to isolate a similar to120-kDa species from bovine and murine brain cytosol, which was identified by mass spectrometry as the divalent Rab4/Rab5 effector Rabenosyn-5. We mapped the sites of interaction to the EH domain of EHD1, and the first two of five NPF motifs of Rabenosyn-5. Immunofluorescence microscopy studies revealed that EHD1 and Rabenosyn-5 partially colocalize to vesicular and tubular structures in vivo. To address the functional roles of EHD1 and Rabenosyn-5, we first demonstrated that RNA interference (RNAi) dramatically reduced the level of expression of each protein, either individually or in combination. Depletion of either EHD1 or Rabenosyn-5 delayed the recycling of transferrin and major histocompatibility complex class I to the plasma membrane. However, whereas depletion of EHD1 caused the accumulation of internalized cargo in a compact juxtanuclear compartment, Rabenosyn-5-RNAi caused its retention within a dispersed peripheral compartment. Simultaneous RNAi depletion of both proteins resulted in a similar phenotype to that observed with Rabenosyn-5-RNAi alone, suggesting that Rabenosyn-5 acts before EHD1 in the regulation of endocytic recycling. Our studies suggest that Rabenosyn-5 and EHD1 act sequentially in the transport of proteins from early endosomes to the endosomal recycling compartment and back to the plasma membrane.