Gokool Suzanne, Tattersall Daniel, Seaman Matthew N J
Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 0XY, UK.
Traffic. 2007 Dec;8(12):1873-1886. doi: 10.1111/j.1600-0854.2007.00652.x. Epub 2007 Oct 7.
Endosome-to-Golgi retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR) requires the function of the retromer complex. Retromer is localized to endosomes and comprises two distinct sub complexes: the vacuolar protein sorting 35/29/26 sub complex that binds cargo and the sorting nexin (SNX)1/2 sub complex that tubulates endosomal membranes. To identify up- or down-stream regulatory factors of retromer, a comparative proteomic strategy was employed. Protein profiles of endosomally enriched membranes, from either wild-type or retromer-deficient mouse cells, were compared to identify proteins with either elevated or reduced expression levels. Eps15 homology domain-containing protein-1 (EHD1) was identified in endosomally enriched membrane fractions from retromer-deficient cells and was found to be approximately threefold upregulated in the absence of retromer. EHD1 is localized to tubular and vesicular endosomes, partially colocalizes with retromer and is associated with retromer in vivo. Mutation of the nucleotide-binding P-loop of EHD1 results in a dominant-negative effect upon retromer localization and endosome-to-Golgi retrieval, while loss of EHD1 expression by RNA interference destabilizes SNX1-positive tubules and inhibits endosome-to-Golgi retrieval. The interaction between EHD1 and retromer and the requirement for EHD1 to stabilize SNX1-tubules establish EHD1 as a novel facilitating component of endosome-to-Golgi retrieval.
阳离子非依赖性甘露糖6-磷酸受体（CIMPR）从内体到高尔基体的回收需要逆转录复合物的功能。逆转录复合物定位于内体，由两个不同的亚复合物组成：结合货物的液泡蛋白分选35/29/26亚复合物和使内体膜形成微管的分选连接蛋白（SNX）1/2亚复合物。为了鉴定逆转录复合物的上游或下游调节因子，采用了比较蛋白质组学策略。比较野生型或逆转录复合物缺陷型小鼠细胞的内体富集膜的蛋白质谱，以鉴定表达水平升高或降低的蛋白质。含Eps15同源结构域蛋白-1（EHD1）在逆转录复合物缺陷型细胞的内体富集膜组分中被鉴定出来，并且发现在没有逆转录复合物的情况下其表达上调约三倍。EHD1定位于管状和囊泡状内体，与逆转录复合物部分共定位，并且在体内与逆转录复合物相关。EHD1核苷酸结合P环的突变对逆转录复合物的定位和内体到高尔基体的回收产生显性负效应，而通过RNA干扰使EHD1表达缺失会破坏SNX1阳性微管的稳定性并抑制内体到高尔基体的回收。EHD1与逆转录复合物之间的相互作用以及EHD1对稳定SNX1微管的需求确立了EHD1作为内体到高尔基体回收的一种新型促进成分。
