McGough Ian J, Steinberg Florian, Gallon Matthew, Yatsu Ayaka, Ohbayashi Norihiko, Heesom Kate J, Fukuda Mitsunori, Cullen Peter J
The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan.
J Cell Sci. 2014 Nov 15;127(Pt 22):4940-53. doi: 10.1242/jcs.156299. Epub 2014 Oct 2.
Retromer is a protein assembly that orchestrates the sorting of transmembrane cargo proteins into endosome-to-Golgi and endosome-to-plasma-membrane transport pathways. Here, we have employed quantitative proteomics to define the interactome of human VPS35, the core retromer component. This has identified a number of new interacting proteins, including ankyrin-repeat domain 50 (ANKRD50), seriologically defined colon cancer antigen 3 (SDCCAG3) and VPS9-ankyrin-repeat protein (VARP, also known as ANKRD27). Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo-specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma-membrane sorting. Extending this, suppression of the retromer-associated WASH complex did not uniformly affect retromer cargo, thereby confirming cargo-specific functions for retromer-interacting proteins. Further analysis of the retromer-VARP interaction identified a role for retromer in endosome-to-melanosome transport. Suppression of VPS35 led to mistrafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway. Overall, these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex.
逆转录酶复合物是一种蛋白质组装体，它协调跨膜货物蛋白进入从内体到高尔基体以及从内体到质膜的运输途径。在此，我们运用定量蛋白质组学来定义人类VPS35（逆转录酶复合物的核心组分）的相互作用组。这鉴定出了许多新的相互作用蛋白，包括锚蛋白重复结构域50（ANKRD50）、血清学定义的结肠癌抗原3（SDCCAG3）和VPS9-锚蛋白重复蛋白（VARP，也称为ANKRD27）。这些蛋白的缺失导致了逆转录酶复合物依赖性货物的运输缺陷，但不同的以及货物特异性的效应表明在逆转录酶复合物介导的从内体到质膜的分选过程中存在惊人程度的功能异质性。进一步拓展这一研究，抑制与逆转录酶复合物相关的WASH复合物并不会一致地影响逆转录酶复合物的货物，从而证实了逆转录酶相互作用蛋白具有货物特异性功能。对逆转录酶复合物-VARP相互作用的进一步分析确定了逆转录酶复合物在从内体到黑素小体运输中的作用。抑制VPS35导致黑素生成酶酪氨酸酶和酪氨酸相关蛋白1（Tyrp1）的运输错误，这表明逆转录酶复合物在这一运输途径中与VARP协同发挥作用。总体而言，这些数据揭示了逆转录酶复合物介导的分选过程中隐藏的复杂性，并为我们对这一重要分选复合物的分子理解开辟了新的方向。
