Trafico vesicular

Published November 3, 2008

JCB: ARTICLE

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7
Raul Rojas,1 Thijs van Vlijmen,3 Gonzalo A. Mardones,1 Yogikala Prabhu,1 Adriana L. Rojas,2 Shabaz Mohammed,4 Albert J.R. Heck,4 Graça Raposo,5 Peter van der Sluijs,3 and Juan S. Bonifacino1
1

Cell Biology and Metabolism Program, Eunice Kennedy Shriver NationalInstitute of Child Health and Human Development and 2Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 3 Department of Cell Biology, University Medical Center Utrecht, 3584 CX Utrecht, Netherlands 4 Department of Biomolecular Mass Spectrometry, Utrecht University, 3584 CA Utrecht, Netherlands 5Institut Curie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 144, Paris 75248, France

THE JOURNAL OF CELL BIOLOGY

T

he retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomalmembranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate

(GTP) andlocalizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.

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Introduction
The retromer is a phylogenetically conserved multisubunit complex that mediates retrograde transport of transmembrane cargofrom endosomes to the TGN (Seaman, 2005; Bonifacino and Rojas, 2006; Bonifacino and Hurley, 2008). The best-characterized cargo for the mammalian retromer is the cation-independent mannose 6-phosphate receptor (MPR [CI-MPR]), one of two intracellular sorting receptors that participates in the delivery of acid hydrolases to lysosomes (Kornfeld, 1992). The CI-MPR binds newly synthesized acidhydrolases at the TGN and carries them within clathrin-coated vesicles to endosomes, where the hydrolases are released for eventual transport to lysosomes. The retromer functions to retrieve the unoccupied receptors to the
R. Rojas and T. van Vlijmen contributed equally to this paper. Correspondence to P. van der Sluijs: p.vandersluijs@umcutrecht.nl; or J.S. Bonifacino: juan@helix.nih.gov Abbreviationsused in this paper: AP, adapter protein; CI-MPR, cation-independent MPR; GMP-PNP, guanylyl-5 -imidodiphosphate; MPR, mannose 6-phosphate receptor; PI3K, phosphatidylinositol 3-kinase; PI3P, phosphatidylinositol 3-phosphate; SNX, sorting nexin; TfR, transferrin receptor; Vps, vacuolar protein sorting. The online version of this article contains supplemental material.

TGN, where they engage infurther cycles of acid hydrolase sorting. Depletion of retromer subunits by RNAi prevents this retrieval, leading to rerouting of the receptors to lysosomes and consequent leakage of newly synthesized acid hydrolases into the extracellular medium (Arighi et al., 2004; Carlton et al., 2004; Seaman, 2004; Rojas et al., 2007). The mammalian retromer comprises two biogenetically distinct subcomplexes of...