RME-8 coordinates the activity of the WASH complex with the function of the retromer SNX dimer to control endosomal tubulation

Journal of Cell Science, May 2014

Retromer is a vital element of the endosomal protein sorting machinery and comprises two subcomplexes that operate together to sort membrane proteins (cargo) and tubulate membranes. Tubules are formed by a dimer of sorting nexins, a key component of which is SNX1. Cargo selection is mediated by the VPS35–VPS29–VPS26 trimer, which additionally recruits the WASH complex through VPS35 binding to the WASH complex subunit FAM21. Loss of function of the WASH complex leads to dysregulation of endosome tubulation, although it is unclear how this occurs. Here, we show that FAM21 also binds to the SNX1-interacting DNAJ protein RME-8. Loss of RME-8 causes altered kinetics of SNX1 membrane association and a pronounced increase in highly branched endosomal tubules. Building on previous observations from other laboratories, we show that these tubules contain membrane proteins that are dependent upon WASH complex activity for their localization to the plasma membrane. Therefore, we propose that the interaction between RME-8 and the WASH complex provides a means to coordinate the activity of the WASH complex with the membrane-tubulating function of the sorting nexins at sites where retromer-mediated endosomal protein sorting occurs.

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RME-8 coordinates the activity of the WASH complex with the function of the retromer SNX dimer to control endosomal tubulation

Caroline L. Freeman 0 Geoffrey Hesketh 0 Matthew N. J. Seaman 0 0 University of Cambridge, Cambridge Institute for Medical Research/Department of Clinical Biochemistry , Wellcome Trust/MRC Building , Addenbrooke's Hospital , Cambridge CB2 0XY , UK Retromer is a vital element of the endosomal protein sorting machinery and comprises two subcomplexes that operate together to sort membrane proteins (cargo) and tubulate membranes. Tubules are formed by a dimer of sorting nexins, a key component of which is SNX1. Cargo selection is mediated by the VPS35-VPS29-VPS26 trimer, which additionally recruits the WASH complex through VPS35 binding to the WASH complex subunit FAM21. Loss of function of the WASH complex leads to dysregulation of endosome tubulation, although it is unclear how this occurs. Here, we show that FAM21 also binds to the SNX1interacting DNAJ protein RME-8. Loss of RME-8 causes altered kinetics of SNX1 membrane association and a pronounced increase in highly branched endosomal tubules. Building on previous observations from other laboratories, we show that these tubules contain membrane proteins that are dependent upon WASH complex activity for their localization to the plasma membrane. Therefore, we propose that the interaction between RME-8 and the WASH complex provides a means to coordinate the activity of the WASH complex with the membrane-tubulating function of the sorting nexins at sites where retromer-mediated endosomal protein sorting occurs. - INTRODUCTION The endocytic system is a major hub for membrane protein sorting in the mammalian cell. Membrane proteins (cargo) in an early endosome are destined for one of a number of fates recycling to the plasma membrane (either directly or through the recycling endosomes), retrieval to the trans-Golgi network or degradation in the lysosome (reviewed in Seaman, 2008). Sophisticated sorting mechanisms are required if all cargoes are to reach their correct destination, and one important aspect of this is the partitioning of cargo into distinct tubular transport intermediates before onward trafficking. This requires the concerted action of several macromolecular protein complexes, which function to both select and package cargo and to generate transport intermediates. The retromer complex, which is composed of two loosely associated subcomplexes [the sorting nexin (SNX) dimer and the cargo-selective complex (CSC)], is a well-established example of protein machinery that combines the roles of membrane deformation and cargo selection. The SNX dimer, currently understood to comprise a combination of SNX1 or SNX2 with SNX5 or SNX6 (Wassmer et al., 2007), binds to phosphoinositol 3-phosphate through its phox homology domains and induces membrane curvature by the action of its C-terminal BAR (BinAmphiphysin-Rvs) domains (Kurten et al., 2001; Cheever et al., 2001; Yu and Lemmon, 2001; Cozier et al., 2002; Carlton et al., 2004). The tubules generated by the SNX dimer are stabilised by EHD1 (also known as RME-1), which associates with the CSC (Gokool et al., 2007b; Zhang et al., 2012). Although SNX5 and SNX6 do not drive membrane tubulation (van Weering et al., 2012), both interact with the p150Glued component of dynactin (Wassmer et al., 2009; Hong et al., 2009) and, therefore, link retromer-mediated protein sorting with microtubules through dynein. The retromer CSC is composed of VPS26, VPS35 and VPS29 and associates transiently with the SNX dimer (Swarbrick et al., 2011) but relies upon Rab7a and SNX3 for interaction with the endosomal membrane (Rojas et al., 2008; Seaman et al., 2009; Harterink et al., 2011; Vardarajan et al., 2012; Harrison et al., 2014). This association is negatively regulated by the Rab GTPase-activating protein TBC1D5 (Seaman et al., 2009). All three subunits of the CSC are required for the assembly and stability of the complex (Collins et al., 2005; Gokool et al., 2007a; Restrepo et al., 2007; Zhao et al., 2007), and cargo recognition is mediated by the VPS35 and VPS26 subunits (Nothwehr et al., 1999; Nothwehr et al., 2000; Arighi et al., 2004; Fjorback et al., 2012). Retromer was first identified as a mediator of endosome-to-Golgi retrieval (Seaman et al., 1997; Seaman et al., 1998), and its cargoes include Vps10p and the cation-independent mannose-6-phosphate receptor (CI-MPR, also known as IGF2R) (reviewed in Seaman, 2012). However, the recent characterisation of the retromer-interacting WASH complex has implicated retromer in the recycling of receptors to the plasma membrane (Gomez and Billadeau, 2009; Derivery et al., 2009; Temkin et al., 2011; Zech et al., 2011; Piotrowski et al., 2013; Steinberg et al., 2013 and reviewed in Seaman et al., 2013). The WASH complex is a pentameric protein complex [comprising WASH1, strumpellin (also known as KIAA0196), KIAA1033 (also known as the strumpellin and wash interacting protein, SWIP), FAM21 and CCDC53] that is responsible for the generation of branched actin networks on endosomes (...truncated)


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Caroline L. Freeman, Geoffrey Hesketh, Matthew N. J. Seaman. RME-8 coordinates the activity of the WASH complex with the function of the retromer SNX dimer to control endosomal tubulation, Journal of Cell Science, 2014, pp. 2053-2070, 127/9, DOI: 10.1242/jcs.144659