Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5

Journal of Cell Science, Jul 2009

Matthew N. J. Seaman, Michael E. Harbour, Daniel Tattersall, Eliot Read, Nicholas Bright

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Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5

Matthew N. J. Seaman 1 Michael E. Harbour 1 Daniel Tattersall 0 1 Eliot Read 1 2 Nicholas Bright 1 0 Present address: Centre for Cutaneous Research, Institute of Cell and Molecular Science , 4 Newark Street, Whitechapel, London E1 2AT , UK 1 University of Cambridge, Cambridge Institute for Medical Research, Department of Clinical Biochemistry, Wellcome Trust/MRC Building, Addenbrookes Hospital , Hills Road, Cambridge CB2 0XY , UK 2 Present address: University of Cambridge, Department of Pathology , Tennis Court Road, Cambridge CB2 1QP , UK Summary Retromer is a membrane-associated heteropentameric coat complex that functions in the endosome-to-Golgi retrieval of the cation-independent mannose-6-phosphate receptor, the Wntless protein and other membrane proteins of physiological significance. Retromer comprises two functional subcomplexes: the cargo-selective subcomplex is a trimer of the VPS35, VPS29, ce VPS26 proteins, whereas the sorting nexin proteins, Snx1 and en Snx2 function to tubulate the endosomal membrane. Unlike the ic sorting nexins, which contain PtdIns3P-binding PX domains, lleS tdhoemcaairngso-asneldecittisverVecPrSu3it5m/2e9n/t26tocotmhpeleexndhoassonmoallipmide-mbibnrdainnge C remains mechanistically uncharacterised. In this study we show fo that the VPS35/29/26 complex interacts with the small GTPase lran RWaebs7hoawndthraetqtuhiereRsaRb7aKb715f7oNrimtsurteacnrtutihtamtecnatustoestthhee epnedriopshoemrael. u o J - Introduction The steady-state localisation of membrane proteins within the secretory and endocytic pathways is the result of dynamic sorting mechanisms in which membrane-associated coat proteins recognise distinct motifs within the cytoplasmic tails of membrane proteins to direct the membrane protein into a specific pathway (reviewed by Mancias and Goldberg, 2005; Bthune et al., 2006; Seaman, 2008). The coatomer/COPI coat recognises di-lysine motifs comprising KKxx or KxKxx to sort membrane proteins into a Golgito-endoplasmic reticulum (ER) pathway (Letourneur et al., 1994). Clathrin with the AP-1 adaptor binds to Yxx motifs at the transGolgi network (TGN) to sort membrane proteins into clathrin-coated vesicles (CCVs) (Bonifacino and Traub, 2003). Before the cytoplasmic coat can recognise and sort cargo proteins, the coat complex must be recruited to the appropriate membrane. This key regulatory step is often mediated by small guanine triphosphate (GTP) hydrolases (GTPases) that regulate coat recruitment through the binding and hydrolysis of GTP (reviewed by Spang, 2008). Both coatomer/COPI and AP-1 are recruited to Golgi or TGN membranes by the small GTPase ADP-ribosylation factor-1 (ARF1) (Stamnes and Rothman, 1993; Palmer et al., 1993; Orci et al., 1993). Interestingly, COPI also interacts with an ARF1 GTPase activating protein (GAP) that promotes the hydrolysis of GTP by ARF1, thereby triggering the release of ARF1 and coatomer/COPI from the membrane (Eugster et al., 2000; Szafer et al., 2001). Through its interactions with ARF1 and the ARF1 neuropathy, Charcot-Marie-Tooth disease, does not interact with the VPS35/29/26 complex, resulting in a weakened association with the membrane. We have also identified a novel retromer-interacting protein, TBC1D5, which is a member of the Rab GAP family of proteins that negatively regulates VPS35/29/26 recruitment and causes Rab7 to dissociate from the membrane. We therefore propose that recruitment of the cargo-selective VPS35/29/26 complex is catalysed by Rab7 and inhibited by the Rab-GAP protein, TBC1D5. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/14/2371/DC1 GAP, coatomer/COP-1 thereby regulates its own membrane association. The ARF1-related small GTPase, Sar1, functions in both yeast and mammalian cells to drive recruitment of the COPII coat to the ER and the Sec23 component of the COPII coat complex acts as a GAP for Sar1 producing a similar self-regulatory loop to that for ARF1 and coatomer/COPI (Yoshihisa et al., 1993; Barlowe et al., 1994). The Rab family of small GTPases also have a crucial regulatory role in the secretory and endocytic pathways by functioning as organisers of discrete regions of the endo-membrane system and by mediating the tethering interactions that precede docking and fusion of donor and acceptor membranes (reviewed by Fukuda, 2008; Zerial and McBride, 2001). Amongst the best characterised of the Rab proteins are Rab5 and Rab7. The Rab5 protein acts early in the endocytic pathway and is required for the fusion of endocytic vesicles and early endosomes (Bucci et al., 1992). Rab5 has also been shown to regulate the dynamics of CCVs at the plasma membrane (McLauchlan et al., 1998; Semerdjieva et al., 2008). Rab7 acts later in the endocytic pathway and is required to mediate the fusion of late-endosomes with lysosomes (Bucci et al., 2000). As early endosomes mature they undergo Rab conversion in which Rab5 is progressively displaced by Rab7 (Rink et al., 200 (...truncated)


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Matthew N. J. Seaman, Michael E. Harbour, Daniel Tattersall, Eliot Read, Nicholas Bright. Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5, Journal of Cell Science, 2009, pp. 2371-2382, 122/14, DOI: 10.1242/jcs.048686