Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN. INTRODUCTIONMannose 6-phosphate receptors (MPRs) bind newly synthesized lysosomal hydrolases in the Golgi complex and deliver them to prelysosomes (Ghosh et al., 2003). There, the enzymes are released from MPRs, which are then recycled to the trans-Golgi network (TGN) for another round of enzyme delivery. There are two MPRs in most cell types: the cationindependent (CI)-MPR of ϳ300 kDa and the cation-dependent (CD)-MPR, a dimer of ϳ45 kDa. Both MPRs seem to require similar cellular components for their transport between compartments within mammalian cells (Ghosh et al., 2003).This laboratory has studied the process by which MPRs are transported from late endosomes to the Golgi. We have shown that MPR recycling requires Rab9 GTPase and its effector p40 (Lombardi et al., 1993;Riederer et al., 1994;Diaz et al., 1997), a cargo adaptor named TIP47 (for tail-interacting protein of 47 kDa; Diaz and Pfeffer, 1998;Burguete et al., 2005) and several other general transport factors (Itin et al., 1997). Using live cell video microscopy, we were able to track yellow fluorescent protein (YFP)-Rab9 -containing transport vesicles and watch them fuse with a cyan fluorescent protein-labeled trans-Golgi compartment (Barbero et al., 2002). That study showed that Rab9 is present on transport vesicles en route to the Golgi complex, but it did not provide additional clues to the mechanisms by which such vesicles are targeted.In an effort to understand the full scope of Rab9 function, we sought to identify additional partners for the active form of this GTPase. We report here the discovery that a Golgilocalized, putative tether of the Golgin protein family binds specifically to Rab9. Golgins contain long, predicted coiledcoil motifs and are important for Golgi organization, vesicle tethering, and secretory protein transport (for reviews, see Gillingham and Munro, 2003;Gleeson et al., 2004;Short et al., 2005). Several ...

Summary Rho GTPases are key regulators of the actin-based cytoskeleton; Rab GTPases are key regulators of membrane traffic. We report here that the atypical Rho GTPase family member, RhoBTB3, binds directly to Rab9 GTPase, and functions with Rab9 in protein transport from endosomes to the trans Golgi network. Gene replacement experiments show that RhoBTB3 function in cultured cells requires both RhoBTB3’s N-terminal, Rho-related domain, and C-terminal sequences that are important for Rab9 interaction.9 Biochemical analysis reveals that RhoBTB3 binds and hydrolyzes ATP rather than GTP. Rab9 binding opens the auto-inhibited RhoBTB3 protein to permit maximal ATP hydroysis. Because RhoBTB3 interacts with TIP47 on membranes, we propose that it may function to release this cargo selection protein from vesicles to permit their efficient docking and fusion at the Golgi.