Jcb_201403026 1..14

Rab18 is one of the most highly conserved Rab GTPase regulators of membrane traffic being present in the last eukaryotic common ancestor of both the plant and animal kingdoms (Elias et al., 2012; Klöpper et al., 2012). A defined biological function has remained elusive, partly because it was lost in the budding yeast lineage used for the genetic screens for regulators of membrane traffic. Rab18 has been linked to lipid droplet formation (Martin et al., 2005; Ozeki et al., 2005), ER–Golgi trafficking (Dejgaard et al., 2008), and the regulation of secretory granules (Vazquez-Martinez et al., 2007) and peroxisomes (Gronemeyer et al., 2013), and may be exploited during hepatitis C infection (Salloum et al., 2013). However, no clear molecular function or site of action has been defined for Rab18, despite the fact that loss-of-function mutations are found in the autosomal-recessive human neurological and developmental disorder Warburg Micro syndrome (Bem et al., 2011). These children suffer from multiple specific developmental abnormalities in brain and eye development, profound global developmental delay, and neurodegeneration (Bem et al., 2011). However, the pattern of Rab18 conservation in both plants and animals indicates that the essential cellular function is unlikely to be specific to neuronal cells (Lütcke et al., 1994; Klöpper et al., 2012). In addition to Rab18, a known Rab regulatory complex is mutated in Warburg Micro syndrome (Aligianis et al., 2005, 2006; Handley and Aligianis, 2012; Handley et al., 2013). This is the Rab3 GTPase-activating protein (GAP) complex originally identified using biochemical purification from brain tissue as a cellular factor promoting GTP hydrolysis by Rab3 (Fukui et al., 1997; Nagano et al., 1998). Like Rab18, the Rab3GAP complex is both more widely conserved and more broadly expressed than Rab3 and is ubiquitously expressed in human tissues (Nagano et al., 1998), raising the possibility that regulation of Rab3 is not its only function. Simple logic suggests that the Rab3GAP complex and Rab18 act in the same pathway because mutations result in the same disease phenotype. We therefore set out to identify the cellular site of action of Rab18 and its functional relationship with Rab3GAP. Two simple alternatives present themselves, either The ancestral Rab GTPase Rab18 and both subunits of the Rab3GAP complex are mutated in the human neurological and developmental disorder Warburg Micro syndrome. Here, we demonstrate that the Rab3GAP complex is a specific Rab18 guanine nucleotide exchange factor (GEF). The Rab3GAP complex localizes to the endo­ plasmic reticulum (ER) and is necessary for ER targeting of Rab18. It is also sufficient to promote membrane re­ cruitment of Rab18. Disease­associated point mutations of conserved residues in either the Rab3GAP1 (T18P and E24V) or Rab3GAP2 (R426C) subunits result in loss of the Rab18 GEF and membrane­targeting activities. Support­ ing the view that Rab18 activity is important for ER struc­ ture, in the absence of either Rab3GAP subunit or Rab18 function, ER tubular networks marked by reticulon 4 were disrupted, and ER sheets defined by CLIMP­63 spread out into the cell periphery. Micro syndrome is therefore a dis­ ease characterized by direct loss of Rab18 function or loss of Rab18 activation at the ER by its GEF Rab3GAP. Rab18 and a Rab18 GEF complex are required for normal ER structure