The Mon1-Ccz1 GEF activates the Rab7 GTPase Ypt7 via a longin fold-Rab interface and association with PI-3-P-positive membranes

Abstract

To function in fusion and signaling, Rab GTPases need to be converted into their active GTP form. We previously identified the conserved Mon1-Ccz1 complex as the guanine nucleotide exchange factor (GEF) of the yeast Rab7 GTPase Ypt7. To address the possible GEF mechanism, we generated a homology model of the predicted longin domains of Mon1 and Ccz1 using the Rabbinding surface of the TRAPP complex as a template. On the basis of this, we identified mutations in both yeast Mon1 and Ccz1 that block Ypt7 activation, without affecting heterodimer formation and intracellular localization of Mon1 and Ccz1 at endosomes. Strikingly, the activity of the isolated Mon1-Ccz1 complex for Ypt7 is highly stimulated on membranes, and is promoted by the same anionic phospholipids such as phosphatidylinositol-3-phosphate (PI3P), which also support membrane association of the GEF complex. Our data imply that the GEF activity of the Mon1-Ccz1 complex towards Rab7/Ypt7 requires the interface formed by their longin domains and profits strongly from its association with the organelle surface.

Structure and function of longin SNAREs

“…The longin domain is also found in GEF multi-protein complexes that are involved in membrane tethering events. In SAND heterodimeric complexes, the longin domain mediates the assembly of the two subunits, as well as being part of the binding interface with the associated Rab GTPase (Cabrera et al, 2014). In multimeric TRAPPI complexes, the longin domain is present in three subunits [sedlin, synbindin (Synb) and Bet5 (also known as TRAPPC2, TRAPPC4 and TRAPPC1, respectively)] (Brunet and Sacher, 2014).…”

Multiple Types of Guanine Nucleotide Exchange Factors (GEFs) for Rab Small GTPases

“…4A). In addition, the Mon1-Ccz1 complex preferentially binds to negatively charged phospholipids, including to phosphatidylinositol 3-phosphate (PI3P), which mainly localizes on early endosomes, and its interaction with the PI3P may facilitate encounters between Ypt7p and the Mon1-Ccz1 complex in specific endosomal compartments (Poteryaev et al, 2010;Cabrera et al, 2014). Similarly, the mammalian Mon1A-Ccz1 complex functions as the GEF for Rab7 on late endosomes, but not on lysosomes Yasuda et al, 2016).…”

“…For example, immobilization of Rabs on certain kinds of liposomes in GDP release assays could dramatically enhance GEF activity. Actually, the GDP release activity of the Mon1-Ccz1 complex for Ypt7p has been reported to increase 1600-fold when Ypt7p is immobilized on vesicles containing NTA (nickel-nitrilotriacetic acid) lipids (Cabrera et al, 2014), and a similar result was obtained for the Rab11-GEF REI-1 (Sakaguchi et al, 2015). Different GEF assay methods, e.g., an in vitro GEF assay as opposed to in vivo GEF assay, may also yield different results (GEF assay methods used to determine GEF activity are summarized in Table I).…”

Formation of Tubulovesicular Carriers from Endosomes and Their Fusion to the trans-Golgi Network

“…The concentration of PtdIns(3)P is also important for the recruitment of SAND/Mon1 to EEs so that it can bind to Rab5 (Poteryaev et al, 2010). Two GEFs for Rab7/ Ypt7 have been proposed in this process; the first one is the Vps39 subunit of the HOPS tethering complex (Rink et al, 2005) which binds to Mon1 in vitro (Nordmann et al, 2010) but does not have GEF activity in mammalian cells (Peralta et al, 2010), and the second is the Mon1-Ccz1 complex itself whose GEF activity in vitro is greatly enhanced when Ypt7 is placed on membranes (Cabrera et al, 2014). Active Rab7 then continues recruiting its own effectors, like Rab-interacting lysosomal protein (RILP), which interacts with the dyneinedynactin motor complex to move LEs along microtubules toward the perinuclear region of the cell ( Jordens et al, 2001), the retromer complex that retrieves receptors and other cargoes from lysosomal degradation (Arighi et al, 2004;Seaman, 2004) and the HOPS tethering complex that participates in lysosomal fusion events Solinger and Spang, 2013).…”