Rab small GTPases are molecular switches that regulate vesicle trafficking in eukaryotic cells via
interactions with effector proteins. Human Rabs comprise about 70 members, while Saccharomyces cerevisiae
contains only 11 Rabs [1
]. Rabs perform a variety of functions including vesicle formation, motility, tethering, fusion, and the various steps are mediated via
recruitment of effector proteins [6
]. Rab GTPases contain a flexible C-terminal tail that is post-translationally modified at Cys residues to enable attachment to lipid bilayers [3
Rabs oscillate between an active (GTP) and inactive (GDP) states, regulated by GDP dissociation inhibitor (GDI), GTPase activating factors (GAPs), and GDP/GTP exchange factors[8
]. Local conformational changes in switch I and switch II, adjacent to the γ-phosphate, distinguish these states. Active Rabs reside in distinct sub-cellular compartments and mediate their biological effects via
recruitment of specific effector proteins. Some understanding of the molecular basis for effector recognition has emerged from the crystal structures of Rabs with the Rab-binding domains (RBDs) of effectors [3
]. Generally, the GTP-sensitive switch regions, switch I and II, as well as an invariant tryptophan residue in the interswitch region (between switch I and II) are important determinants of binding to α-helical motifs of RBDs. The exceptions are Early Endosomal Autoantigen 1 [EEA1; [10
]] and the Lowe Syndrome protein OCRL1[9
], which have non-helical RBDs.
proteins Ypt31 and Ypt32 are homologs with 87% sequence identities (95% homology) in their globular Ras fold. They regulate vesicle exit from late Golgi compartments [11
]. In the current model, Ypt31/32 together with phosphatidylinositol 4-phosphate recruit Sec2, which is also an exchange factor for Sec4. The nucleotide exchange action of Sec2 converts Sec4(GDP) to Sec4(GTP), and Sec2 also binds to Sec15, concomitantly releasing Ypt31/32 from the complex, thus facilitating the latter steps of vesicle delivery[13
]. Both Sec4 and Ypt31/32 also bind to the globular tail domain of the actin-based class V myosin, Myo2 [15
]. Sec15 is an effector of Sec4 and a component of the multi-protein exocyst, a docking complex that captures vesicles from the Golgi and promotes their fusion[18
]. The equivalent interaction in mammalian cells is the Rab11/Sec15, which has been observed in photoreceptor cells of Drosophila melanogaster
]. Thus, Ypt31/32 and Sec4 regulate consecutive steps in a complex cascade that involves Sec2, myosin V and the exocyst.
Here, we present the crystal structures of Ypt32 in the GTP and GDP bound states. The structure of active Ypt32 is compared with Sec4, which is required for a later step in the trafficking of secretory vesicles [20
]. Comparisons of their structures reveal significant differences in the conformation of switch II that are influenced by the underlying network of interactions with Rab sub-family specific (RabSF) regions. The structure of Ypt32(GDP) together with the previously determined structure of the Ypt31/GDI complex facilitates a complete description of the pathway leading from an active (membrane-bound) Ypt31/32 conformation to the GDI-bound (cytosolic) structure. Strikingly, the structures reveal that GDI induces a remodeling of the switch II helix prior to membrane extraction.