Vps9, Rabex-5 and DSS4: proteins with weak but distinct nucleotide-exchange activities for Rab proteins.

The activities of three Rab-specific factors with GDP/GTP exchange activity, Vps9p, Rabex-5 and DSS4, with their cognate GTPases, Ypt51p, Rab5 and Ypt1p, have been analysed quantitatively. In contrast to other exchange factors examined and to DSS4, Vps9p, and by analogy probably Rabex-5, have considerably lower affinity than GDP to the respective GTPases. In keeping with this, they are relatively weak exchangers, with a maximal rate constant for GDP release from the ternary complex between exchange factor, GTPase and GDP of ca 0.01 s(-1), which is several orders of magnitude lower than for other exchange factors examined. If interaction with these proteins is a mandatory aspect of the Rab cycle, this suggests that the overall rate of cycling might be controlled at this point of the cycle. Surprisingly, DSS4, which has the thermodynamic potential to displace GDP effectively from Ypt1p, also does this very slowly, again with a maximal rate constant of ca 0.01 s(-1). An additional, and based on present knowledge, unique, feature of the Ypt1p.DSS4 complex, is that the association of GTP (or GDP) is more than 10(3)-fold slower than to Ypt1p, thus leading to a long life-time of the binary complex between the two proteins, even at the high nucleotide concentrations that prevail in the cell. This leads to the conclusion that the protein-protein complex is likely to have an important biological significance in addition to its probable role in GTP/GDP exchange.

High-resolution crystal structure of S. cerevisiae Ypt51(DeltaC15)-GppNHp, a small GTP-binding protein involved in regulation of endocytosis.

Ypt/Rab proteins are membrane-associated small GTP-binding proteins which play a central role in the coordination, activation and regulation of vesicle-mediated transport in eukaryotic cells. We present the 1.5 A high-resolution crystal structure of Ypt51 in its active, GppNHp-bound conformation. Ypt51 is an important regulator involved in the endocytic membrane traffic of Saccharomyces cerevisiae. The structure reveals small but significant structural differences compared with H-Ras p21. The effector loop and the catalytic loop are well defined and stabilized by extensive hydrophobic interactions. The switch I and switch II regions form a well-defined epitope for hypothetical effector protein binding. Sequence comparisons between the different isoforms Ypt51, Ypt52 and Ypt53 provide the first insights into determinants for specific effector binding and for fine-tuning of the intrinsic GTP-hydrolysis rate.