Kinetic and Thermodynamic Basis of Guanine Nucleotide Exchange on the Rab Ypt1p by the Membrane Tethering Complex TRAPPI

Abstract

TRAPPI is a multiprotein tethering complex involved in mediating the initial interaction of endoplasmic reticulum (ER)-derived vesicles at the cis-Golgi, that serves as a guanine nucleotide exchange factor (GEF) for the Rab GTPase, Ypt1p. Activation of Ypt1p by TRAPPI is required for membrane fusion, and thus the mechanism of nucleotide exchange by TRAPPI is a crucial step of regulation in vesicular transport. A minimal complex of five proteins (Bet5p, Trs23p, Trs31p, and 2 copies of Bet3p) is required for GEF activity, suggesting a complex interaction between TRAPPI and Ypt1p.The large majority of GEFs accelerate nucleotide exchange from G-proteins by inserting a glutamate finger wedge into the nucleotide binding pocket. We recently solved the crystal structure of nucleotide-free Ypt1p in complex with TRAPPI, which revealed the absence of the canonical glutamate finger and suggested a novel mechanism of GEF activity by TRAPPI.In this study, we measured the rate and equilibrium constants for nucleotide and TRAPPI binding to Ypt1p. Nucleotide binding is accelerated ∼30-fold and dissociation more than three orders of magnitude, suggesting that the nucleotide binding site is more accessible in the presence of TRAPPI. Analysis of various mutant TRAPPI complexes, a Ypt1 mutant defective in nucleotide binding, and the Mg2+-dependence of exchange do not support a glutamate finger mechanism, and favor nucleotide exchange through a novel mechanism in which TRAPPI accelerates nucleotide dissociation by opening the nucleotide binding site through interactions with switch I and II. Thermodynamic linkage analysis indicates that TRAPPI binds nucleotide-free Ypt1 with greater affinity than Ypt1 with bound nucleotide. However, this increased stability cannot account for the >1000-fold increase in the rate of nucleotide exchange, indicating that GEF activity of TRAPPI is under kinetic control.