Kinetic analysis of the guanine nucleotide exchange activity of TRAPP, a multimeric Ypt1p exchange factor.
TRAPP complexes, which are large multimeric assemblies that function in membrane traffic, are guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1p. Here we measured rate and equilibrium constants that define the interaction of Ypt1p with guanine nucleotide (guanosine 5'-diphosphate and guanosine 5'-triphosphate/guanosine 5'-(beta,gamma-imido)triphosphate) and the core TRAPP subunits required for GEF activity. These parameters allowed us to identify the kinetic and thermodynamic bases by which TRAPP catalyzes nucleotide exchange from Ypt1p. Nucleotide dissociation from Ypt1p is slow (approximately 10(-4) s(-1)) and accelerated >1000-fold by TRAPP. Acceleration of nucleotide exchange by TRAPP occurs via a predominantly Mg(2+)-independent pathway. Thermodynamic linkage analysis indicates that TRAPP weakens nucleotide affinity by <80-fold and vice versa, in contrast to most other characterized GEF systems that weaken nucleotide binding affinities by 4-6 orders of magnitude. The overall net changes in nucleotide binding affinities are small because TRAPP accelerates both nucleotide binding and dissociation from Ypt1p. Weak thermodynamic coupling allows TRAPP, Ypt1p, and nucleotide to exist as a stable ternary complex, analogous to strain-sensing cytoskeleton motors. These results illustrate a novel strategy of guanine nucleotide exchange by TRAPP that is particularly suited for a multifunctional GEF involved in membrane traffic.
Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, 260 Whitney Avenue, New Haven, CT 06520-8114, USA.
Harvey F
HF
Chin
Yiying
Y
Cai
Shekar
S
Menon
Susan
S
Ferro-Novick
Karin M
KM
Reinisch
Enrique M
EM
De La Cruz
D004926:Escherichia coli / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013816:Thermodynamics; D014161:Transduction, Genetic; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C406327:transport protein particle, TRAPP; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins