Kinetic analysis of the guanine nucleotide exchange activity of TRAPP, a multimeric Ypt1p exchange factor

Abstract

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.

Figure 1. TRAPP activation of nucleotide exchange from Ypt1p

(A) Time courses of mantdGDP dissociation from 0.25 μM Ypt1p in the presence of (right to left) 1 μM, 1.5 μM, 2 μM or 2.5 μM TRAPP. Inset. Time courses of mantGDP dissociation from 0.25 μM Ypt1p in the presence of (right to left) 1 μM, 3 μM, 4.5 μM, 6 μM or 15 μM TRAPP. The smooth black lines through the data (red) represent the best fits to single (panel) or double (inset) exponentials. (B) [TRAPP]-dependence of the observed rate constant for nucleotide dissociation. The smooth line is the best fit of the data to Eq. 1 with k_-4 = 0.17 s^-1, and K₃ = 4.8 μM for mantGDP (red) and k_-4 = 0.095 s^-1, and K₃ = 8 μM for mant-GMPPNP dissociation (blue). Dissociation of [³H]GDP from Ypt1p

Figure 2. MantGDP binding Ypt1p and TRAPP-Ypt1p

(A) Time courses of (right to left) 12.5, 20, 40 or 60 μM mantGDP binding Ypt1p-TRAPP complex. (B) mantGDP binding to nucleotide-free Ypt1p (green) or nucleotide-free TRAPP-Ypt1p (red). The smooth black lines through the data in panels A and B represent the best fits to single (Ypt1p) or double (TRAPP-Ypt1p) exponentials. (C) [Mant-GDP]-dependence of the observed rate constant for mantGDP binding to Ypt1p-TRAPP complex. (D) Concentration-dependence of slow phase for mantGDP binding Ypt1p-TRAPP complex. The solid lines through the data in panels C and D represent the best fit to a straight line (Eq. 3) and hyperbola (Eq. 4) for fast and slow phases, respectively. The dashed lines represent the global fit for all data to the quadratic rate equation (Eq. 2). MantGDP binding to Ypt1p alone (green triangles) with the best fit of data to a straight line is shown for comparison.

Figure 3. Mg²⁺-dependence of GDP dissociation from Ypt1p and TRAPP-Ypt1p

(A) Time courses of mant-dGDP dissociation after mixing 250 nM Ypt1p-mdGDP with excess GDP in buffer containing (left to right), 0.06, 0.44, 0.8, 7.9, 26.6, or 90.3 μM free Mg²⁺ (final). (B)[Mg²⁺]-dependence of the observed rate constants for mant-dGDP (squares) or mantGDP (circles) dissociation from Ypt1p. The smooth black lines through the data (green) represents the best fit to a hyperbola (Eq. 6) yielding $K_{\mathrm{Mg}}^{\mathrm{YD}}=3.7\mu \mathrm{M}$, k_-1^' = 0.0073 s, k = 0.00032 s^-1-1 for mant-dGDP and $K_{\mathrm{Mg}}^{\mathrm{YD}}=2.2\mu \mathrm{M}$, k_-1^' = 0.0104 s^-1, k_-1 = 0.0011 s^-1-1 for mantGDP. (C) Time courses of mantGDP dissociation after mixing 500nM Ypt1p-mGDP with excess GDP2 in buffer containing (left to right) 0.87, 2.5, 4.9, 10.5, or 130 μM free Mg²⁺ (final) and 2.5 μM TRAPP. The smooth black lines through the data (red) represent the best fits to double exponentials; the small fast phase shows little [Mg²⁺]-dependence and is observed only with mixed 2' and 3'-mant isomers. (D) [Mg²⁺]-dependence of mantGDP dissociation from Ypt1p in the presence of 2.5 μM TRAPP. The smooth black lines through the data (red) represents the best fit to a hyperbola (Eq. 7) yielding K_0.5 = 1.0 μM, k_∞ = 0. 0014 s^-1, and k₀ = 0.55 s^-1 for mant-GDP.

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Scheme A1