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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.

SUBMITTER: Chin HF

PROVIDER: S-EPMC2770256 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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

Chin Harvey F HF Cai Yiying Y Menon Shekar S Ferro-Novick Susan S Reinisch Karin M KM De La Cruz Enrique M EM

Journal of molecular biology 20090408 2

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 thermo ...[more]

PMID: 19361519

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Project description:Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4.IMPORTANCE Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4.

Dataset Information

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

Publications

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

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