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The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin.

ABSTRACT: Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E)) being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E)-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E)-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.

SUBMITTER: Ahmad KF

PROVIDER: S-EPMC2892021 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin.

Ahmad K Farid KF Lim Wendell A WA

PloS one 20100624 6

Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To inves ...[more]

PMID: 20585582

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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

The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin.

Publications

The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin.

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