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Loss of the Sec1/Munc18-family proteins VPS-33.2 and VPS-33.1 bypasses a block in endosome maturation in Caenorhabditis elegans.

ABSTRACT: The end of the life of a transport vesicle requires a complex series of tethering, docking, and fusion events. Tethering complexes play a crucial role in the recognition of membrane entities and bringing them into close opposition, thereby coordinating and controlling cellular trafficking events. Here we provide a comprehensive RNA interference analysis of the CORVET and HOPS tethering complexes in metazoans. Knockdown of CORVET components promoted RAB-7 recruitment to subapical membranes, whereas in HOPS knockdowns, RAB-5 was found also on membrane structures close to the cell center, indicating the RAB conversion might be impaired in the absence of these tethering complexes. Unlike in yeast, metazoans have two VPS33 homologues, which are Sec1/Munc18 (SM)-family proteins involved in the regulation of membrane fusion. We assume that in wild type, each tethering complex contains a specific SM protein but that they may be able to substitute for each other in case of absence of the other. Of importance, knockdown of both SM proteins allowed bypass of the endosome maturation block in sand-1 mutants. We propose a model in which the SM proteins in tethering complexes are required for coordinated flux of material through the endosomal system.

SUBMITTER: Solinger JA

PROVIDER: S-EPMC4244200 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Loss of the Sec1/Munc18-family proteins VPS-33.2 and VPS-33.1 bypasses a block in endosome maturation in Caenorhabditis elegans.

Solinger Jachen A JA Spang Anne A

Molecular biology of the cell 20141001 24

The end of the life of a transport vesicle requires a complex series of tethering, docking, and fusion events. Tethering complexes play a crucial role in the recognition of membrane entities and bringing them into close opposition, thereby coordinating and controlling cellular trafficking events. Here we provide a comprehensive RNA interference analysis of the CORVET and HOPS tethering complexes in metazoans. Knockdown of CORVET components promoted RAB-7 recruitment to subapical membranes, where ...[more]

PMID: 25273556

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Project description:ObjectiveGenetic variants in STXBP1, which encodes the conserved exocytosis protein Munc18-1, are associated with a variety of infantile epilepsy syndromes. We aimed to develop an in vivo Caenorhabditis elegans model that could be used to test the pathogenicity of such variants in a cost-effective manner.MethodsThe CRISPR/Cas9 method was used to introduce a null mutation into the unc-18 gene (the C. elegans orthologue of STXBP1), thereby creating a paralyzed worm strain. We subsequently rescued this strain with transgenes encoding the human STXBP1/Munc18-1 protein (wild-type and eight different epilepsy-associated missense variants). The resulting humanized worm strains were then analyzed via behavioral, electrophysiological, and biochemical approaches.ResultsTransgenic expression of wild-type human STXBP1 protein fully rescued locomotion in both solid and liquid media to the same level as the standard wild-type worm strain, Bristol N2. Six variant strains (E59K, V84D, C180Y, R292H, L341P, R551C) exhibited impaired locomotion, whereas two (P335L, R406H) were no different from worms expressing wild-type STXBP1. Electrophysiological recordings revealed that all eight variant strains displayed less frequent and more irregular pharyngeal pumping in comparison to wild-type STXBP1-expressing strains. Four strains (V84D, C180Y, R292H, P335L) exhibited pentylenetetrazol-induced convulsions in an acute assay of seizure-like activity, in contrast to worms expressing wild-type STXBP1. No differences were seen between wild-type and variant STXBP1 strains in terms of mRNA abundance. However, STXBP1 protein levels were reduced to 20%-30% of wild-type in all variants, suggesting that the mutations result in STXBP1 protein instability.SignificanceThe approach described here is a cost-effective in vivo method for establishing the pathogenicity of genetic variants in STXBP1 and potentially other conserved neuronal proteins. Furthermore, the humanized strains we created could potentially be used in the future for high-throughput drug screens to identify novel therapeutics.

Dataset Information

Loss of the Sec1/Munc18-family proteins VPS-33.2 and VPS-33.1 bypasses a block in endosome maturation in Caenorhabditis elegans.

Publications

Loss of the Sec1/Munc18-family proteins VPS-33.2 and VPS-33.1 bypasses a block in endosome maturation in Caenorhabditis elegans.

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