title: A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.
abstract: Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.
authors.affiliation: Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Palmer
authors.affiliation: Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Iwona I
authors.initials: II
authors.lastname: Smaczynska-de Rooij
authors.affiliation: Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Marklew
authors.affiliation: Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Ellen G
authors.initials: EG
authors.lastname: Allwood
authors.affiliation: Department of Biological Science, Durham University, Durham DH1 3LE, UK.
authors.forename: Ritu
authors.initials: R
authors.lastname: Mishra
authors.affiliation: Department of Biological Science, Durham University, Durham DH1 3LE, UK.
authors.forename: Simeon
authors.initials: S
authors.lastname: Johnson
authors.affiliation: Department of Biological Science, Durham University, Durham DH1 3LE, UK.
authors.forename: Martin W
authors.initials: MW
authors.lastname: Goldberg
authors.affiliation: Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK. Electronic address: k.ayscough@sheffield.ac.uk.
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Ayscough
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism*; D034281:Dynamins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000235:genetics*; D008840:Microfilament Proteins / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D021381:Protein Transport / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D022161:Transport Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins / Q000235:genetics*; D015003:Yeasts
chemical_list: C062612:ABP1 protein, S cerevisiae; D000199:Actins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C075637:amphiphysin; D019204:GTP-Binding Proteins; C064553:VPS1 protein, S cerevisiae; D034281:Dynamins