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tra12156-sup-0001-FigureS1.docWord document522KFigure S1: Structure and sequence comparisons among dynamin family members. A) Homology-based structural model comparing bacterial dynamin and yeast Vps1 (both as monomers). The following image depicts an alignment of the known crystal structure of bacterial dynamin shown in pink and the predicted structure of yeast Vps1 shown in grey, with a portion of the GED of Vps1 highlighted in the yellow box. The residues Y628, K642 and I649 are situated within this region. B) Sequence alignment of yeast Vps1 (residues 619–686) with dynamin family members. Organisms include Drosophila melanogaster (dDynamin), Caenorhabditis elegans (ceDynamin) and Rattus norvegicus/Homo sapiens (mDynamin). Residues conserved across multiple species and chosen for mutation are highlighted in green.
tra12156-sup-0002-FigureS2.docWord document197KFigure S2: Comparison of protein levels and reporter activity among Vps1 wild type and mutant vacuoles. A) Vacuoles were purified from Vps1 variant strains, resuspended in PS buffer (20 mm Sorbitol, 10 mm PIPES pH 6.8) and resolved on SDS–PAGE. Western blot analysis was then performed to compare protein levels of Vps1, Vam3, Nyv1, Vam7, Vti1 and Vps11. B) To control reporter enzyme (Alkaline phosphatase) activity, fusion reactions were carried out in the presence of TX-100.
tra12156-sup-0003-FigureS3.docWord document60KFigure S3: Fusion efficiency of Vps1 wild type and mutant vacuoles. Content mixing between different strain combinations of Vps1 mutant and wild type vacuoles was measured under standard assay conditions.
tra12156-sup-0004-FigureS4.docWord document360KFigure S4: Lipid mixing of Vps1 wild type and mutant vacuoles after SNARE antibody treatment. Vacuoles were isolated from the ΔVps1 BJ strain reconstituted with plasmid expressing either (A) wild type Vps1 or Vps1 mutants (B) I649K, (C) K642L or (D) Y628F. A population of the vacuoles was labeled with Rh-PE and incubated with the unlabeled population under standard hemifusion conditions. Normalized relative fluorescence units were plotted versus time for three independent experiments and are shown as mean ± SD. Prior to the addition of ATP, the vacuole mixture was incubated with antibodies to Vam3, Vam7, Vti1 and Nyv1. Treatment with each antibody diminished the extent of lipid mixing.
tra12156-sup-0005-FigureS5.docWord document247KFigure S5: Coomassie staining of purified recombinant Vps1 variants.
tra12156-sup-0006-FigureS6.docWord document59KFigure S6: Strategies to rescue fusion defects of Vps1 mutant vacuoles. Standard fusion reactions of Vps1 wild type or mutant vacuoles were set up in the presence of following agents: red bars indicating 10-fold Vam3 overexpression on each fusion partner, green bars indicating addition of 100 nm recombinant Vam7, purple bars indicating addition of 150 µm Chlorpromazine and grey bars indicating addition of 4 µm recombinant wild type Vps1. Normalized OD400 values were plotted for three independent experiments and are shown as mean ± SD.

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