title: The yeast ATP-binding cassette (ABC) transporter Ycf1p enhances the recruitment of the soluble SNARE Vam7p to vacuoles for efficient membrane fusion.
abstract: The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd(2+) transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p(K669M)) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.
authors: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors: Terry L
authors: TL
authors: Sasser
authors: Gus
authors: G
authors: Lawrence
authors: Surya
authors: S
authors: Karunakaran
authors: Christopher
authors: C
authors: Brown
authors: Rutilio A
authors: RA
authors: Fratti
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D017136:Ion Transport; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D018129:Phosphatidylinositol Phosphates; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; C494205:VAM7 protein, S cerevisiae; C089179:YCF1 protein, S cerevisiae; C055525:phosphatidylinositol 3-phosphate; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; D002118:Calcium
keywords: ABC Transporter; Bpt1; Calcium Transport; Intracellular Trafficking; Lysosomes; Membrane Fusion; Phosphatidylinositol 3-Phosphate; SNARE; Ybt1
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