Purification and in vitro analysis of yeast vacuoles.
The purification of eukaryotic organelles is a prerequisite for the detailed analysis of protein sorting, localization and translocation, membrane fusion and vesicle budding. Yeast vacuoles receive cargo from the exocytic, endocytic, and autophagic pathways and hence represent an excellent model system for the study of organelle biogenesis and protein sorting. Yeast vacuoles undergo fission and fusion in vivo, events that can be monitored in vitro by an assay that employs purified vacuoles from two tester strains. Here, we describe the methodology of yeast vacuole purification, and provide protocols for the detailed analysis of the fusion reaction. We also include methods to analyze protein dynamics on yeast vacuoles and the controls required to ensure their reliability.
Cabrera
Margarita
M
Department of Biology, Biochemistry Section, University of Osnabrück, Osnabrück, Germany.
Ungermann
Christian
C
D001681:Biological Assay / Q000379:methods*; D001692:Biological Transport / Q000502:physiology; D002458:Cell Fractionation / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods; D008561:Membrane Fusion / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014617:Vacuoles / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D029701:Saccharomyces cerevisiae Proteins