Enolase activates homotypic vacuole fusion and protein transport to the vacuole in yeast.
Membrane fusion and protein trafficking to the vacuole are complex processes involving many proteins and lipids. Cytosol from Saccharomyces cerevisiae contains a high Mr activity, which stimulates the in vitro homotypic fusion of isolated yeast vacuoles. Here we purify this activity and identify it as enolase (Eno1p and Eno2p). Enolase is a cytosolic glycolytic enzyme, but a small portion of enolase is bound to vacuoles. Recombinant Eno1p or Eno2p stimulates in vitro vacuole fusion, as does a catalytically inactive mutant enolase, suggesting a role for enolase in fusion that is separate from its glycolytic function. Either deletion of the non-essential ENO1 gene or diminished expression of the essential ENO2 gene causes vacuole fragmentation in vivo, reflecting reduced fusion. Combining an ENO1 deletion with ENO2-deficient expression causes a more severe fragmentation phenotype. Vacuoles from enolase 1 and 2-deficient cells are unable to fuse in vitro. Immunoblots of vacuoles from wild type and mutant strains reveal that enolase deficiency also prevents normal protein sorting to the vacuole, exacerbating the fusion defect. Band 3 has been shown to bind glycolytic enzymes to membranes of mammalian erythrocytes. Bor1p, the yeast band 3 homolog, localizes to the vacuole. Its loss results in the mislocalization of enolase and other vacuole fusion proteins. These studies show that enolase stimulates vacuole fusion and that enolase and Bor1p regulate selective protein trafficking to the vacuole.
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844, USA.
Bridget L
BL
Decker
William T
WT
Wickner
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry; D001692:Biological Transport; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000737:chemistry*; D005786:Gene Expression Regulation; D026901:Membrane Transport Proteins; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000502:physiology*; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D013752:Tetracycline / Q000494:pharmacology; D014617:Vacuoles / Q000737:chemistry*
D001457:Anion Exchange Protein 1, Erythrocyte; C511808:Bor1 protein, S cerevisiae; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D010751:Phosphopyruvate Hydratase; D013752:Tetracycline