Enolase Activates Homotypic Vacuole Fusion and Protein Transport to the Vacuole in Yeast

Bridget L Decker,William T Wickner

doi:10.1074/jbc.m600911200

Abstract

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.

Highlights

Because band 3, an integral membrane protein, binds glycolytic proteins to the membrane of human erythrocytes [9], we looked for a similar protein in yeast that might be responsible for the localization of cytosolic enolase to the vacuole
To identify factors involved in yeast vacuole fusion, we fractionated yeast cytosol and monitored the ability of fractions to stimulate in vitro vacuole fusion
Fractions were tested for their ability to stimulate in vitro vacuole fusion

Summary

EXPERIMENTAL PROCEDURES

Vacuoles were isolated from yeast strains BJ3505 (MATa ura trp101 his3-D 200 lys801 trp1-D 101 (gal3) can gal prb1-D1.6R pep4::HIS3) [11] and DKY6281 (Mata ura leu leu112 trp901 his200 lys801 suc pho8::TRP1) [12] or derivatives of R11258 (URA::CMV-tTA MATa his leu0 met15-0) (OpenBiosystems) for in vitro fusion reactions. BDY1 and BDY2 were transformed with the integrating vector pRS403 containing MET15 flanked by upstream and downstream regions of ENO1. Vacuole suspensions were mixed with 0.2 volume of 50% glycerol and 10 mM Pipes-KOH, pH 6.8, 200 mM sorbitol to final concentration of 3 ␮g/␮l and frozen dropwise in liquid N2. Each fraction was assayed for stimulation of in vitro vacuole fusion, and the active fractions were pooled These fractions were lyophilized, resuspended in 0.1 volume of water, and dialyzed into 10 mM Pipes-KOH, pH 6.8, using regenerated cellulose dialysis tubing with a nominal Mr cut off of 3,500 (Fisherbrand). Fractions were dialyzed in to 200 mM sorbitol, 10 mM Pipes, pH 6.8, using regenerated cellulose dialysis tubing and tested for activity in the in vitro fusion assay. One unit of fusion stimulatory activity increases the in vitro fusion assay signal by 0.1 A400 nm

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RESULTS

DISCUSSION