The human synaptic vesicle protein, SV2A, functions as a galactose transporter in Saccharomyces cerevisiae

Abstract

SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function.

Keywords: Drug Action; Galactose; Levetiracetam; SV2A; Saccharomyces cerevisiae; Sugar Transport; Synapse.

FIGURE 1.

The growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A is galactose-dependent. A, the EBY.VW4000 yeast strain, which lacks all hexose transporters, shows normal growth on lactate-containing (YPL) medium and defective growth on glucose-containing (YPD) medium, and expression of hSV2A does not rescue the growth defect on YPD. 10-fold serial dilutions of EBY.VW4000 yeast cells, EBY.VW4000 cells harboring the empty centromeric plasmid pCM188, or EBY.VW4000 cells expressing hSV2A in the pCM188 plasmid were plated on rich YP medium (1% yeast extract, 2% peptone) containing either 3% lactate (YPL) or 2% glucose (YPD). B, hexose transport-deficient EBY.VW4000 yeast cells expressing hSV2A are able to grow on synthetic medium containing 2% galactose. SV2A expression does not rescue the growth defect if the synthetic medium contains other 6-carbon sugars. 10-fold serial dilutions of EBY.VW4000 yeast cells and EBY.VW4000 cells harboring the empty centromeric plasmid pCM188 or expressing human SV2A in the pCM188 plasmid were plated onto SC medium lacking uracil (−ura) and supplemented with 2% glucose, galactose, raffinose, sucrose, mannose, and fructose, respectively. The same strains were plated on synthetic complete medium containing either 3% lactate or 3% glycerol to test hexose-independent, normal growth. Plates were incubated at 30 °C for 48 h.

FIGURE 2.

The antiepileptic drug, levetiracetam, inhibits the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. The growth of SV2A-expressing EBY.VW4000 yeast cells on 2% galactose-containing YP medium was concentration-dependently inhibited by levetiracetam, an antiepileptic drug, which specifically binds to SV2A.

FIGURE 3.

Human SV2A expressed in yeast cells transports galactose. The cellular uptake of [¹⁴C]galactose was measured in hexose transport-deficient EBY.VW4000 yeast cells either harboring the empty centromeric plasmid pCM188 or expressing human SV2A in the pCM188 vector. Cells were grown in liquid synthetic complete medium lacking uracil (SC−ura) and supplemented with 3% lactate, to A₆₀₀ of 0.6, and then harvested and resuspended in liquid SC−ura medium supplemented with 3% glycerol. Galactose uptake was initiated by adding [¹⁴C]galactose to the medium, and radioactivity in the cells was measured at different time points. A, time-dependent, significant cellular uptake of [¹⁴C]galactose could only be measured in hSV2A-expressing cells. Symbols and bars represent mean ± S.D. (n = 5). B, the hSV2A-mediated galactose uptake is coupled to proton transport. [¹⁴C]Galactose uptake by hSV2A was measured in the absence or presence of the protonophore, CCCP (10 μm), for 15 and 30 min. Columns and bars represent mean ± S.D. (n = 5). C, Michaelis-Menten plot of hSV2A-mediated galactose uptake. Symbols and bars represent mean ± S.D. (n = 4–5). In A and C, curve fittings by nonlinear regression were performed using GraphPad Prism 5.