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Abstract
Mal11 catalyzes proton-coupled maltose transport across the plasma membrane of Saccharomyces cerevisiae. We used structure-based design of mutants and a kinetic analysis of maltose transport to determine the energy coupling mechanism of transport. We find that wildtype Mal11 is extremely well coupled and allows yeast to rapidly accumulate maltose to dangerous levels, resulting under some conditions in self-lysis. Three protonatable residues lining the central membrane-embedded cavity of Mal11 were identified as having potential roles in proton translocation. We probed the mechanistic basis for proton coupling with uphill and downhill transport assays and found that single mutants can still accumulate maltose but with a lower coupling efficiency than the wildtype. Next, we combined the individual mutations and created double and triple mutants. We found some redundancy in the functions of the acidic residues in proton coupling and that no single residue is most critical for proton coupling to maltose uptake, unlike what is usually observed in related transporters. Importantly, the triple mutants were completely uncoupled but still fully active in downhill efflux and equilibrium exchange. Together, these results depict a concerted mechanism of proton transport in Mal11 involving multiple charged residues.
Highlights
The first step of sugar metabolism in yeast typically involves transport of the molecule into the cell
We found a maltose concentration-dependent drop in pHin upon addition of the disaccharide to galactose-energized cells, which is consistent with maltose-proton symport (Supplementary Fig. 1b)
We found that the forward scatter (FSC), side scatter (SSC), relative fluorescence, and number of cells remained constant in the maltose preloading conditions for at least 21 h (Supplementary Fig. 5d–f)
Summary
The first step of sugar metabolism in yeast typically involves transport of the molecule into the cell. The canonical model of MFS symport is the alternating access mechanism, whereby binding of both substrates triggers a conformational change in the protein to alternately expose the substrate binding site(s) to the outside and inside of the cell. Mutation of a single amino acid residue can significantly alter the coupling properties of a transporter, changing the apparent stoichiometry of transported substrate to co-substrate This is caused by “leak” pathways, whereby the locked binary complex of substrate (or ion) with transporter becomes statistically more likely to unlock and transport one substrate down its concentration gradient in the absence of the other (Fig. 1b)[8]. These results suggest a mechanism involving at least three acidic residues to ensure proper proton coupling to maltose transport
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