On the mechanism of proton-coupled transport by the maltose permease of Saccharomyces cerevisiae

Ryan Henderson

Research output: ThesisThesis fully internal (DIV)

446 Downloads (Pure)

Abstract

Biological membranes enclose all cells and regulate the entry or exit of molecules. Symporters are proteins that span the membrane and can move specific molecules into or out of the cell by using energy stored in electrochemical ion gradients. These transporters are flexible machines that bind solutes on one side of the membrane and change their shape to release them on the other. To allow coupled transport, a symporter should be "locked" when only one of the two molecules is bound. However, some misbehaving proteins don't follow this rule, whereby one of the solutes can "leak" across the membrane without the other, blurring the lines between symporter and uniporter. For Mal11, a proton-coupled sugar symporter from yeast, we found that transport is extremely well coupled. To better understand the nature of proton coupling in symporters, we mutated numerous amino acids in Mal11 and found three to be most critical for function. When all three were mutated, Mal11 could still transport sugar but not protons. However, yeast strains with these “triple mutants” could not grow on certain sugars, so we fed cells with sucrose until they grew rapidly: directed evolution. To our surprise, additional mutations in Mal11 were responsible for the growth, some of which restored proton-coupled transport activity. We then changed focus to examine the sugar-binding site. Through mutagenesis, we found the most essential amino acids for maltose transport. This thesis represents an improved understanding of transport by Mal11 and related transporters and provides a foundation for further study.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • University of Groningen
Supervisors/Advisors
  • Poolman, Berend, Supervisor
  • Slotboom, Dirk, Supervisor
Award date11-Jan-2019
Place of Publication[Groningen]
Publisher
Print ISBNs978-94-034-1280-1
Electronic ISBNs978-94-034-1279-5
Publication statusPublished - 2019

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