Quantifying secondary transport at single-molecule resolution

Nature. 2019 Nov;575(7783):528-534. doi: 10.1038/s41586-019-1747-5. Epub 2019 Nov 13.


Secondary active transporters, which are vital for a multitude of physiological processes, use the energy of electrochemical ion gradients to power substrate transport across cell membranes1,2. Efforts to investigate their mechanisms of action have been hampered by their slow transport rates and the inherent limitations of ensemble methods. Here we quantify the activity of individual MhsT transporters, which are representative of the neurotransmitter:sodium symporter family of secondary transporters3, by imaging the transport of individual substrate molecules across lipid bilayers at both single- and multi-turnover resolution. We show that MhsT is active only when physiologically oriented and that the rate-limiting step of the transport cycle varies with the nature of the transported substrate. These findings are consistent with an extracellular allosteric substrate-binding site that modulates the rate-limiting aspects of the transport mechanism4,5, including the rate at which the transporter returns to an outward-facing state after the transported substrate is released.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Allosteric Site
  • Amino Acids / analysis
  • Amino Acids / chemistry
  • Amino Acids / metabolism*
  • Bacterial Proteins / analysis
  • Bacterial Proteins / metabolism
  • Biological Transport
  • Cell Survival
  • Fluorescence Resonance Energy Transfer
  • Hydrophobic and Hydrophilic Interactions
  • Kinetics
  • Lipid Bilayers / metabolism
  • Protein Conformation
  • Single Molecule Imaging*
  • Symporters / analysis*
  • Symporters / chemistry
  • Symporters / metabolism*


  • Amino Acids
  • Bacterial Proteins
  • Lipid Bilayers
  • Symporters