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Published on: June 24, 2015
Electrophysiological characterization of LacY
Juan J Garcia-Celma1, Irina N Smirnova, H Ronald Kaback
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, D-60438 Frankfurt am Main, Germany.
Summary
Wild-type lactose permease (LacY) activity generates currents via proton release during sugar transport. Mutants show minimal electrogenic events, suggesting proton release is key to LacY function.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Lactose permease (LacY) is a crucial transporter in Escherichia coli.
- Understanding LacY's electrogenic events is key to elucidating its transport mechanism.
Purpose of the Study:
- To investigate the electrogenic events associated with wild-type and mutant LacY activity.
- To determine the primary electrogenic step in downhill sugar/H(+) symport.
Main Methods:
- Utilized proteoliposomes with purified LacY on a solid-supported membrane electrode.
- Measured transient currents generated during symport.
- Performed studies at varying lipid-to-protein ratios and pH, and used N-ethylmaleimide for inactivation.
Main Results:
- Wild-type LacY generated significant currents, primarily attributed to H(+) release during symport.
- LacY mutants (Glu-325-->Ala, Cys-154-->Gly) exhibited minimal electrogenic activity (6% of wild-type).
- Mutant activity is linked to substrate binding or conformational changes, not symport.
Conclusions:
- LacY turnover involves at least two electrogenic steps: a minor one upon sugar binding and a major one likely involving cytoplasmic H(+) release.
- H(+) release during downhill symport is the predominant and likely rate-limiting electrogenic event.
