Electrophysiological characterization of LacY

Department of Biophysical Chemistry, Max Planck Institute of Biophysics, D-60438 Frankfurt am Main, Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 05/2009; 106(18):7373-8. DOI: 10.1073/pnas.0902471106
Source: PubMed

ABSTRACT Electrogenic events due to the activity of wild-type lactose permease from Escherichia coli (LacY) were investigated with proteoliposomes containing purified LacY adsorbed on a solid-supported membrane electrode. Downhill sugar/H(+) symport into the proteoliposomes generates transient currents. Studies at different lipid-to-protein ratios and at different pH values, as well as inactivation by N-ethylmaleimide, show that the currents are due specifically to the activity of LacY. From analysis of the currents under different conditions and comparison with biochemical data, it is suggested that the predominant electrogenic event in downhill sugar/H(+) symport is H(+) release. In contrast, LacY mutants Glu-325-->Ala and Cys-154-->Gly, which bind ligand normally, but are severely defective with respect to lactose/H(+) symport, exhibit only a small electrogenic event on addition of LacY-specific substrates, representing 6% of the total charge displacement of the wild-type. This activity is due either to substrate binding per se or to a conformational transition after substrate binding, and is not due to sugar/H(+) symport. We propose that turnover of LacY involves at least 2 electrogenic reactions: (i) a minor electrogenic step that occurs on sugar binding and is due to a conformational transition in LacY; and (ii) a major electrogenic step probably due to cytoplasmic release of H(+) during downhill sugar/H(+) symport, which is the limiting step for this mode of transport.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H(+) across the Escherichia coli membrane (galactoside/H(+) symport). Initial X-ray structures reveal N- and C-terminal domains, each with six largely irregular transmembrane helices surrounding an aqueous cavity open to the cytoplasm. Recently, a structure with a narrow periplasmic opening and an occluded galactoside was obtained, confirming many observations and indicating that sugar binding involves induced fit. LacY catalyzes symport by an alternating access mechanism. Experimental findings garnered over 45 y indicate the following: (i) The limiting step for lactose/H(+) symport in the absence of the H(+) electrochemical gradient ([Formula: see text]) is deprotonation, whereas in the presence of [Formula: see text], the limiting step is opening of apo LacY on the other side of the membrane; (ii) LacY must be protonated to bind galactoside (the pK for binding is ∼10.5); (iii) galactoside binding and dissociation, not [Formula: see text], are the driving forces for alternating access; (iv) galactoside binding involves induced fit, causing transition to an occluded intermediate that undergoes alternating access; (v) galactoside dissociates, releasing the energy of binding; and (vi) Arg302 comes into proximity with protonated Glu325, causing deprotonation. Accumulation of galactoside against a concentration gradient does not involve a change in Kd for sugar on either side of the membrane, but the pKa (the affinity for H(+)) decreases markedly. Thus, transport is driven chemiosmotically but, contrary to expectation, [Formula: see text] acts kinetically to control the rate of the process.
    Proceedings of the National Academy of Sciences 01/2015; 112(5). DOI:10.1073/pnas.1419325112 · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Major Facilitator Superfamily (MFS) is a diverse group of secondary transporters with over 10,000 members, found in all kingdoms of life, including Homo sapiens. A paradigm for MFS is the lactose permease (LacY) of Escherichia coli, which couples the stoichiometric translocation of a galactopyranoside and an H+ across the cytoplasmic membrane. LacY has been the test bed for the development of many methods routinely applied for the analysis of transport proteins. X-ray structures of an inward-facing conformation and most recently, an almost occluded conformation with a narrow periplasmic opening have been solved, which confirm many conclusions from biochemical and biophysical studies. Although structure models are critical, they are not sufficient to explain the catalysis of transport. The clues to understanding transport mechanisms are based on the principles of enzyme kinetics. Secondary transport is a dynamic process that can be described only partially by the static snapshots provided by X-ray crystallography. However, without structural information it is virtually impossible to deduce the chemistry underlying ion-coupled transport. By combining a large body of biochemical/biophysical data derived from systematic studies of site-directed mutants in LacY, residues involved in substrate binding and H+ translocation have been identified. On the basis of the functional properties of the mutants and the X-ray structures, a working model for the symport mechanism that involves alternating access of the binding site is presented. The general concepts derived from the bacterial LacY and other MFS transporters are examined for their relevance to the human D-glucose transporter, GLUT1, by comparing conservation of functionally critical residues.
    09/2014; 2014:523591. DOI:10.1155/2014/523591
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4 (+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4 (+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4 (+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.
    Proceedings of the National Academy of Sciences 07/2014; 111(27). DOI:10.1073/pnas.1406409111 · 9.81 Impact Factor


Available from