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The kamikaze approach to membrane transport

Nature Reviews Molecular Cell Biology volume 2, pages 610–620 (2001)Cite this article

Key Points

  • Membrane transport proteins mediate the movement of molecules into, or out of, cells, intracellular organelles and across epithelia. Despite their abundance in the genome and evident importance for the cell, we know little about their structure and function, largely because their hydrophobic and metastable nature makes them difficult to study.

  • Recent developments have allowed initial insight into the structure and mechanism of membrane transport proteins. One example is the lactose permease from Escherichia coli, a member of the major facilitator superfamily. This membrane protein uses free energy released from the energetically downhill translocation of H+ in response to an electrochemical H+ gradient to drive the accumulation of specific sugars against a concentration gradient.

  • Extensive use of site-directed mutagenesis demonstrates that only six amino acid residues are irreplaceable with respect to active lactose transport. Furthermore, mutants engineered for various biochemical and biophysical approaches provide structural information about how the helices are packed and how the irreplaceable residues interact to catalyse transport. Through this work, charge pairs have been identified that mediate substrate binding and H+ translocation.

  • The residues that are irreplaceable for activity are conserved in other members of the oligosaccharide/H+ symport subfamily, but are not found in other members of the major facilitator superfamily. Despite this, it is thought that relatively few residues will be critical for transport in these other families of membrane transport proteins and that the conformational changes involved will be largely rigid body movements of the transmembrane helices.

Abstract

Membrane transport proteins catalyse the movement of molecules into and out of cells and organelles, but their hydrophobic and metastable nature often makes them difficult to study by traditional means. Novel approaches that have been developed and applied to one membrane transport protein, the lactose permease from Escherichia coli, are now being used to study various other membrane proteins.

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Figure 1: Secondary structure model of lac permease.
Figure 2: Uphill and downhill lactose/H+ symport.
Figure 3: Helix packing of lactose permease.
Figure 4: Putative substrate-binding site in lactose permease.
Figure 5: Efflux, exchange and counterflow.
Figure 6: Kinetic scheme for lactose efflux, exchange and counterflow.
Figure 7: Mechanism of lactose/H+ symport by wild-type lactose permease.

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Acknowledgements

We are indebted to Mark Girvin for computer modelling and to Ernest Wright for helpful discussions. The 'Kamikaze approach' to membrane transport is taken from a review by G. von Heijne76.

Author information

Authors and Affiliations

  1. Departments of Physiology, Howard Hughes Medical Institute, and Microbiology, Immunology and Molecular Genetics, and the Molecular Biology Institute, University of California, Los Angeles, 90095-1662, California, USA

    H. Ronald Kaback, Miklós Sahin-Tóth & Adam B. Weinglass

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Supplementary information

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DATABASE LINKS

lacY

GLUT1

EmrE

cystic fibrosis

CFTR

connexin 26

Pendred syndrome

diastrophic dysplasia

long QT syndrome

Menkes disease

Wilson's disease

congenital renal glycosuria

GLUT2

Fanconi–Bickel syndrome

hyperkalemic periodic paralysis

spinocerebellar ataxia type 6

familial hemiplegic migraine

central core disease

congenital stationary night blindness

myotonia congenitas

autosomal dominant frontal lobe

nocturnal epilepsy

congenital myasthenic syndromes

hyperekplexia

FURTHER INFORMATION

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Kaback lab

Glossary

ELECTROCHEMICAL H+ GRADIENT

(Δ\(\overline{μ}\)H+). When two aqueous phases are separated by a membrane, the electrochemical potential difference of H+ between the two phases is expressed as Δ\(\overline{μ}\)H+/F = ΔΨ − 2.3RT/FΔpH, where F is the Faraday constant, ΔΨ is the electric potential difference between two phases, R is the gas constant, T is the absolute temperature, and ΔpH is the difference in the concentration of H+ across the membrane.

CYSTEINE-SCANNING MUTAGENESIS

The use of site-directed mutagenesis to replace individual residues in a given protein, or a region of that protein, with cysteine (preferably where all native cysteines have been replaced without loss of function).

CUT AND PASTE

Unique, engineered restriction enzyme sites in the lacY DNA are used to replace a portion of a mutant with a given cysteine replacement with a homologous segment encoding another single cysteine mutant, to construct a lacY gene encoding a protein with two cysteine residues.

SECOND-SITE-SUPPRESSOR ANALYSIS

Involves selection of a second mutation that corrects (suppresses) the phenotype of an original inactivating mutation. This can be done by classical or site-directed mutagenesis.

EXCIMER FLUORESCENCE

When two four-membered conjugated pyrene moieties are within ∼3.5 Å of each other and are in the correct orientation, an excited-state dimer (excimer) is observed in the fluorescence-emission spectrum, which emits at a higher wavelength than the monomer fluorescence.

DIVALENT-METAL-BINDING SITE

The simplest requirement for this consists of two imidazole side chains (histidine residues) within close proximity. Mn2+ binding is then measured directly by electron paramagnetic resonance.

THIOL CROSSLINKING

A technique that uses two cysteine residues, which can either be oxidized to form a disulphide bond or crosslinked with a homobifunctional crosslinking agent.

DISCONTINUOUS MONOCLONAL ANTIBODY EPITOPE

Refers to an antibody-binding site that is composed of portions of the protein that are not continuous with respect to the primary amino-acid sequence.

POLARIZED ATTENUATED TOTAL REFLECTION–FOURIER TRANSFORM INFRARED SPECTROSCOPY

A technique used to measure the average helix tilt angle of a membrane protein. Attenuated reflection refers to the use of a reflection element on which a protein–phospholipid mixture is dried. Multiple reflections enhance the sensitivity of the technique.

LIPID ORDER PARAMETER

The extent to which the fatty-acyl side chains of the membrane phospholipids are ordered.

HYDROGEN/DEUTERIUM (H/D) EXCHANGE

The rate at which the backbone amide protons exchange with deuterons. This can be measured either by infrared spectroscopy or by studying the amide II vibration.

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Kaback, H., Sahin-Tóth, M. & Weinglass, A. The kamikaze approach to membrane transport. Nat Rev Mol Cell Biol 2, 610–620 (2001). https://doi.org/10.1038/35085077

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