Function and Structure of Membrane Transport Proteins

Peter J.F. Henderson    (Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK)

INTRODUCTION

The hydrophobic bilayer membrane that bounds cells is inherently impermeable to the great majority of hydrophilic solutes required for cell nutrition and to many of the waste products and/or toxins that must be excreted. Accordingly, the membrane contains proteins, the sole function of which is to catalyze the translocation of substrates through the membrane. As the substrates for many membrane processes can be obtained in radioisotope-labeled form, it has been technically feasible to characterize the functions of many of these transport proteins. The structures of the proteins themselves, however, have proved to be difficult to elucidate: they are of low natural abundance in the membrane; they are very hydrophobic and refractory to isolation methods in aqueous solutions; and, even when purified, usually in non-denaturing detergents, they are very difficult to crystallize. Where the proteins happen to be abundant - bacteriorhodopsin from Halobacterium halobium, K+/Na+ ATPases in nerve and Ca2+ ATPase from muscle, cytochrome oxidases in bacteria and mitochondria, glucose transporter from human erythrocytes, for example – progress has been made in elucidating the structure–function relationship. Yet, of these proteins the three-dimensional structure has only been determined for bacteriorhodopsin and the oxidases1–3, and this is just the beginning of determining their molecular mechanisms of operation.

Free-living microorganisms (bacteria, algae, yeasts, parasitic protozoa) often inhabit environments where nutrients are in short supply, and different species must compete with each other for the available metabolites. Accordingly, they couple expenditure of metabolic energy to inward transport of essential nutrients (K+, NH+4, Pi, SO2−4, sugars, vitamins, etc.) to achieve intracellular concentrations sufficient for optimal growth rates. This expenditure can amount to 20–30% of the organism's available energy when a carbohydrate is fermented under anaerobic conditions to yield only 2–3 moles ATP per mole sugar4,5. Since the efficiencies of the transport steps may therefore influence cell yield and growth rate4,6,7 an understanding of the transport processes is important to both the academic researcher seeking to understand bacterial cell physiology, and the industrial manager trying to maintain the profitability of a fermentation process. Furthermore, the process of eliminating metabolic wastes and/or toxins such as antibiotics is often coupled to the expenditure of metabolic energy, an indication of its importance for survival. Motility appears to be driven by transport processes also, although this may not consume so much energy8.

In higher organisms, where survival functions are distributed between different organs, the energization of nutrient capture and waste efflux may be confined to specific tissues, e.g. the gut and the kidney. As a result of their activities, cells in other tissues enjoy an unchallenging environment in which their energy reserves can be channeled into other functions. Thus, their transport processes more often occur by facilitated diffusion.

As approximately 5–15% of all proteins, revealed by the current efforts in genome sequencing, are membrane transport proteins9, we anticipate the need for a huge effort in the new millennium to determine the structures of these proteins that are vital for the capture of nutrients and hence the first stage in cell growth. Their additional roles in antibiotic resistance, toxin secretion, ATP synthesis, ion balance, generation of action potentials, synaptic neurotransmission, kidney function, intestinal absorption, tumor growth and other diverse cell functions in organisms from microbe to man presage a major investigative effort to elucidate their molecular mechanisms of action. This effort to elucidate vectorial processes can be compared to the continuing efforts to understand enzyme-mediated catalysis, though there is the possibility of an underlying uniformity of translocation mechanism despite the huge numbers of independent transport proteins that exist.

The advent of recombinant DNA technology has enabled the study of membrane transport proteins to be furthered in at least four major directions. The first is the burgeoning appearance of an enormous number of amino acid sequences of the proteins predicted from the DNA sequences of their genes in the genome mapping projects. This sequence information has enabled a second advance: the unambivalent exposure of the evolutionary relationships between proteins not thought hitherto to be related. The third is the manipulation of the genes to expedite amplified expression and purification of the proteins. Finally the ability to mutagenize individual amino acids and to make chimeric proteins is being used to elucidate the relationship of function to structure.

A number of transport proteins play a role in human health and disease. The study of “ABC” transport systems (see later) in mammalian cells was intensified with the discovery that cystic fibrosis, the commonest inherited disease in the western world, was caused by a defect in the Cl− transport protein10. The significance of a multidrug resistance protein, “Mdr” that catalyzes secretion of cytotoxins and the failure of anti-tumor chemotherapy similarly focused attention on a different ABC system. In both cases their similarity as ABC-type systems would have been completely obscured without the amino acid sequence information derived from the cloning and sequencing of their genes. Other transport proteins are involved in glucose/galactose malabsorption, albinism, adrenoleukodystrophy.

This FactsBook is intended to catalyze this new age of exploration of membrane transport protein structure. It is our major goal to arrive at a sensible classification of transport systems based upon both evolutionary and mechanistic considerations. The numbers of protein sequences now known is too large to include them all, and the expected appearance of legions more from the genome sequencing programs makes it timely to formulate a systematic approach to their classification. First it is important to describe current concepts of their functions. The treatment below is necessarily brief, and the reader is referred to the appropriate chapters in standard biochemistry textbooks11,12 for a fuller introduction.

A watershed in the field occurred when Peter Mitchell13–15 showed that transport processes were intimately associated with the mechanism of oxidative and photosynthetic ATP synthesis, a process which is central to energy metabolism in almost all organisms. However, because of the difficulties in studying the hydrophobic membrane proteins involved we know very little about the molecular mechanism of such vectorial events; this contrasts with the wealth of information on the molecular mechanisms of chemical events catalyzed by water-soluble enzymes. It is quite possible that there is an underlying unity in the molecular mechanism of the translocation process, even when the direction of solute movement and any energization steps are completely different. This question is likely to be illuminated only when we elucidate the 3D structures and determine the structure-activity relationships of the transport proteins. By far the most central question in the transport field is precisely this – what are the 3D structures of the proteins involved?

Before reaching this question it is useful to define some terms often used in the characterization of transport processes.

USEFUL CONCEPTS

Passive diffusion

Passive diffusion is the translocation of a solute across a membrane down its electrochemical gradient without the participation of a transport protein. The process follows Fick's law, and so obeys the relationship below in which the velocity has a linear relationship to the [solute]:

=PAc

where v is velocity, P is the permeability coefficient for the particular solute, A is the area, and c is the difference in solute concentration across the cell membrane.

Diffusion has a low temperature coefficient (v ∝ absolute temperature) and is non-specific. Typical biologically important compounds that follow this mechanism are O2, CO2, NH3, HCO2H, CH3CO2H, CH2OH.CHOH.CH2OH – small, neutral molecules that are soluble in lipid membranes.

Facilitated diffusion

Facilitated diffusion is the translocation of a solute across a membrane down its electrochemical gradient catalyzed by a transport protein. The Michaelis–Menten relationship11,12 often adequately relates the initial rate of transport (v) to initial substrate concentration ([S] = c at zero time):

=Vmax.[S]/(Km+[S])

(Vmax is maximum velocity, Km = [S] where v is Vmax/2). As with enzyme reactions, there is a high temperature coefficient and, usually, strong substrate specificity. Biological substrates that follow this mechanism are typically charged and/or larger than about the size of glycerol, with a very low inherent solubility in biological membranes. Mitchell classified such transport of a single substrate as “uniport”, and glycerol transport is an example of...