Molecular biology of carrier proteins.

ferent presentations and approaches was the need for more information on the structure and membrane topology of these proteins. It is evident that the hard-won knowledge of the detailed functional properties of these proteins will only lead to genuine insights when our structural informa- tion is comparably advanced. Introduction Structure and Dynamics The revolution in the study of biological systems at the molecular level has irreversibly altered our approach to fundamental questions and dramatically increased the rate of progress in many fields. One such area is the study of the way in which certain membrane proteins mediate the transfer of solutes across cell membranes. Progress has been remarkable in identifying the primary structure of such proteins; only a decade ago, knowledge of these proteins was often limited to an apparent molecular weight from a gel and a detailed knowledge of the processes mediated, but little of the composition or structure of the protein. Transporters are membrane proteins so that although they share many properties with soluble proteins, their location within the membrane phase separating intracellu- lar and extracellular aqueous compartments adds several levels of complexity to the principles governing their struc- ture and function. The standard progression of events in studying these systems consists of deducing the amino acid sequence of the particular transport protein from its cDNA and then speculating on its transmembrane topol- ogy using an appropriate algorithm (see, for example, Kyte and Doolittle, 1982). Thus, an early milestone on the road to understanding the protein is a model for how it is incor- porated into the membrane. This relies on determining the number of transmembrane crossings within the polypep- tide and their location within the primary structure. If appro- priate expression systems are available, various forms of mutagenic analysis are performed in the hope of obtaining structure-function relations for the protein and its interac- tions with the transported substrates. The goal is to under- stand how the protein is able to achieve its catalytic (trans- port) function via changes in conformation. Unfortunately, since the functions of membrane transport proteins are much more readily accessible for study than their struc- ture, progress toward this goal has been less dramatic. The similarities in structure and function of transporters have encouraged scientists working in diverse systems that a great deal is to be learned by listening to each other. A recent meeting at Woods Hole, Massachusetts(Septem- ber, 1992) sponsored by the Society of General Physiolo- gists and organized by L. Reuss and M. Jennings (both University of Texas Medical Branch at Galveston) and J. Russell (Medical College of Pennsylvania), was dedicated to this purpose. The meeting provided abundant evidence of a commonality in approach in many different systems as well as a rapid convergence toward the same limits to further advances. The overwhelming theme that emerged from many dif- In his keynote address, Ft. Kaback (University of California at Los Angeles) offered lac permease of Escherichia coli as a paradigm for membrane transport proteins. Over the past 20 years or so, work on this protein has pro- gressed through arguments about the energy coupling of lactose uptake (ultimately resolved by Mitchell’s chemi- osmotic hypothesis) to the current concerns of identifying specific roles for specific amino acid residues. Lac per- mease is responsible for accumulating 8-galactosides against an electrochemical potential gradient by utilizing the inwardly directed proton electrochemical potential gradient. This is the prokaryotic analogy to eukaryotic Na+dependent transporters where the energy currency of transport is an inwardly directed gradient for Na+ ions. Using methods developed by J. Beckwith and colleagues (Harvard Medical School) (see below), it has been shown that lac permease comprises 12 transmembrane helices (Calamia and Manoil, 1990). In recent years, Kaback’s laboratory has been engaged in the wholesale mutagenesis of lac permease, resulting in proposals for important intra- membrane charge-pairing interactions, as well as for the spatial relationship between specific transmembrane helices. Although this work has certainly enabled the elimination of many earlier suggestions for the identity essential residues involved in active 8galactoside trans- port, the vital structural information that will allow us to distill the data into mechanistic insights is still beyond reach. The initial step in converting deduced amino acid sequence of a membrane protein into structure consists of a proposal for the folding protein within mem- brane, including a decision on how many times the protein crosses the membrane. There are many alternative ap- proaches to this question, but in essence they all consist of identifying likely membrane-spanning regions on the basis of their highly hydrophobic nature, their usual a-helical structure, and their length of around 20-22 amino acids. This analysis provides a first approximation, but the subsequent experimental confirmation is an essential area of study. There are several approaches available, includ- ing chemical modification (using impermeant reagents in sided systems), proteolysis, immunochemical methods, and gene fusion approaches. This latter strategy has been largely developed in the laboratory of Beckwith, and he described progress using the gene fusion approach to membrane protein topology questions. In this approach, reporter proteins are fused to the test protein at different sites along the length of the protein, replacing the C-ter-