(Current topics in membranes and transport 4) felix bronner and arnost kleinzeller (eds ) elsevier, academic press (1974)

Kedem and Katchdsky were concerned primarily a t that time with the permeability of biological membranes to nonelectrolytes; Spiegler waa concerned with transport processes in ion-exchan

Current Topics

in Membranes and Transport

Vdllllls 4

and

Arnost Kleinzeller

Graduate Division of Medicine University of Pennsylvania Philadelphia, Pennsylvania

Academic Press New York and London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1973, BY ACADEMIC PRESS, INC

ALL RIGHTS RESERVED

NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR

TRANSMllTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC

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United Kingdom Edition published by

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24/28 Oval Road, London NWI

LIBRARY OF CONGRESS CATALOQ CARD NUMBER: 70-117091

List of Contributors, vii

Preface, ix

Contents of Previous Volumes, xi

Aharon Katzir-Katchalsky, 1913-1972, xiii

Bibliography of the Principal Publications of Aharon Katzir-Katchalsky on Membrane Phenomena, xix

The Genetic Control of Membrane Transplant

CAROLYN W SLAYMAN

I Introduction, 1

11 Isolation of Transport Mutants, 3

111 Criteria for Identifying Genes That Affect Transport Directly, 136

IV Linkage Relationships of Transport Mutants, 140

VI Regulation of Transport Systems, 150

V Dominance and Recessiveness, 146

VII Usefulness of Mutants in Understanding Transport Mechanisms, 151

11 Action of Hydrolytic Enzymes on Model Systems, 184

111 Effect of Enzymic Hydrolysis on System Properties of Biomembranes, 191

IV Effect of Enzymic Hydrolysis on Transport Systems, 217

V Catabolism of Membrane Components by Endogenous Enzymes or

Intracellular Catabolism, 233

References, 238

VI Conclusions and Epilog, 236

Regulation of Sugar Transport in Eukaryotic Cells

HOWARD E MORGAN AND CAROL F WHITFIELD

I Kinetic Characterization of Passive Transport, 256

11 Nonhormonal Regulation of Sugar Transport, 261

111 Hormonal Control of Sugar Transport, 274

IV Mechanisms of the Regulation of Transport, 287

V Summary, 296

References, 297

List of Contributors

Richard P Durbin, Cardiovascular Research Institute and Department of Physiology,

Mahendra Kumar Join, Department of Chemistry, Indiana University, Bloomington,

Howard E Morgan, Department of Physiology, The Milton S Hershey Medical Center,

Carolyn w Slayman, Departments of Human Genetics, Microbiology, and Physiology,

Carol F Whitfleld, Department of Physiology, The Milton S Hershey Medical Center,

University of California, San Francisco, California

Indiana*

The Pennsylvania State University, Hershey, Pennsylvania

Yale University School of Medicine, New Haven, Connecticut

The Pennsylvania State University, Hershey, Pennsylvania

* Present address: Department of Chemistry, University of Delaware, Newark, Delaware

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The fourth volume of Current Topics in Membranes and Transport ex- tends the analysis of significant transport processes and structures Carolyn Slayman presents a comprehensive summary of the genetics of transport, Jain reviews some aspects of the bilayer nature of the biological membrane, Morgan and Whitfield deal with sugar transport and its control in eukary- otes, and Durbin reviews some problems of gastric ion secretion The editors hope that all interested in biological transport will find these re- views rewarding and provocative

Last year we dedicated the volume to the memory of Aharon Katair- Katchalsky Caplan’s thoughful appreciation in this volume reminds us of the breadth of Aharon’s work and of the human qualities that went into it

We hope this series will continue to reflect the scientific ideal of Aharon Katdr-Katchalsky, namely a passionate belief in the dispassionate search for scientific truth

FELIX BRONNER ARNOST KLEINZELLER

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Contents of Previous Volumes

Volume 1

Some Considerations about the Structure of Cellular Membranes

The Transport of Sugars across Isolated Bacterial Membranes

Galactoside Permease of Escherichia coli

Sulfhydryl Groups in Membrane Structure and Function

Molecular Architecture of the Mitochondrion

DAVID H MACLENNAN

Author Index-Subject Index

MAYNARD M DEWEY AND LLOYD BARR

Ion-Translocation in Energy-Conserving Membrane Systems

Structure and Biosynthesis of the Membrane Adenosine Triphosphatase

of Mitochondria

Mitochondria1 Compartments : A Comparison of Two Models

HENRY TEDESCHI

Author Index-Subject Index

W R LIEB AND W D STEIN

ROBERT E FORSTER

B CHANCE AND M MONTAL

xii CONTENTS OF PREVIOUS VOLUMES

Properties of the Isolated Nerve Endings

W J ADELMAN, JR AND Y PALTI

GEORGINA RODR~GUEZ DE LORES ARNAIZ AND

EDUARDO DE ROBERTIS

Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells : I n Vitro Studies

The Movement of Water Across Vasopressin-Sensitive Epithelia

Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm

Author Index-Subject Index

J D JAMIESON

RICHARD M HAYS

WILLIAM R HARVEY AND KARL ZERAHN

Aharon Katzir-Katchalrky

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or discussion around the coffee table; the sense of excitement-sometimes bordering on euphoria-he never failed to communicate to the audience during his lectures; the in- tense pleasure he took (and gave) in debating a theoretical point, chalk in hand He loved elegance and style wherever he found them, but especially in a page of mathematics;

a blackboard covered with equations in his own graceful handwriting often possessed something of the richness of a medieval manuscript Of what manner of a man he was- his warmth, humanity, and largeness of spirit-others have written eloquently For those

of us who had found inspiration in his teaching, including his oldest colleagues, he always remained in the profoundest sense of the word a mentor

In a way it is an invidious task to separate Katchalsky’s studies on membranes from the totality of his work Viewed as a whole, his work is as impressive in its unity as it is astonishing in its variety The twin themes of mechanochemical coupling and chemo- diffusional coupling dominate large parts of it, both being intimately related to the most characteristic properties of biological systems His contributions to the understanding

of such processes do not, however, stand apart from his contributions to polymer chem- istry and nonequilibrium thermodynamics His ideas on membranes encompassed many dimensions of the problem and had wide ramifications The earliest papers, written in the late ~ O ’ S , were directed towards the proper understanding of existing experimental techniques and the proper interpretation of permeability measurements commonly made on biological membranes The implications were speedily realized to have impor- tance in the technological application of membranes-especially in desalination For Katchalsky, however, synthetic membranes remained models for biological systems, and

by the mid-60’s the problem of the coupling of chemical reaction and transport had become paramount in his thoughts In his last years he was deeply concerned with the mechanism of memory recording, which involved considerations relating to hysteretic effects in both biopolymers and biomembranes As always the thermodynamic implica- tions of the phenomena intrigued him, and frequently led him far into the realm of philosophical speculation

Katchalsky’s early work on membranes and transport phenomena in general was a natural outgrowth of his studies, covering more than a decade, of the chemical physics and biophysics of macromolecules At the outset of his career he was profoundly im- pressed by the notion that large polymeric molecules might play an essential role in all processes occurring in living systems-as a consequence of, among other things, their conformational degrees of freedom This was a t a time when little was known of protein structure and DNA was still to be recognized as the carrier of genetic information However, the work of Staudinger and later Kern in the 30’s had suggested that charged synthetic polymers would be useful as models of biological macromolecules I n the brief period between the end of World War I1 and the beginning of the Israeli War of In-

xvi

dependence, Katchalsky developed his ideas during a seminal spell in the laboratory of the redoubtable Werner Kuhn in Basel, and returned to Palestine (as it then was) to found a school of polyelectrolyte chemistry a t the Hebrew University on Mount SCOPUS With the conclusion of hostilities this school shifted to Rehovot, where he was called upon to establish a polymer department a t the newly-created Weizmann Institute of Science Starting with papers in the first volumes of the Journal of Polymer Science in

1946 and 1947, Katchalsky and his colleagues produced a veritable avalanche of theo- retical and experimental publications, making an explosive impact on the rapidly growing field of polyelectrolyte solutions and gels The impact of these studies was also felt in the equally rapidly developing technology of ion-exchange resins and membranes, stimulating the concerns which led ultimately to Katchalsky’s later work on the per- meation of salt through charged membranes In the papers written during this “poly- electrolyte” period the seeds of his deep and lasting interest in mechanochemistry are

to be found, and the profound influence of Gibbsian thermodynamics on his thinking is already strikingly in evidence

In the early 50’s Staverman demonstrated the utility of nonequilibrium thermo- dynamics in describing membrane processes, and introduced the concept of a reflection coefficient in relation to the osmometry of polymer solutions Kirkwood showed that local force equations (written in terms of resistance coefficients), in contrast to local flow equations, may be integrated across a membrane t o give global phenomenological relations But very few workers appreciated the power of the method until 1958,

when Kedem and Katchalsky, and also Spiegler, published almost simultaneously and quite independently their classical papers on the application of nonequilibrium thermo- dynamics to membrane transport Kedem and Katchdsky were concerned primarily a t that time with the permeability of biological membranes to nonelectrolytes; Spiegler waa concerned with transport processes in ion-exchange membranes Katchalsky had always understood the importance of thermodynamics as an organizing principle, often claiming that it “plays the role of scientific logics.” It was therefore characteristic of his thinking

to invoke nonequilibrium thermodynamics when his attention turned t o membranes and transport

Undoubtedly the contribution for which Katchalsky is chiefly known (and may well

be chiefly remembered) among workers in the membrane field is precisely his collabora- tive study of permeability with Kedem, which led t o the derivation of what they called

“practical” phenomenological equations These Kedem-Katchalsky (or K-K) equations already possess a time-honored air As Richardson has pointed out, it is little over a decade since the most complete versions of the equations were publbhed, yet they have already taken their place alongside the Nernst-Planck and the Goldman equations as standard working models for physiologists and biophysicists I n fact the Kedem- Katchalsky equations are familiar to a generation of young biophysicists who may never read the original papers In one simple form, applicable to systems involving a single solute, the equations describe volume flow (Jy) and solute flow (J.) through a homoge- neous membrane in the absence of electric current:

Here A p and AT are the hydrostatic and osmotic pressure differences respectively, and

cs is an average of the solute concentrations in the solutions on either side of the mem- brane The “practical” phenomenological coefficients appearing in Eqs (1) and (2) are the filtration coefficient L,, the solute permeability U , and the reflection coefficient Q

AHARON KATZIR-KATCHALSKY, 191 3-1 972 xvii

originally introduced by Staverman Prior to the derivation of these equations there existed no self-consistent treatment of permeability, and certainly none general enough

to cover the whole range of physiological phenomena Conventional descriptions of

membrane transport made use of two independent flow equations, one for volume and one for solute, with only two coefficients Thus volume flow was represented by an

expression of the type

J v = L p ( A p - A T )

and solute flow by the Fickian form

both manifestly incomplete in the light of present-day concepts, since the possibility of coupling between flows is entirely ignored Although experimentalists had become progressively aware of the inadequacy of expressions such as these, and although non- equilibrium thermodynamic treatments of transport were in the air and indeed osmotic pressure measurement had been characterized in terms of the reflection coefficient, it remained for the Kedem-Katchalsky equations to link the phenomena, for the first time, into a single gestalt capable of yielding an internally coherent description in physiological

terms This literally revolutionized membrane studies

Equation (2) indicates that the solute permeability w is to be determined under condi- tions of zero volume flow, by measuring the ratio ( J s / A ~ ) ~ v 4 In an alternative version

of the equations a “second permeability” w‘ appears, to be determined under conditions such that the hydrostatic and osmotic pressure differences just balance, i.e., by meas- uring the ratio ( J a / A ~ ) ~ 9 - ~ I This version can be written conveniently in a form

such that the all-important property of Onsager symmetry (identity of the cross-coeffi-

Equations (1) and (3) are identical, and the last term in Eq (4) is obviously just o’Aa

Clumsy though they are, these expressions bring out the nature of the thermodynamic forces conjugate to the flows J, and J., which are seen to be ( A p - AT) and ( A r / c )

respectively Such conjugate flux-force pairs are generated, according to the methodology

of nonequilibrium thermodynamics, by first deriving the so-called “dissipation function,” i.e., the temperature times the rate of entropy production due to irreversible processes

in the membrane This takes the form

T ( d i S / d t ) = J v ( A p - A T ) J&a0 ( 5 )

where the quantity Apec denotes the concentration-dependent part of the chemical potential difference of the solute across the membrane Equation ( 5 ) is exact for dilute solutions providing that local processes within the membrane obey linear relations

Kedem and Katchalsky sought t o elicit, from the global phenomenological relations

corresponding to this dissipation function, a set of transport coefficients which t o some extent would have the virtue of familiarity to researchers in the field (and thus be readily accessible experimentally and compatible with existing data), to some extent would be insensitive to concentration changes, but above all would be thermodynamically self- consistent To reconcile these requirements they were compelled t o introduce the

xviii AHARON KATZIR-KATCHALSKY, 191 3-1 972

appropriate average concentration csr such that

Consequently, cs is a logarithmic average This transformation of the dissipation function has the virtue of “saving the phenomenon,” and led directly to the formulation of the practical flow equations

It is perhaps a reflection of the importance attached to the Kedem-Katchalsky equations that they did not fail to attract their share of criticism It has been variously claimed that they are misleading on a t least two grounds: that their range of validity (the linear regime) must be vanishingly small, and that global Onsager symmetry cannot actually hold when bulk flow occurs This is not the place to analyze such criticisms in depth, but a comment seems to be called for It is basic to the nonequilibrium thermo- dynamic approach that the forces be small enough to ensure linearity of the flux equa- tions The transformation from ApBc to AT is itself nonlinear since it invokes the logarith- mic average concentration-but it is the key to the entire representation Certainly this limits the applicability of Eqs (1) and ( 2) to very small values of J , or A T , but it does not by any means render them invalid (especially for the description of physiological membrane processes) On the contrary, it has been shown repeatedly that the Kedem- Katchalsky equations represent a first-order expansion of the integrals of the local frictional equations; indeed the expansion indicates that when the concentration ratio exceeds (say) 2:1, so that c8 departs by more than a few percent from the arithmetic

average, use of the latter can preserve the linear formalism if J , is not too large I t also

emerges from such calculations that the reflection coefficients appearing in Eqs (1) and

( 2 ) [or Eqs (3) and (4)] are identical, and hence global Onsager symmetry does in fact obtain, a result which has been verified experimentally in several systems But clearly

it is in the nature of linear phenomenological equations that they represent behavior within certain limits-they are not expected to be exact under all circumstances How- ever small the range of the Kedem-Katchalsky relations may be, their heuristic and practical importance remains unquestionably immense

Perhaps the turning point in the realization by biologists of the significance of the new approach was the memorable symposium on membrane transport and metabolism held in Prague in 1961, when by all accounts Katchalsky electrified the audience by his presentation At any rate, from then on the biological literature reflects a growing interest

in the formalism Hard on the heels of this meeting appeared an interpretation of the practical phenomenological coefficients in terms of friction and distribution coefficients Here Kedem and Katchalsky turned their attention to charged membranes and demon- strated that for electrolyte permeation through such membranes, the reflection coefficient

u may assume negative values, indicating “anomalous” osmosis The mechanism of anomalous osmosis had been a controversial topic: its elucidation in phenomenological terms was thus a vivid demonstration of the scope of the method The analysis was per- formed for a membrane conforming to the well-known Teorell-MeyerSievers fixed- charge model, yielding results in good agreement with the experimental data of Loeb and of Grim and Sollner This study led naturally to the consideration of electric current flow through charged membranes In an elegant treatment it was shown that if the potential across the system is applied by means of electrodes reversible to one of the ions present, the dissipation function requires just one more term-current times the potential difference between the electrodes

A fully developed treatment of the permeability of highly charged membranes t o

electrolytes was presented in a series of three successive papers by Kedem and Katchalsky

in 1963 These papers analyzed the properties of composite membranes in terms of series

AHARON KATZIR-KATCHALSKY, 1 9 1 3-1 9 7 2 xix

and parallel arrays, emphasizing the fundamental importance of polarity and circulation (respectively) in understanding the behavior of such structures I n particular, this analysis showed that mosaic membranes may exhibit pronounced negative anomalous osmosis, i.e., high negative values of u, an effect which had been predicted qualitatively much earlier by Sollner and demonstrated by Neihof and Sollner The substance of Katchalsky’s work on membrane and other transport processes up to this point was incorporated in the book “Nonequilibrium Thermodynamics in Biophysics” by

Katchalsky and Curran, published in 1965

I n the late 60’s Katchalsky became increasingly absorbed in the problem of coupling

between diffusion and chemical reaction, especially as manifested in biological mem- branes An examination of the thermodynamics and kinetics of active transport in erythrocytes (with Blumenthal and Ginzburg) was followed by an analysis of facilitated diffusion (with Blumenthal) which modeled, inter a h , the allosteric transitions of a

carrier protein At this time periodicity in membranes assumed an important place in his thinking-especially periodicity related to the presence of chemical reaction To- gether with Spangler he showed that if an autocatalytic system undergoes a phase transition with metastable states, oscillatory behavior can be achieved by appropriately coupling the reaction t o a membrane transport process regulating the flow of product or reactant These considerations invoked the concept of thermodynamically metastable

ateady states and suggested the possibility that hysteresis loops might exist in transitions

between steady states Such hysteresis cycles in biomembranes were linked t o similar phenomena in biopolymers and t o the phenomenon of memory

I n 1969 Katchalsky wrote the following credo*:

I believe that the ultimate goal of biological study is to “translate” the phenomena

of life into meaningful physical concepts It is rather clear that present-day physics is still unable t o deal adequately with the complex and diversified expressions of life, and many years of research and contemplation await the scientists before they will be able to fit animate and inanimate matter into a common, unified conceptual framework The driving force of quantitative biological study is, however, our mystical conviction that “Nature” is one and that future generations will comprehend life within an integrated

“Natural Philosophy of the Physical World.”

The profound belief in the unity of nature expressed here led Katchalsky t o pondex deeply the chemical basis of morphogenesis and the origin of life His ideas were in- fluenced primarily by the early work of Turing on the ability of homogeneous chemical processes to develop structure spontaneously (as a consequence of a random disturb- ance) and the more recent work of Prigogine on the thermodynamic theory of structure and stability Structures which survive only by the dissipation of an energy input were termed by Prigogine “dissipative structures.” They can appear in systems maintained far from thermodynamic equilibrium, and are characterized by an unstable transition point (a “symmetry-breaking” transition or instability) Such transitions in chemical systems require nonlinear reaction schemes such as occur in the autocatalytic process referred to above Katchalsky considered that dissipative structures may not only play a major role in the maintenance of certain cellular patterns, but that they may also have participated in the development of the earliest structures from which life arose Prior to his end Katchalsky was preoccupied with several complementary interests: the nature of prebiotic peptide synthesis, the consequences of chemodiffusional coupling,

* I n “Biology and the Physical Sciences” (S Devons, ed.) Columbia Univ Press, New York

xx AHARON KATZIR-KATCHALSKY, 1 9 1 3-1972

and the basis of the molecular memory record Through all these there runs as a unifying thread the notion of generation, storage, and retrieval of information This notion is strongly reflected in his later writings concerning membrane phenomena He viewed chemodiffusional coupling as a structuring agent closely related to biological mor- phogenesis, and this led him to speculate that active membranes might be dissipative structures whose dynamics are governed by the very chemodiffusional processes par- ticipating in their action Indeed he conjectured, in agreement with Prigogine, that all living organization may be based on dissipative structures, fixed to dxerent degrees by covalent bonds His deep concern with information flow, energy flow, and the establish- ment of pattern and shape led him t o break fresh ground in the application of thenno- dynamics to complex systems A love of thermodynamic rigor expressed itself in everything he did, and his final major work-which was cut short even as it began to flourish-was an attempt to develop a “network thermodynamics” especially suited to the organizational complexity of biological systems By generalizing network theory t o include irreversible thermodynamic systems, thermodynamics was to be brought within the framework of modern dynamic systems analysis The bond graph approach to systems analysis, still in its infancy, impressed Katchalsky with its versatility as a representation of arbitrarily complex networks In his last paper on this subject, com- pleted just before he died, he showed, together with Oster and Perelson, that the bond graph technique can be used to describe diffusion-reaction systems including facilitated and active transport, the rectification properties of complex membranes, and relaxation oscillations in coupled membrane systems

Many of Katchalsky’s ideas on these and other matters live on in the minds of his colleagues, and will ultimately see the light of day But although the direction in which the broader thrust of his thinking lay may be known, we are denied the pleasure of ever witneesing its realization His work will be carried forward, but we shall never know which path Katchalsky himself would have trodden The particular flavor and style that was his will be missing, and so will the insight and the flair for finding connections among

a bewildering variety of seemingly unrelated concepts

Katchalsky was born in Lodz, Poland, came to Israel in 1925, and received his M.Sc and Ph.D degrees from the Hebrew University in 1937 and 1940 He had many honors, and engaged in a host of activities He was the first President of the Israel National Academy of Sciences and Humanities, President and then Honorary Vice-president of the International Union for Pure and Applied Biophysics, a Council Member of the International Council of Scientific Unions, a Foreign Member of the United States National Academy of Sciences, a Council Member of the European Molecular Biology Organization, and Visiting Miller Professor of the University of California a t Berkeley

He was a true Renaissance man, a citizen of the world, renowned for his charismatic charm, his encyclopedic knowledge of topics ranging from history to philosophy and metaphysics, his love of the cut and thrust of scientific dialogue In his work he constantly emphasized historical perspective, and in his passing he symbolized the historic con- frontation of his people-creativity versus hatred and destruction The cruel snuffing out of his life deprived us all of a very precious source of enlightenment, but he left a legacy of riches to build on for a long time

S R CAPLAN

Bibliography of the Principal Publications of

Aharon Katzir-Katchalsky on Membrane Phenomena

Kedem, O., and Katchalsky, A (1958) Thermodynamic Analysis of the Permeability

of Biological Membranes to Non-electrolytes Biochim Biophys Acta 27, 229 Katchalsky, A (1961) Membrane Permeability and the Thermodynamics of Irreversible Processes “Membrane Transport and Metabolism” (A Kleinzeller and A Kotyk, eds.), p 69 Academic Press, New York

Kedem, O., and Katchalsky, A (1961) A Physical Interpretation of the Phenomeno-

logical Coefficients of Membrane Permeability J Gen Physiol 45, 143

Katchalsky, A,, and Kedem, 0 (1962) Thermodynamics of Flow Processes in Biological

Systems Biophys J 2, 53

Kedem, O., and Katchalsky, A (1963) Permeability of Composite Membranes Part I Electric Current, Volume Flow and Flow of Solute Through Membranes Trans Faraday SOC 59, 1918

Kedem, O., and Katchalsky, A (1963) Permeability of Composite Membranes Part 11 Parallel Elements Trans Faraday SOC 59, 1931

Kedem, O., and Katchalsky, A (1963) Permeability of Composite Membranes Part 111 Series Array of Elements Trans Faraday SOC 59, 1941

Ginaburg, B Z., and Katchalsky, A (1963) The Frictional Coefficients of the Flows of Nonelectrolytes through Artificial Membranes J Gen Physiol 47, 403

Katchalsky, A., and Curran, P F (1966) “Nonequilibrium Thermodynamics in Bio- physics,” Harvard Univ Press, Cambridge, Massachusetts

Blwnenthal, R., Ginzburg, B Z., and Katchalsky, A (1967) Thermodynamic and Model Treatment of Active Ion Transport in Erythrocytes “Hemorheology” (Proc First Intern Conf.), p 91, Pergamon, New York

Katchalsky, A ( 1967) Membrane Thermodynamics “The Neurosciences, A Study Program” (G C Quarton, T Melnechuk, and F 0 Schmitt, eds.), p 326 The Rockefeller Univ Press, New York

Katchalsky, A., and Spangler, R (1968) Dynamics of Membrane Processes Quart Rev Biophys 1, 127

Katchalsky, A ( 1968) Thermodynamic Treatment of Membrane Transport Pure Appl Chem 16,229

Katchalsky, A (1968) Thermodynamic Consideration of Biological Membranes “Mem- brane Models and the Formation of Biological Membranes” (L Bolis and B A Pethica, eds.), p 318 North-Holland, Amsterdam

Blumenthal, R., and Katchalsky, A (1969) The Effect of the Carrier Association-

Dissociation Rate on Membrane Permeation Biochim Biophys Acta 173, 367 Katchalsky, A (1969) Membrane Thermodynamics “Membranes $ Permeabilite SBlective,” p 19 Editions du Centre National de la Recherche Scientifique, Paris Katchalsky, A ( 1969) Non-equilibrium Thermodynamics of Bio-Membrane Processes

“Theoretical Physics and Biology” (M Marois, ed.), p 188 North-Holland, Amsterdam

Katchalsky, A., and Oster, G (1969) Chemico-Diffusional Coupling in Biomembranes

xxii

“The Molecular Basis of Membrane Function” (D C Tosteson, ed.), p 1 Prentice-

Hall, Englewood Cliffs

Katchalsky, A (1970) Thermodynamic Consideration of Active Transport “Perme- ability and Function of Biological Membranes” (L Bolis, A Katchalsky, R 1)

Keynes, W R Loewenstein, and B A Pethica, eds.), p 20 North-Holland, Amsterdam

Katchalsky, A (1971) Thermodynamics of Flow and Biological Organization ZYGON:

J Relig Sci 6, 99

Katchalsky, A (1971) Biological Flow Structures and Their Relation to Chemico-

Diffusional Coupling Neurosci Res Prog Bull 9, 397

Oster, G., Perelson, A., and Katchalsky, A (1971) Network Thermodynamics Nature (London) 234, 393

Katchalsky, A., and Neumann, E (1972) Hysteresis and Molecular Memory Record

Int J Neurosci 3, 175

Oster, G F., Perelson, A S., and Katchalsky, A (1973) Thermodynamics of Biological

Networks Quart Rev Biophys 6, 1

The Genetic Control of Membrane Transport

CAROLYN W SLAYMAN

Departments of Human Genetics Microbiology and Physiology

Yale Univwsily School of Medicine New Haven Connecticut

B Selection of Nongrowing Cells 129

C Direct Screening for Uptake 132

E The Strategy of Recovering Transport Mutants 133

F The Problem of Multiple Transport Systems for a Single Substrate 134

136

Linkage Relationships of Transport Mutants 140

A Escherichia coliand Salmonella typhimurium 140

B Neurospora crassa 144

Dominance and Recessiveness 146

Regulation of Transport Systems 150

A In Determining the Number of Separate Transport Systems for a Particular Substrate 151

C In Determining the Functionof a Component 152

D Recognition of Transport Defects in Higher Organisms 133

Criteria for Identifying Genes that Affect Transport Directly

Usefulness of Mutants in Understanding Transport Mechanisms 151

B In Identifying the Components of a Transport System 152

1 INTRODUCTION

The genetic approach to the study of membrane transport can be an extremely useful one By isolating and mapping mutants defective in the transport of a particular substrate it is possible to determine the number

of systems involved and even the number of subunits in each system;

by analyzing the kinetics and the biochemistry of transport in the mutants

2 CAROLYN W SLAYMAN

one can often obt,ain information about molecular mechanisms Progress

in both of these areas is discussed in this article, together with some of the problems that remain

Membrane genetics is still a relatively young field The first realization that transport systems, like simple cytoplasmic enzymes, are under

genetic control came in the 1950s with work on cystinuria in man and

lactose transport in Escherichia coli Cystinuria had long been known to

be an inherited disease, and in fact was one of the “inborn errors of metabolism” (together with pentosuria, alkaptonuria, and albinism)

discussed by Archibald Garrod in his famous Croonian lectures in 1908

By 1951 cystinuria had been shown to involve the excretion of abnormal

amounts of arginine, lysine, and ornithinc, as well as cystine The structural

relationship among this group of amino acids led Dent and Rose (1951)

to postulate that the disease was caused by a primary defect in transport across the renal tubules Since that time cystinuria has been found to affect the intestinal mucosa as well, and the transport defect has been well characterized i n vitro in intestinal tissue obtained by biopsy (Thier

et al., 1964, 1965; McCarthy et al., 1964) I n addition, other inherited

diseases have been shown to lead to transport defects in man (for example, Hartnup disease, iminoglycinuria, methionine malabsorption, tryptophan malabsorption, X-linked hypophosphatemia), and the idea that all mammalian transport systems are under genetic control-even though mutations may often be lethal or otherwise undetectable-is now widely accepted

The genetic analysis of transport in microorganisms also dates to the

1950s Although the earlier results of Davis and of Doudoroff had drawn

attention to the cryptic nature of certain enzyme systems in bacteria (in which intact cells were unable to metabolize a given substrate even though cell-free extracts contained the necessary enzymes), i t was the work of Cohen and Rickenberg (1955) and Rickenberg et al (1956) that

first demonstrated the existence of a specific bacterial transport system These investigators showed that the product of the lacy gene is required for the transport of p-galactosides in E coli, and that its synthesis is regulated jointly with the synthesis of the enzyme 8-galactosidase The kinetics of the transport system have since been examined in detail, both in wild-type E coli and in l a c y mutants, and more recently the

l a c y gene product ([%I protein”) has been isolated by Fox and Kennedy

(1965) I n microorganisms, and particularly in E coli, this initial work has been followed by the isolation of many different kinds of transport mutants, and the notion that transport systems are genetically determined has been amply documented

This article discusses tfhe techniques that have been developed to isolate

GENETIC CONTROL OF MEMBRANE TRANSPORT 3

and characterize transport mutants, the criteria for identifying genes that affect transport directly, the linkage relationships of transport mutants in several organisms (E coli, Salmonella typhimurium, Neurospora crassa) ,

the regulation of synthesis of transport systems, and the usefulness of mutants in understanding transport mechanisms I n order t o simplify the discussion, basic information about the properties of existing transport mutants has been collected in Table I, leaving the text free to consider the general topics just listed [For further information on microbial trans- port systems, the reader is referred to recent reviews by Heppel (1971), Kaback (1970a, b, 1972), Lin (1970, 1971), Oxender (1972a, b), Roseman (1972), and Simoni (1972) ; and for a more complete description of genetic defects affecting transport in man, to reviews by Rosenberg (1969), Rosenberg and Scriver (1969) , Scriver (1969) , Thier and Alpers (1969), Scriver and Hechtman (1970), and t o several chapters in Stanbury et al

(1972) ]

II ISOLATION OF TRANSPORT MUTANTS

As interest in the genetic analysis of transport has increased, a variety of

methods havc been developed for the isolation of transport mutants, particularly in microorganisms Some are selection methods, making use of conditions under which the transport mutant can grow but the wild-type cell cannot (or the mutant survives and the wild type is killed); others are merely screening methods, permitting the rapid identification of transport mutants among large numbcrs of wild-type cells The particular strategy to be used in a given instance depends, as discussed below, on the function of the transport system in question and on whether or not there are alternate routes for the substrate to enter the cell

A Resistance to Analogs

One of the most powerful methods involves selecting mutants whose growth is resistant t o an appropriate analog This approach has been particularly successful in the isolation of amino acid, purine, and pyri- midine transport mutants (Table 11), but has also been used for carbo- hydrate, cation, and anion transport mutants

Cclls can, of course become resistant to analogs through several kinds

of mutations-not only those reducing the uptake of the analog, but very frequently those affecting later steps in metabolism (see review by Umbarger, 1971) I n studying the effects of D-cycloserine (an analog of alanine) in Streptococcus strain Challis, for example, Reitz et al (1967)

found two classes of resistant mutants The first possessed a defective

TABLE I Mutations Affecting Membrane Transport

Amino Acids and Peptides Organism Transport System Specificity Gene Escherichia Aspartate A constitutive, high-affinity transport system which is asf

c o l i fairly specific f o r L-aspartate (K, = 3.7 x 10-6 M I

Ki's f o r D-aspartate, L-glutamate, L-glutamine, and a series o f aspartate analogs (N-formyl-L-aspartate, N-methyl-DL-aspartate, a-methyl-DL-aspartate,

p-methyl-DL-aspartate, DL-eryfhro-p-hydroxyespartate, and DL-fhreo-p-hydroxyaspartate) are substantially

higher, ranging f r o m 1.8 t o 8.4 x lo4 M (Kay, 1971 )

Glutamate A transport system, partially repressed in wild-type

E coli (Marcus and Halpern, 1969) which i s fairly specific f o r L-glutamate (K, = 7.7 x 10-6 M; Halpern and Even-Shoshan, 1967) Competitively inhibited by D-glutamate, L-glutamine, and several glutamate derivatives (L-glutamate-y-methyl and -y-ethyl esters, p-hydroxy-DL-glutamate, a-methyl -DL-glutamate)

w i t h Ki's o f 2.10 x 10-5 t o 3.35 x 10-3 M (Halpern and Even-Shoshan, 1967) Shows complex kinetics

w i t h nonlinear double reciprocal plots under some

glfC

Linkage Method of Isolating Mutants Transport Defect in Mutants

Near xyl (Kay and

Kornberg, 1969; Lo

etal., 1972) May be

allelic with the FM-

mutant of E coli

isolated by its inabil-

ity to grow on suc-

cinate and later

shown to be deficient

in the uptake of di-

carboxylic acids and

strain (which lacks the general dicarboxylic acid transport system; see below) by selecting for the inability to use aspar- tate as sole nitrogen source;

several mutants with this phe- notype were found but proved

t o transport aspartate as well

as the parent strain (Kay, 1971)

Resistance to 3-fluoromalate (Kay and Kornberg, 1969; Lo

era/., 1972) or L(-)-tartrate (Kay and Kornberg, 1971)

dct mutants are also unable

to grow on malate, succinate,

or fumarate as sole carbon source (Kay and Kornberg,

1969, 1971) Revertants, selected for the ability t o grow on any one of the C4

dicarboxylic acids, have simultaneously recovered the ability to grow on a l l the other acids (Kay and Kornberg, 1969,1971)

dct mutants must be distin- guished from strains with a

primary block in the tricarbox- ylic acid cycle; such strains often showa secondary impair- ment of the ability to take up

one or more C4 acids (Kay

and Kornberg, 1971)

Isolated, in wild-type E coli

W, H,or K-12 (site), by the ability to grow o n glutamate

as carbon source (Halpern and Umbarger, 1961; Halpern and Lupo, 1965; Marcus and Halpern, 1967).g/tcC mutants also show increased sensitivity

to 2methyl-DLglutamate (Halpernand Umbarger, 1961)

Lack the high-affinity aspartate trans-

port system The V,,, for aspartate uptake in one ast mutant, HA3, was decreased from 39 to 25 nmolesl minute per milligram dry weight, and and a single Km (30 x 1 0 6 MI was observed, compared with two Km's

(39 and 3.7 x 10-6M) in the wild- type The defect is also seen in iso- lated membrane vesicles from ast

mutants (Kay, 1971)

Lack tho low-affinity dicarboxylic acid uptake system I n one dct mutant, F M l , the V,,, for aspartate uptake was decreased to 1.5 inmolesl

minute per milligram dry weight, and there was a single K, of 3.5 x 10-6M (Kay, 1971)

gltc is believed to be the operator for gltS (see below).gltC mutants show quantitative but not qualitative alterations in glutamate transport;

in a series of such strains the V,,,

was increased by a factor of 2 to 7, while the K,,, was unchanged (Halpern and Lupo, 1965; Marcus and Halpern, 1969) Furthermore,gltC andgltS are very closely linked, and gltC mutants

(Con tinued)

TABLE I Mutations Affecting Membrane Transport (Continued)

Amino Acids and Peptides Oraanism TransDort Svstem SDecificitv Gene

by binding a t a second site (Halpern and EvenShoshan,

1967)

A glutamate-binding protein, released from g l C

mutants during spheroplast formation, is thought to

be involved in glutamate transport It has a KD of

6.7 x 1 0 6 M (close to the K, of the transport system),

is inhibited competitively by L-glutamate-y-methyl ester and noncompetitively by alanine, and can restore the capacity of spheroplasts for glutamate uptake (Barash and Halpern, 1971)

g/fS

gltR

A highly specific transport system for glutamine -

(K, = 0.8 x l o 7 M ) , competitively inhibited by y-glutamylhydrazide and y-glutamylhydroxamate but not by any naturally occurring amino acids

Transport is thought to involve a glutamine-binding protein, which is released by osmotic shock and has

a K D (for glutamine) of 3 x M Both transport and the binding protein are repressed by growth in a

rich medium (Weineret a/., 1971; Weiner and Heppel,

Linkage Method of Isolating Mutants Transport Defect in Mutants

19691 Might also be isolated from gltCc by resistance to 2methyl-D L-glutamate (Halpern and Umbarger, 1961;

Halpern and Lupo, 1965)

Temperature-sensitive g/tR

mutants were isolated, from wild-type E coli, by their ability to grow on glutamate

at 42' but not a t 30" (Marcus and Halpern, 1969)

Ability to grow on glutamine

as sole carbon source (Weiner

e t al 1971 ; Weiner and Heppel, 1971)

Resistance to y-glutamylhy- drazide (Weiner and Heppel, 1971)

are derepressed (with high levels of glutamate transport activity) even in

the presence of a normal g/tR gene

(the postulated repressor gene; see below) The kinetics of transport in

gitCC strains are discussed further in Halpern (19671 Halpern and Even- Shoshan (1967) and Frank and Hopkins (1969)

g/tS i s thought to be the structural gene for the glutamate transport sys- tem.g/rS mutants with qualitatively altered transport systems have been isolated; in strain CS 7/50, for e x - ample, the Vmax was decreased by a

factor of 3,and the K,,, was increased

by a factor of 20 (from 5 x 10.6 M to

1 x 1 0 4 M ) (Marcusand Halpern, 1969) No mutants with altered allo- steric properties (see Specificity) have yet been reported

g/tR i s thought to be the repressor gene for the glutamate transport operon In one temperature-sensitive

g/tR mutant (CS ZTC), the V,,, of transport a t 42' was increased by a

factor of 4.5, with no change in Km

Furthermore, brief periods of heating

a t 44' in the absence of growth

increased the differential rate of syn- thesis of glutamate transport activity during subsequent growth a t 30°, suggesting that the repressor i s ther- molabile in this mutant (Marcus and Halpern, 1969)

Increased transport of glutamine Mutant strain GLNP 1 showed a

3-fold higher initial uptake rate (with

a normal Km) and had three times more binding protein than the parent strain (Weiner and Heppel, 1971) Decreased transport of glutamine In mutant strain GH 20, both the initial

uptake rate and the amount of bind- ing protein were decreased by about 90% (and the small amount of uptake

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued)

Amino Acids and Peptides Organism Transport System Specificity Gene Escherichia

(K,,, = 2.6 x lo-* M ) ;

(2) a specific system for lysine (K, = 1 x 10-5 M ) ,

inhibited by thiosine; and

(3) a general system (LAO) for lysine (K, = 0.5 x 10-6 M ) , ornithine (K,,, = 1.4 x 10-6 M ) , arginine, and canavanine

Osmotic shock causes the release of several arginine- binding proteins, one of which may play a role i n the arginine-specific transport system (Wilson and Holden, 1969a, b; Rosen, 1971a) and i n addition a lysine-

arginine-ornithine-binding protein (LAO), which appears to be associated with the general transport system The L A O protein has a molecular weight of 30,000 and KD's of 3.0 x 10-6 M for lysine, 1.5 x

1 0 - 6 M for arginine, and 5.0 x 10'6M for ornithine (Rosen, 1971a)

argf

The lysine-specific system i s not affected by osmotic shock under the usual conditions I n cells in which the general system has been repressed, however, lysine transport becomes sensitive to osmotic shock

and a lysine-binding (LS) protein i s released This protein is labile at 4" and i s rapidly inactivated at ionic strengths above 0.02 (Rosen, 1971 b)

Linkage Method o f Isolating Mutants Transport Defect i n Mutants

Near serA (Taylor,

1970)

that remained may have been medi- ated by another system, since it was inhibited b y glutamate) The genetic relationship between strains GLNP 1

and GH 20 has n o t yet been estab- lished, b u t the quantitative corre- spondence b t w e e n uptake rates and amounts o f binding protein i n the

t w o strains has been used t o support the idea that binding i s involved in

transport (Weiner e r a / , 1971; Weiner

Rosen, 1971a; Maas cited i n Rosen,

1971a), b u t the exact function o f the

argP locus i s n o t clear

Rosen (1971a) reported, i n one such

mutant (Can R22) that t w o transport systems were affected; arginine trans-

p o r t was completely missing, and ornithine and lysine transport via the

L A O system were partially reduced Both the arginine-specific binding protein and the LAO-binding protein have now been found t o be present

i n Can R22, and the mutant shows normal facilitated diffusion of argi- nine (assayed by coupling arginine uptake to arginine decarboxylase, and measuring COP production)

(Rosen personal communication)

Rosen has therefore postulated that the lesion i s in energy coupling, pre- sumably i n a factor common t o both the arginine-specific and L A O trans-

p o r t systems

However, Maas (personal cornmunica-

t i o n ) found that another argP mutant,

JC 182-5, produces an altered LAO-

binding protein with a lowered bind- ing capacity for arginine, ornithine, and lysine; the arginine-specific binding proteins appear normal i n

JC 182-5 Further work will be

Resistance t o canavanine

(€ coli W, Schwartz e t a l ,

1959; E coli K - I 2, Maas, 1965; Rosen, 1971a)

(Continued)

TABLE I Mutations Affecting Membrane Transport Konrinuedl

Amino Acids and Peptidm

Organism Transport System Specificity Gene

4.0 x lO-7M) tyrosine (K, = 5.7 x l o 7 M1,and phenylalanine (K,,, = 4.7 x 10-7 M I , with much lower affinities for several other amino acids (histidine, leucine, methionine, alanine, cysteine, and aspertate) The general system is inhibited by p-fluorophenyl- alanine, @-2-thienylalanine, and 5-methyltryptophan;

aroP

(2) a specific system for tyrosine (K,,, = 2.2 x 1 0 - 6 ~ ) inhibited by p-fluorophenylalanine (and sewral other analogs) and b y high concentrations of phenylelanine;

(3) a specific system for phenylalanine (K,,, = 2.0 x 10-6 M I , similarly inhibited by p-fluorophenylalanine (and several other analogs) and by high concentrations

of tyrosine;

(41 a specific system for tryptophan (K, = 3.0 x 10-6 M I , inhibited by 4methyltryptophan (and other analogs); and

(5) an inducible system for tryptophan (Boezi and

DaMoss, 1961 ; Burrous and DeMoss, 1963)

trpf

Linkage Method of Isolating Mutants Transport Defect in Mutants

Resistance to thiosine (Rosen, personal communication)

Between leu and pan

(Brown, 1970)

Resistance to p-2-thienylala- nine (E coli K-12; Brown, 1970) The aroP mutants were also resistant to p-fluorophe- nylalanine and 5-rnethyltryp- tophan, and additional aroma- tic transport mutants might

be isolated using these analogs

Isolated in an aroP mutant of

E coli K-12 strain W31 10, which has a high level of the tryptophan-specific transport system, by resistance to 4-

methyltryptophan (Yanofsky, cited in Oxender, 1972a)

Between trpA and ton6

(Thorne and Corwin,

in some argP mutants (Can R22) but

apparently not in others (JC 182-5) Lack the specific transport system for lysine but contain the lysine- binding protein (Rosen, personal communication)

Lack the general aromatic transport system (Brown, 1970).aroP mutants retain the specific tryptophan, tyro- sine, and phenylalanine systems, with

K;s similar to those of the wild-type strain but with somewhat lower

V,,,'s This latter finding, if signifi- cant, may mean that the general and specific transport systems share the component determined by the gene aroP (Brown 19711

Not discussed

Two lines of evidence have led to the view that a gene for tryptophan trans-

port is located between trpA and

ton6 (Thorne and Corwin, 1970,

1971 ): (1) Deletions in this region cause a 10-fold decrease in trypto- phan uptake, and ( 2 ) low uptake in

point mutants, selected by resistance

to indole acrylic acid, can be restored

to normal by introduction of the F'trp episome

However, the uptake of other amino acids was not measured in these experiments, and the results might be explained by the observation that deletions extending into the ton6

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued)

Amino Acids and Peptides Organism Transport System Specificity Gene Eschen'chia

Wild-type E coli K-12 has t w o (or three) kinetically

distinct transport systems for glycine atanine, and serine (Wargel etal., 1970; Qxender, 1972a):

(1) a system for L-alanine (Km = 5.7 x 10-5 M ) and probably L-serine inhibited by L-cycloserine and 0-carbamyl-Dserine; and

(2) and (3?) a system for glycine, D- and L-alanine

and D-serine with nonlinear double reciprocal plots which have been resolved into t w o sets of Km's and VmaX's (Km's = 2.5 and 9.1 x 10-5 M for glycine, and 2.5 and 8.2 x 1 0 % M for D-alanine) This system is inhibited by D-cycloserine

picture i s not yet clear Guardiola and laccarino (1971)

reported a single set of Km's for laucine, isoleucine,

and valine (7 x 10-6 M, 4.8 x l o 6 M and 12 x l o 6 M,

respectively); but Piperno and Oxender (19681 ob-

served two K,'s for valine (0.7 and 8 x 1 0 6 MI,

Furlong and Weiner (1970) observed t w o Km'S for leucine (0.2 and 2 x 10-6 M I , and more recently

brnP

brnQ

Linkage Method of Isolating Mutants Transport Defect i n Mutants

region lead t o decreased transport of

a variety of amino acids, as i f tun8

influences general membrane perme- ability (Yanofsky, cited in Oxender, 1972a)

c y c r l (cycA) maps near

purA (Russell 1972);

and cyc'2 and cycr3

which are cotransduci-

ble with one another,

map at least 0.5 minute

away (Curtiss et a/.,

Resistance t o D-cycloserine;

three successive steps in resis- tance (cycrl, cycr2 and cycr3) have been identified in € coli

1972a), or by penicillin treat-

ment of glycine-requiring strains I€ culi W and B) grow- ing in lowglycine medium (Kessel and Lubin 1965) The relationship of these mutants

t o the cyc loci has not been renorted

Isolated, i n a Dserine-resistant mutant that lacked the glycine

D- and L-alanine D-serine sys-

temls), by penicillin salection

on L-alanine (Oxender, 1972a)

Resistance t o valine (Guardiola and laccarino, 1971 1 The growth of wild-type E coli

K-12 i s inhibited by valine, which blocks isoleucine bio- synthesis through feedback inhibition o f acetolactate syn- thetase (Leavitt and Umbarger

and Stadtman, 1968; Wargel et at

1971; Oxender, 1972a) Thecycrl strains lack the high-affinity com- ponent of transport, and the cycr2 and cycr3 strains lack the low- affinity component (Wargel er &., 1971)

The double mutant has lost about 95% of the L-alanine transport capac-

ity and i s assumed t o lack the L-alanine L-serina system as well as

as the glycine D- and L-alanine, D-serine system (Oxender, 1972al

brnP mutant M I 183a showsd

abnormal Km's for isoleucine ( 3 6 x

106M.comparedwith4.8 x 1 0 - 6 M

in the parent strain) and for leucine (0.4 and 7 x 1 0 6 M, respectively), although not for valine (12 and 12.5

x 1 0 8 M ) ; Guardiola and laccarino 11971) concluded that it might h a w

a qualitatively altered transport sys-

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued)

Amino Acids and Peptides Organism Transport System Specificity Gene

Escherichia

coli (Con't.)

Guardiola, De Felice, and laccarino (oersonal com- munication) detected four systems for the branched- chain amino acids, a shared system with K,'s of

about 2 x 10.6 M for leucine, isoleucine, and valine, and specific systems for each of the three amino acids with Km's of about 50 x

At least two binding proteins have been isolated One has comparable affinities for leucine, isoleucine, and valine, and presumably is associated with the shared LIV transport system It has a molecular weight of about 36,000, and both i t and LIV transport are repressed when leucine is present in the growth medi-

um (Piperno and Oxender, 1968; Anraku, 1968a, b, c;

Nakane eta/., 1968; Penrose eta/., 1968, 19701

The second protein binds only leucine It is remarkably similar to the first in molecular weight, amino acid composition and K D for leucine; it, like the first, is repressed by leucine; and the two proteins cross-react immunologically, suggesting a common developmental origin (Furlong and Weiner, 1970; Furlong, personal communication I

M

dlu

Linkage Method of Isolating Mutants Transport Defect in Mutants

valine-resistant mutants defec- tive i n transport, others have been described that possess either a valine-resistant aceto- lactate synthetase o r an increased rate o f isoleucine biosynthesis (Glover, 1962;

Ramakrishnan and Adelberg, 1964,1965)

Isolated, in a leucine-requiring strain, b y the ability t o use D-leucine as a source o f L-

leucine (Rahmanian and Oxender, 1972a.b)

Isolated, in a dlu parent strain

(see above), by resistance t o azaleucine (Rahmanian and

Oxender, 1972b)

t e m for t h e branched-chain a m i n o acids, and thus that brnP might be the structural gene f o r this transport system

By contrast,brnO m u t a n t M I 1 7 4 b showed decreased V,,,'s f o r iso-

leucine, valine, and leucine (by a

factor of 5 t o 10) a n d it was sug- gested t h a t brnO m i g h t be a regula-

t o r y gene (Guardiola and laccarino, 1971)

With the discovery o f m u l t i p l e trans-

p o r t systems f o r leucine, isoleucine, and valine (see Specificity), however,

the kinetic analysis o f b r n P a n d brnO

mutants w i l l have to be carried o u t

in greater detail before one can con- clude which system o r systems are altered b y these mutations and w h i c h are unaffected

Recently Guardiola, De Felice, and laccarino (personal communication) measured t h e amounts and t h e dis- sociation constants of b o t h the L I V and the leucine-binding proteins in

6rnP and b r n 0 mutants, and have

f o u n d the proteins to be normal They conclude tentatively t h a t brnP and brn0 d o n o t code f o r either

binding protein

Increased transport o f D- and L-

leucine, isoleucine, a n d valine via t h e shared system, and increased amounts

o f t h e LIV-binding protein dlu is concluded t o be a regulatory gene f o r the L I V system (Rahmanian and Oxender, 1972b)

One class o f azaleucine-resistant mutants lacks L I V transport activity

b u t retains the LIV-binding protein; another class shows reduced transport activity f o r branched+hain amino acids and also f o r other, unrelated amino acids (Rahmanian and Oxender, 1972b)

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued

Amino Acids and Peptides Organism Transport System Specificity Gene Escherichia Cysti ne , Wild-type E coli W has two transport systems for -

co/i (Con%) diaminopimelic cystine: (1) a general system (K,,, = 3 x lo-' M ) ,

inhibited by diaminopimelic acid (DAP), and (2) a specific system (K, = 2 x 1 0 8 M ) , not inhibited by DAP The activity of the general system i s reduced by osmotic shock, and a cystine- and DAP-binding pro- tein (molecular weight 28,000; K D for cystine = 2 x

l o 7 M ) has been partially purified from the shock fluid (BergereraL, 1971)

acid

Proline Wild-type E coli takes up proline with a K,,, of 6.4 x

10-7 M Uptake is inhibited by azetidine-2-carboxylic acid and 3.4-dehydroproline (with Ki's of 2.4 x 10-5 M, and 2.6 x 1 0 6 M, respectively) and by several other proline analogs (Tristram and Neale, 1968) A proline- binding activity has recently been partially purified from membrane vesicles (Gordon era/., 1972)

-

Dipeptides A transport system for dipeptides containing two -

L-amino acids or glycine plus one L-amino acid

(Kessel and Lubin, 1963;Sussman and Gilvarg, 1971)

Oligopeptides A transport system for oligopeptides containing lysine, -

omithine, glycine, tyrosine, and perhaps other amino acids (Payne, 1968;Sussman & Gilvarg, 1971)

Linkage Method of Isolating Mutants Transport Defect in Mutants

Diaminopimelic acid-requiring mutants of E coli W normally grow slowli with DAP as sole

supplement, and require lysine,

i n addition, for normal growth;

"D" mutants were isolated which had lost the partial requirement for lysine (Leive and Davis 19651

Penicillin treatment of a pro- line-requiring strain of € coli

W or B in low-proline medium

(Lubin etal., 1960; Kessel and Lubin, 1962)

Resistance t o 3.4-dehydropro- line or L-azetidine-2-carbox- ylic acid ( E coli strain C4;

Tristram and Neale, 1968)

Al I deh ydroprol i ne-resistan t

strains tested showed cross- resistance t o azetidine, b u t by contrast the azetidine-resistant strains (14 tested) were sensi- tive t o dehydroproline

Penicillin treatment of a gly- auxotroph of E col; W o n glycylglycine medium (Kessel and Lubin, 19631

in the passive diffusion of proline across the membrane (Kessel and Lubin, 1962; Kaback and Stadtman, 1966)

Some of the dehydroproline-resistant mutants were defective i n proline uptake; others showed normal uptake

b u t excreted large quantities of pro- line (Tristram and Neale, 1968) Most azetidine-resistant mutants were reduced i n proline uptake One mutant was of particular interest because i t s transport system had normal affinities for proline and dehydroproline but a 10-fold re- duced affinity for azetidine In a few azetidine-resistant mutants, proline uptake was normal, proline was not excreted in large quantities, and the mechanism o f resistance is unknown (Tristram and Neale, 19681 The genetic relationship among the various mutants has not yet been determined

Defective uptake of glycylglycine and other dipeptides (Kessel and Lubin, 1963)

Indirect evidence (from growth experiments) for a defect i n the transport of oligopeptides (Payne and Gilvarg, 1968; Payne, 1968)

(Continued)