LrST OF CONTRIBUTORS Susanne M. Bockholt Department of Biology University of Utah Keith Burridge Department of Cell Biology and Anatomy University of North Carolina, Chapel Hill David G. Capco Molecular and Cellular Biology Program Arizona State University Coralie A. Carothers Carraway Department of Biochemistry and Molecular Biology University of Miami School of Medicine Kermit L. Carraway Department of Cell Biology and Anatomy University of Miami School of Medicine Detlev Drencl<liahn Institute of Anatomy University of WiJrzburg Wurzburg, Germany Jo tin E. Hesl<eth Rowett Research Institute Aberdeen, Scotland Thomas Jons Institute of Anatomy University of Wiirzburg Wurzburg, Germany Colin Masters Faculty of Science and Technology Griffith University Brisbane, Australia VII LIST OF CONTRIBUTORS Ian F. Pryme Department of Biochemistry and Molecular Biology University of Bergen Bergen, Norway Bernd Puschel Institute of Anatomy University of Wurzburg Wurzburg, Germany Jaakko Saraste Department of Biochemistry and Molecular Biology University of Bergen Bergen, Norway Frank Schmitz Institute of Anatomy University of Wurzburg Wurzburg, Germany Howard Stebbings Department of Biological Sciences Washington Singer Laboratories University of Exeter Johan Thyberg Department of Cell and Molecular Biology Medical Nobel Institute Karolinska Institute Stockholm, Sweden INTRODUCTION During the last 10 years it has become evident that the cytoskeleton is intimately involved in different aspects of cell physiology. The chapters in this second volume of the The Cytoskeleton, A Multi-Volume Treatise, describe a wide variety of cell functions in which the cytoskeleton has been either implicated or shown to have a role; the emphasis is on its role in general cell processes rather than specialized aspects in particular cells or tissues which will be described in volume three. A persistent theme throughout this volume is the important role that the cytoskeleton plays in compartmentation, targeting, and subcellular organization. For many years cell biologists and biochemists have speculated as to whether there is compartmentation and spatial organization of metabolic reactions within the cytoplasm. The discovery of the cytoskeleton provided a possible mechanism for such subcellular organization, but to date conclusive evidence for a role of the cytoskeleton in metabolic compartmentation has remained elusive. The possible association of glycol3^ic enzymes with actin is discussed in the context of metabolic compartmentation by Masters. As reviewed by Hesketh and Pryme in the second chapter, there is also an increasing body of evidence that a proportion of both polyribosomes and mRNAs is associated with the cytoskeleton; such interactions may have important roles in mRNA localization and the spatial organization of the protein synthetic apparatus. Compartmentation and spatial organization is particu larly evident in embryonic development and, as described by Capco, the cytoskele ton plays an important role in the early stages of development. ix X INTRODUCTION Cell organization involves transport of material within the cell, as particularly illustrated by axonal transport. The cytoskeleton has well defined roles in organelle transport and this is discussed in the chapter by Stebbings. As described in detail by Drenckhahn and colleagues, such transport by the cytoskeleton can be highly specific in a spatial sense and this allows the cytoskeleton to contribute to the generation of cell polarity; furthermore the interaction of membrane proteins with the cytoskeleton may be the basis of domains within the membrane. The interaction of the cytoskeleton with membrane proteins also has other important functions: as discussed by Bockholt and Burridge, the best-characterized is the interaction of the cytoskeleton with the extracellular matrix which brings about cell adhesion and movement; there is also increasing evidence that links between membrane receptors and the cytoskeleton are important in signaling processes between extracellular and intracellular environments (Carraway and Carraway); finally, Saraste and Thyberg discuss the evidence that the cytoskeleton is involved in secretion. John Hesketh and Ian Pryme Editors ON THE ROLE OF THE CYTOSKELETON IN METABOLIC COMPARTMENTATION Colin Masters I. Introduction 2 II. Evidence for the Micro-Compartmentation of Carbohydrate Metabolism . . .. 3 III. Enzyme Multiplicity and Interactions with Cellular Structure 9 IV. Variation of Structure Within the Cytoskeleton 11 V CovalentModificationof Enzymes and Cellular Structure 13 VI. Energy Requirements for Signal Transduction 16 VII. Matrical Compartmentation 18 VIII. Perturbations of Compartmentation During Cellular Differentiation and Dysfunction 21 IX. Compartmentation in Intermediary Metabolism 25 X. Concluding Comments 26 References 28 The Cytoskeleton, Volume 2 Role in Cell Physiology, pages 1—30 Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-688-6 1 2 COLIN MASTERS I. INTRODUCTION There is increasing evidence that many of the cytoplasmic enzymes exist in vivo as part of an organized structural system that provides a framework for the coordina tion of metabolic activities (Masters, 1981; Clegg, 1984; Srere and Ovadi, 1990). This evidence has been derived from a number of divergent techniques, but probably the most complete and systematic series of investigations into this phenomena in recent years has been those studying the interactions between glycolytic enzymes and the cytoskeleton (Humphreys and Masters, 1986; Chen et al., 1986; Chen and Masters, 1988; Shearwin et al, 1990 a, 1990b; Masters, 1991, 1992). Using this data as an appropriate base, this review seeks to draw attention to the need for modification of many of the classical views of cytoskeletal function and the regulation of intermediary metabolism. Historically, for example early research into intermediary metabolism assumed that individual metabolic pathways were controlled by single regulatory enzymes— an assumption which is no longer recognized as valid (Keleti and Ovadi, 1988). In addition, recent studies have served to emphasize other regions of potential inap- propriateness in the classical biochemical approaches to this topic: many classical studies have tended to rely on investigations with purified enzymes in dilute aqueous solution, and assume that cell metabolism is merely a linear superposition of the kinetic characteristics of single enzymes established under these conditions; alternatively, many other metabolic investigations have been carried out using cytosolic fractions, prepared by the classical methods of subcellular fractionation, and viewed as closely approximating metabolism in the cytoplasmic compartment of the cell. Without wishing to decry the substantial advances achieved in the past by these methodologies, and their contribution to our understanding of normal and abnormal carbohydrate metabolism, it needs to be recognized that, in the light of present knowledge, these procedures do not provide a fully satisfactory simulation of cellular conditions in vivo. For example, the first of these methodologies does not allow for the high protein concentration in cells, nor the marked influence of this molecular crowding on the interactions and kinetic characteristics of individual enzymes, while the second approach also generally involves a dilution effect. As well, and central to the thrust of this review, both methods disregard any contribu tion of the cytoskeleton towards the compartmentation of carbohydrate metabo lism. With regard to the cytoplasmic compartment, for example, where a major part of intermediary metabolism is located, the concept of self-organization via transient macromolecular associations has received increasing support of late (Srere and Ovadi, 1990). There is now compelling evidence that much of the intermediary metabolism in living cells is carried out within the confines of microenvironments such as that engendered by enzyme-cytomatrix assemblages, and a growing reali zation of the critical importance of such positional factors to our understanding of the living state (Masters, 1981, 1992; Clegg, 1984). Such assemblies of enzymes The Cytoskeleton and Metabolism 3 offer the possibilities of increased efficiency of the overall processes due to proximal juxtaposition of active sites, reductions in substrate transit times, variable localization within the cell, and ready response to variations in metabolic status. The micro-compartmentation of glycolysis deserves especial consideration in this context as a central element of control in carbohydrate metabolism, and with this in mind, this article reviews the available data on the microcompartmentation of carbohydrate metabolism, and comments on localized enzyme associations, the heterogeneity of cytoskeletal structure, the covalent modification of enzymes and structure, energy requirements during signal transduction, the perturbations of micro-organization during cellular dysfunction, and the role of the cytoskeleton in modulating intermediary metabolism, in general. 11. EVIDENCE FOR THE MICRO-COMPARTMENTATION OF CARBOHYDRATE METABOLISM It has been clearly established by a variety of techniques that an extensive, differential binding of glycolytic enzymes to the cytoskeleton exists in most mammalian cells (Masters, 1984, 1992). Certain of the glycolytic enzymes bind more-readily than others and included in this category are the specific binding characteristics of phosphofructokinase, aldolase, glyceraldehydephosphate dehy drogenase, pyruvate kinase, and lactate dehydrogenase. Many of these enzymes have been shown to possess structurally distinct binding sites for substrate and for actin (Humphrey et al., 1986), and interactions with intracellular structures allow these enzyme activities to be positioned in the cell near regions involved in dynamic activities (e.g., the contractile units of muscle, or the cytoskeleton), and hence to contribute to rapid energy production at just those positions in the cell where it is most required. Adding to the potential metabolic advantage of these associations are two other features of the glycolytic enzyme-actin interactions which have emerged from previous studies. These are the modification of enzyme kinetics which occur concomitantly with binding, and the interrelationship between the degree of binding and the emphasis of cellular metabolism. When aldolase binds F-actin-tropomyosin-troponin, for example, the K^ value is increased by two orders of magnitude, whereas Vj^^^ ^^^^^ four-fold relative to free aldolase. Again, in conditions of elevated glycolysis (such as during muscle contraction), a markedly increased degree of binding of the glycolytic enzymes to cellular structure was observed (Masters, 1981, 1984; Figure 1). A number of workers have noted that micro-compartmentation of this type appears to be of major consequence in the regulation of carbohydrate metabolism, and intimately involved in aspects such as the balance of the aerobic versus the anaerobic glycolytic rate (Masters, 1981; Storey, 1985). While aerobic metabolism is of major importance in energy production, for example, many organisms retain anaerobic pathways of metabolism, which despite their low energy yields, can be of great value in periods of stress where demand outstrips the aerobic capacity (e.g., COLIN MASTERS o I (D 40 80 120 160 200 Fructose 1,6 - bisphosphate, mM Figure 1. Substrate saturation curves of free and bound aldolase A4. A. Free Enzyme; B. Enzyme bound to actin-tropomyosin-troponin. periods of intense muscular contraction). The strategy here involves a greatly increased catabolism of substrate to compensate for the relatively lov^ energy yields, and the experimental data indicates that the aerobic to anaerobic transition is accompanied by a shift in the soluble: particulate association of glycolytic enzymes which generally over-rides allosteric type controls and allov^s for unfettered, maximal expression of activities of these enzymes (Masters, 1984; Storey, 1985). There is also evidence that in the cell the sequence of glycolytic enzymes may often operate as a composite of shorter metabolic sequences, each v^ith a degree of independent function and associated v^ith the cytoskeleton, rather than as an entire and soluble sequence as described in most textbooks, or as a complex of all the glycolytic enzymes as postulated by Kurganov and coworkers (1985). It has been proposed, for example, that aldolase (ALD), glyceraldehydephosphate dehydro genase (GAPDH), triosephosphate isomerase (TPI) and phosphoglycerokinase (PGK) form one cluster of enzymes which may function on its own to produce energy—^and indeed may possibly be the most important anaerobic energy source available to most cells (Masters, 1981; Figure 2, Masters et al., 1987). Studies with purified components have indicated that both aldolase and GAPDH bind to actin with comparatively high affinity, and the binding sites for these two enzymes are closely adjacent and periodically spaced on the actin filaments. Thus, a mechanism The Cytoskeleton and Metabolism ,3PGA Figure 2. Diagrammatic representation of the association between a segment of the glycolytic sequence and an actin-containing filament, with indications of the attendant possibilities of metabolic channelling. for the anchoring of the metabolic segments to the structural elements is readily available (Masters 1981, 1984; Humphreys et al., 1986). With regard to the other two components of this segment, TPI and PGK, it has been shown that they bind to actin with far lower efficiency than aldolase and GAPDH; but interestingly, once this complex is an achored by an initial binding of ALD and GAPDH to cytoskeletal components, then TPI and GPK may add on and bind quite firmly—the aptly termed phenomenon of piggy-backing or facilitated binding. Thus the formation and stabilization of this particular cluster of four enzymes can occur, and this in turn allows the possibilities of metabolic channeling and the advantageous positioning of this cluster in appropriate locations within the cell. While the original proposal for the existence of this enzyme cluster was mainly based on binding characteristics such as those outlined above (Masters 1981), it is of interest to note that this concept has received independent kinetic confirmation recently. Han et al. (1992) have demonstrated by kinetic means that skeletal muscle contains a compartmentalized reaction sequence consisting of these four glycolytic enzymes (aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehy drogenase, and phosphoglycerate kinase) which in the structure-associated state (i.e., bound to actin containing filaments) is active in the synthesis of ATP in the triadic junction. They also provided evidence that fructose-1,6-bisphosphate was especially effectively channelled in this system, and that the function of this enzyme cluster was linked to some of the major cell signalling systems (e.g., the phospholi- pase C and protein kinase systems). Other enzyme clusters in the glycolytic sequence—^four in all—^are similarly indicated as being able to function with increased efficiency under appropriate 6 COLIN MASTERS cellular conditions, and these clusters are illustrated in Figure 3. Several studies of micro-environmental and ontogenic variation have pointed to the independent functioning and positioning of these clusters within the cell, and to the associated consequences of an increased flexibility and appropriateness of the glycolytic processes with regard to their many interrelated physiological roles (Masters, 1992). As has been mentioned in preceding sections, there are major difficulties in defining the detail of biphasic interactions between enzymes and cell structure by means of classical procedures such as subcellular fractionation. Novel experimental ATP-N ADPV GLUCOSE I I HK ENERGY CONSUMING GLUCOSE -6 - PHOSPHATE (PRIMING) I GPI SEGMENT FRUCTOSE - 6 - PHOSPHATE ATP ^ PFK ADP I r\ur^^^ I FRUCTOSE -1,6 - BISPHOSPHATE I ALDOLASE + TPI n ENERGY GLYCERALDEHYDE - 3 - PHOSPHATE PSREOGDMUECNINTG 22N NAADDH Z3 i GAPDH 3 - PHOSPHOGLYCEROYL PHOSPHATE 2 ATP -^ 22 AADDPP --^4^> ' I P^G^K^ I 3 - PHOSPHO - GLYCERATE i PGM m 2 - PHOSPHOGLYCERATE HO ENERGY i PRODUCING ENOLASE SEGMENT PHOSPHOENOUPYRUVATE 2 ADP 2 ATP^ EN, C PK PYRUVATE 2NADH-^ •-ir- IV 2 NAD ^ j LDH ANAEROBIC SEGMENT LACTATE Figure 3. Representation of the glycolytic sequence as a series of segments.