Table Of ContentLrST 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.
