Metabolite Channeling: Creatine Kinase Microcompartments

Uwe Schlattner and Theo Wallimann Institute of Cell Biology, Swiss Federal Institute of Technology (ETH), Zu¨rich, Switzerland

Subcellular microcompartments, consisting of multienzyme organization of pathway components is a general complexes embedded within the cellular, highly viscous prerequisite for metabolite channeling. It may involve matrix, associated with the cytoskeleton, or situated along (1) huge covalently linked enzyme-complexes (or multi- membranes, are operating according to exclusion principles functional enzymes) such as fatty acid synthase (FAS), and favor preferred pathways of intermediates. This process, (2) kinetically stable multienzyme complexes like called metabolite or substrate channeling, is defined as transfer pyruvate dehydrogenase (PDH) or bacterial and plant of intermediates between sequential enzymes without equili- tryptophan synthase (TS), (iii) more dynamic, reversibly bration of these metabolites with the surrounding bulk associating enzymes such as glycolytic complexes con- solution. Such an association between two or more sequential taining glyceraldehyde phosphate dehydrogenase enzyme- or transport reactions in a microcompartment, (GAPDH) or glycerol phosphate dehydrogenase forming a distinct functional pool of intermediates, is also (GPDH), or (4) colocalization on subcellular particles. called functional coupling. It can be considered as a general These associations allow the transfer of intermediates mechanism to increase efficiency of sequential reactions in a between the channeling components by different mech- metabolic pathway. Since metabolite channeling leads to anisms: (1) sequential covalent binding to very close segregation of a metabolic pathway from other cellular active sites in the reaction sequence (e.g., PDH), (2) reactions, it represents a specific kind of metabolic compart- physical hindrance or electrostatic effects prevent mix- mentation similar to that operating within membrane-separ- ing with bulk solution and drive a directed diffusion ated organelles or by restricted two-dimensional diffusion at (e.g., TS), (3) transfer of noncovalently bound inter- surface boundary layers. Here, metabolite channeling is mediates between active sites (e.g., NADH dehydrogen- described with special emphasis on high-energy phosphate ase), (4) transfer in an unstirred surface layer. These channeling by creatine kinase (CK), the phosphocreatine mechanisms can be fulfilled in both, static and dynamic circuit or shuttle, and the mitochondrial CK isoenzyme. enzyme associations. However, while static associations allow for an almost perfect or “tight” channeling of metabolites, dynamic channeling is often only partial Subcellular Microcompartments or “leaky.” and Mechanisms of Metabolite Channeling Advantages of Metabolite Life most likely originated autotrophically de novo in Channeling metabolic complexes organized on FeS2 (pyrite) mineral surfaces, the earliest form of microcompart- Sequestering of intermediates in a microcompartment ments. Likewise, a cell is by no means represented best through channeling provides kinetic and regulatory by a well-mixed bag of enzymes, behaving in complete advantages for not only the reaction sequence itself equilibrium according to solution kinetics. Because of (see Figure 1), but also for cellular metabolism. In the intricate structural and functional organization of general: (1) enzyme reaction rates and equilibria are living cells, enzymes and metabolites do not behave as if controlled by local and enzyme bound substrates, rather they were freely diffusible in solution. Instead, they may than bulk phase substrate concentrations, (2) for a form structurally, functionally, and temporally defined readily reversible reaction, local supply of substrate and subcellular microcompartments, either via strong static, removal of product may drive the reaction in the desired or via fickle, dynamic interactions with other enzymes, direction, (3) sequestered intermediates are present at proteins, or subcellular structures. Such a structural high local concentrations and an apparently low Km for

FIGURE 1 Free diffusion versus metabolite channeling. Compartmentation of a reaction sequence without equilibration with bulk solution leads to shorter transition times and further advantages (see text). these intermediates can be observed with the channeling Compartmentation of Creatine complex compared to the nonchanneling situation measured with isolated components, (4) metabolites Kinase Isoenzymes and Channeling are isolated from competing reactions, e.g., between of High-Energy Phosphates anabolic and catabolic pathways, (5) the life-time of the intermediate in the solvent phase is shortened relative to THE CREATINE free diffusion, which may be essential in case of unstable KINASE/PHOSPHOCREATINE intermediates, (6) in certain cases, the unfavorable IRCUIT OR HUTTLE energetics of desolvating the substrate that precedes C S binding to the enzyme is avoided, (7) channeling One fundamental requirement of life is energy supply. components can be regulated by modulators that affect Cellular energy demand and supply are balanced, and enzyme associations, and (8) a larger degree of tightly regulated for economy and efficiency of energy metabolic control of the over-all-flux of the reactions use. CK is a major enzyme of higher eukaryotes that copes can be achieved, e.g., via feed-back regulatory mechan- with high and fluctuating energy demands to maintain isms such as substrate activation, product inhibition, cellular energy homeostasis in general and to guarantee and cooperativity. stable, locally buffered ATP/ADP ratios in particular. The enzyme catalyzes the reversible phosphoryl transfer from ATP to creatine (Cr) to generate ADP and phosphocreatine (PCr). Thus, CK is able to conserve Subcellular Targeting of Glycolytic energy in the form of metabolically inert PCr and vice versa, to use PCr to replenish global as well as local Multienzyme Complexes cellular ATP pools. Since PCr can accumulate to much higher cellular concentrations than ATP, the CK/PCr- In muscle, glycolytic enzymes are targeted to the actin- system constitutes an efficient and immediately available containing thin filaments at the sarcomeric I-band region cellular “energy buffer.” In addition, tissue-specific CK where they form highly complex metabolons. The I- isoenzymes are located in the cytosol (dimeric muscle- band in Drosophila flight muscle contains a multi- type MM-CK and brain-type BB-CK) and the mito- enzyme complex consisting of GDPH-1, aldolase, and chondrial intermembrane space (sarcomeric sMtCK and GAPDH. By elegant experiments with transgenic ubiquitous uMtCK, both forming octamers and dimers). Drosophila expressing GDPH-3 instead of GDPH-1, it CK isoenzymes are often associated with sites of ATP could be shown that all three glycolytic enzymes no supply, where they generate PCr, or with sites of ATP longer colocalize in the I-band to form a microcompart- consumption, where they regenerate ATP by using PCr. ment. Even though the full complement of active Thus, together with the faster diffusion rate of PCr as glycolytic enzymes was still present, their failure to compared to ATP, the CK/PCr system also supports an colocalize in the sarcomer resulted in the inability to fly. intrinsic energy transfer system (CK/PCr-circuit or Thus, correct targeting and formation of multienzyme -shuttle), coupling sites of energy generation (oxidative complexes that lead to functionally coupled microcom- phosphorylation or glycolysis) with sites of energy partments and substrate/product channeling seem to be consumption (Figure 2). This circuit is particularly a prerequisite for proper function of glycolysis and important in large and/or polar cells, such as spermato- ultimately for correct muscle function. In mammalian zoa where diffusional limitations of adenine nucleotides, cells, CK is also participating in the glycolytic especially ADP, along the sperm tail become especially metabolon. apparent. 648 METABOLITE CHANNELING: CREATINE KINASE MICROCOMPARTMENTS

FIGURE 2 The creatine kinase (CK)/phosphocreatine (PCr) system. Isoenzymes of CK are found in different compartments like mitochondria (a) and cytosol (b–d) in soluble form (c) or associated to a different degree to ATP-delivering (a,b) or -consuming processes (d). A large cytosolic PCr pool up to 30 mM is built up by CK using ATP from oxidative phosphorylation like in heart (a) or glycolysis like in fast-twitch glycolytic muscle (b). PCr is then used to buffer global (c) and local (d) ATP/ADP ratios. In cells that are polarized and/or have very high or localized ATP consumption, these CK isoenzymes, together with easily diffusible PCr, also maintain an energy shuttle between ATP-providing or -consuming processes (a,d). Metabolite channeling occurs where CK is associated with ATP-providing or -consuming transporters, pumps or enzymes (a,b,d). Creatine (Cr) is synthesized in only few cell types (e.g., liver and kidney) and has to be taken up from the blood stream by a speciﬁc Cr transporter (CRT).

þ CHANNELING WITH CYTOSOLIC CK inhibition of the ATPase by ADP and H is avoided, since the latter are both substrates of the CK reaction Cytosolic CK is only partially soluble. A significant (PCr þ ADP þ Hþ $ Cr þ ATP) and (2) that the high fraction is structurally and functionally associated or free energy of ATP hydrolysis (DG ) at sites of ATP co-localized with different, structurally bound ATPases ATP hydrolysis is preserved by keeping locally very high or ATP-regulated processes. These include (1) different ATP/ADP ratios due to coupling of CK with those ion pumps in the plasma membrane, (2) the sarcomeric ATPases in situ and thus preventing energy dissipation M-band of the myofibrils in muscle, (3) the calcium caused by transport of ATP and mixing it with the bulk pump of the muscular sarcoplasmic reticulum, and þ surrounding. Interestingly, the strongest phenotype of (4) the ATP-gated K -channel. In all these cases, PCr is CK double knockout mice, which no longer express used for the local regeneration of ATP, which is directly cytosolic MM-CK and mitochondrial MtCK in muscle, channeled from CK to the consuming ATPase without is characterized by significant difficulties with intracellu- major dilution by the surrounding bulk solution. Only in lar calcium handling and muscle relaxation, emphasiz- some cases, the physical basis for the metabolite ing the physiological importance of the CK system for channeling is known. For example, MM-CK uses a the energetics of intracellular calcium homeostasis in “charge clamp” consisting of four lysine residues to general and the delivery of ATP to the energetically specifically bind to partner molecules, myomesin and demanding Ca2þ-ATPase, in particular. M-protein in the M-band, Cytosolic CK is structurally associated with the key regulatory enzyme of glycolysis, phosphofructokinase CHANNELING IN ENERGY (PFK), which itself is regulated by ATP, Likewise, TRANSDUCING MITOCHONDRIAL structural and functional interaction of cytosolic CK with the PAR-1 receptor of the thrombin-signaling MICROCOMPARTMENTS pathway and with the ATP-gated Kþ-channel, respect- MtCK forms mainly large, cuboidal octamers (Figure 3) ively, has been demonstrated. A tight functional that are present (1) between the outer and inner mito- coupling of CK to ATPases, e.g., acto-myosin ATPase chondrial membrane (the so-called “intermembrane and ion pumps, such as the Kþ/Naþ-ATPase or the space” of mitochondria) and preferentially localized at Ca2þ-ATPase, has the advantage (1) that product the so-called “mitochondrial contact sites” between METABOLITE CHANNELING: CREATINE KINASE MICROCOMPARTMENTS 649 outer and inner mitochondrial membrane, as well as ANT is an obligatory antiporter for ATP/ADP (2) in the “cristae space” (see Figure 3 inset). exchange across the inner mitochondrial membrane, The kinase catalyzes the direct transphosphorylation while VDAC is a nonspecific, potential-dependent pore of intramitochondrial produced ATP and Cr from the in the outer mitochondrial membrane. The MtCK- cytosol into ADP and PCr. ADP enters the matrix linked metabolite channeling is based on (1) colocali- space to stimulate oxidative phosphorylation, giving zation, (2) direct interactions, and (3) diffusion rise to mitochondrial recycling of a specific pool of barriers. MtCK tightly binds to cardiolipin, an acidic ATP and ADP, while PCr is the primary “high energy” phospholipid that is specific for the mitochondrial phosphoryl compound that leaves mitochondria into inner membrane. Since ANT is situated in a cardio- the cytosol. The molecular basis for such directed lipin patch, this leads to colocalization and meta- metabolite flux is channeling between the large, bolite channeling between both proteins in cristae cuboidal MtCK octamer and two transmembrane and intermembrane space (Figure 3). MtCK in the proteins, adenine translocator (ANT) and mitochon- intermembrane space further interacts with outer drial porin or voltage-gated anion channel (VDAC). membrane phospholipids and VDAC, thus virtually

FIGURE 3 Inset: Dual localization of mitochondrial CK by electron microscopy. Post-embedding immuno-gold labeling of MtCK in a mitochondrion from photoreceptor cells of chicken retina, showing localization of MtCK in the peripheral intermembrane space (arrows) and along the cristae membranes. Scheme: Microcompartments and metabolite channeling of mitochondrial CK. After the import of nascent MtCK over the mitochondrial outer membrane and cleavage of the targeting sequence, MtCK first assembles into dimers. Dimers rapidly associate into octamers (top left), and although this reaction is reversible, octamer formation is strongly favored by high MtCK concentrations and MtCK binding to acidic phospholipids. MtCK is then found in two locations; (1) in the so-called mitochondrial contact sites associated with ANT and VDAC (top center), and (2) in the cristae associated with ANT only (at right). In contact sites, MtCK simultaneously binds to inner and outer membrane due to the identical top and bottom faces of the octamer. Binding partner in the inner membrane is the twofold negatively charged cardiolipin, which allows a functional interaction with adenine nucleotide translocator that is situated in cardiolipin membrane patches. In the outer membrane, MtCK interacts with other acidic phospholipids and, in a calcium-dependent manner, directly with VDAC. Substrate and product fluxes between MtCK and the associated proteins are depicted as arrows. In contact sites, this substrate channeling allows for a constant supply of substrates and removal of products at the active sites of MtCK. In cristae, only ATP/ADP exchange is facilitated through direct channeling to the MtCK active site, while Cr and PCr have to diffuse along the cristae space to reach VDAC. 650 METABOLITE CHANNELING: CREATINE KINASE MICROCOMPARTMENTS cross-linking inner and outer membrane and contribu- ting to the “mitochondrial contact sites.” Increasing the external calcium concentrations strengthens the interaction of MtCK with VDAC, which may improve channeling under cytosolic calcium overload as occur- ring at low cellular energy state. Some studies suggest that only the membrane-bound, octameric form of MtCK is able to maintain the metabolite channeling described above. Finally, the limited permeability of VDAC and thus of the entire outer membrane creates a dynamic microcompartmentation of metabolites in the intermembrane space that contributes to MtCK-linked channeling and separate mitochondrial ATP- and ADP pools. Similar to MtCK, hexokinase is able to use intramitochondrially produced ATP by binding to VDAC from the cytosolic mitochondrial surface at contact sites containing only ANT and VDAC. The direct functional coupling of MtCK to oxidative phosphorylation can be demonstrated with oxygraph respirometry on skinned muscle fibers from normal and transgenic mice lacking MtCK. FIGURE 4 Intramitochondrial inclusions in patients with mitochon- Oxidative damage of MtCK, induced by reactive drial myopathies consist of mitochondrial MtCK. Immuno-electron oxygen and nitrogen species, generated under cellular histochemistry of human intra-mitochondrial crystalline “railway- stress situations, e.g., in infarcted heart or under track” inclusions seen in patients with mitochondrial myopathies showing “ragged-red” skeletal fibers as a hallmark of the disease. Note chemotherapeutic intervention by anthracyclines lead the grossly enlarged mitochondrion, here from a patient with Kearns- to inactivation and dimerization of MtCK, as well as to Sayre syndrome, displaying the typical regularly spaced intra-cristae dissociation of the enzyme from the mitochondrial inner inclusions (dark) surrounded by mitochondrial inner cristae mem- membrane. Thus, important prerequisites for efficient branes. The section has been stained by rabbit anti-MtCK antibodies, channeling of high-energy phosphates by MtCK are followed by colloidal gold-conjugated second antibody (in collabor- ation with Dr. A. M. Stadhouders, Nijmegen, The Netherlands). Note negatively affected. These events contribute to cardiac that the dark inclusion bodies are heavily and specifically stained by the energy failure and specific anthracycline cardiotoxicity. small 10 nm gold particles, indicating a high propensity of MtCK at these locations. Isolation of such inclusions, plus image processing of sections through them revealed that they consist mainly of crystalline MtCK octamers, which crystallize in sheet-like structures. MITOCHONDRIAL CK, INTRAMITOCHONDRIAL INCLUSIONS, AND LOW CELLULAR ENERGY STATE overexpression is such that it leads to crystallization of the enzyme in a pathological state as described above. MtCK is an indicator for cellular low-energy stress, that is, the expression of this enzyme is highly up-regulated in patients with mitochondrial myopathies in the so called ITOCHONDRIAL AND THE “ragged-red” skeletal muscle fibers, where mitochon- M CK drial volume and size are markedly increased and where MITOCHONDRIAL PERMEABILITY characteristic intra-mitochondrial “railway-track TRANSITION PORE inclusions” are observed as a hallmark of pathology. MtCK, together with its substrate Cr, has been recently The latter were shown to consist of crystalline sheets of implicated in regulation of the mitochondrial per- MtCK (see Figure 4). Similar MtCK inclusions can also meability transition pore that is crucially involved in be induced in animal by chronic Cr depletion leading to triggering apoptosis, This seems to be due to metabolite cellular low-energy state, Even more generally, cellular channeling in the MtCK/ANT microcompartment also, low-energy stress, be it chronic endurance training, which maintains high ADP concentrations in the matrix fasting, Cr depletion, or pathologies in ATP generation space that are inhibitory for permeability transition. like mitochondrial dysfunction seen in patients with Thus, the CK-system and its substrates seem to exert mitochondrial cytopathies, induces a coordinated additional effects that are not necessarily directly induction of “energy gene expression” to compensate coupled to improving cellular energetics. This may for impaired energy supply and transport. In case of explain the remarkable cell- and neuro-protective effects MtCK, one of the most prominent among these genes, of Cr that have been reported. METABOLITE CHANNELING: CREATINE KINASE MICROCOMPARTMENTS 651

SEE ALSO THE FOLLOWING ARTICLES Jacobus, W. E., and Lehninger, A. L. (1973). Creatine kinase of rat heart mitochondria. Coupling of creatine phosphorylation to Free Radicals, Sources and Targets of: Mitochondria † electron transport. J. Biol. Chem. 248, 4803–4810. Mitochondrial Channels † Mitochondrial Membranes, Kay, L., Nicolay, K., Wieringa, B., Saks, V., and Wallimann, T. (2000). Structural Organization † Mitochondrial Metabolite Direct evidence for the control of mitochondrial respiration by Transporter Family † Mitochondrial Outer Mem- mitochondrial creatine kinase in oxidative muscle cells in situ. þ J. Biol. Chem. 275, 6937–6944. brane and the VDAC Channel † P-Type Pumps: Na / Ovadi, J. (1995). Cell Architecture and Metabolic Channeling. þ ´ K Pump Springer, Heidelberg. Ova´di, J., and Srere, P. A. (2000). Macromolecular compartmentation and channeling. Int. Rev. Cytol. 192, 255–280. GLOSSARY Schlattner, U., Forstner, M., Eder, M., Stachowiak, O., Fritz-Wolf, K., metabolite channeling Local transfer of metabolic intermediates and Wallimann, T. (1998). Functional aspects of the X-ray between sequential enzyme or transport reactions without equili- structure of mitochondrial creatine kinase: a molecular physiology bration with bulk solution. approach. Mol. Cell. Biochem. 184, 125–140. metabolic compartmentation Segregation of intermediates and Srere, P. A., and Knull, H. R. (1998). Location-location-location. enzymes of a metabolic pathway by membranes, binding to a Trends Biochem. Sci. 23, 319–320. speciﬁc surface or direct interaction in protein complexes allowing Srivastava, D. K., and Bernhard, S. A. (1986). Metabolite transfer via metabolite channeling. enzyme–enzyme complexes. Science 234, 1081–1086. microcompartment Structural unit allowing metabolic compartmen- Wallimann, T., Wyss, M., Brdiczka, D., Nicolay, K., and Eppenberger, tation, also called “metabolon.” H. M. (1992). Intracellular compartmentation, structure and mitochondrial contact sites Close adhesions of inner and outer function of creatine kinase isoenzymes in tissues with high and mitochondrial membrane that can be observed by electron ﬂuctuating energy demands: the ‘phosphocreatine circuit’ for microscopy and can be isolated as a separate microcompart- cellular energy homeostasis. Biochem. J. 281, 21–40. ment. Contact sites consist of multi-lipid/protein complexes with variable composition and are involved in energy transduc- tion (e.g., containing ANT/VDAC or ANT/MtCK/VDAC) or BIOGRAPHY protein import. Uwe Schlattner is Docent at the Institute of Cell Biology, Swiss Federal mitochondrial permeability transition pore A multienzyme complex, Institute of Technology (ETH), Zu¨ rich, Switzerland. He received his probably composed of VDAC, ANT, Bax, cyclophilin, MtCK and masters in biology at the University of Freiburg, Germany, and his others, that is crucially involved in early events that trigger Ph.D. in Natural Science at the University of Geneva, Switzerland. His apoptosis like release of cytochrome c and other apoptosis-inducing research interest is in cellular energetics, in particular the molecular factors into the cytosol. structure and physiology of kinases involved in regulating the cellular energy state. URTHER EADING F R Theo Walliman is a Professor at the Institute of Cell Biology at Agius, L., and Sherratt, H. S. A. (eds.) (1997). Channelling in Swiss Federal Institute of Technology (ETH) in Zu¨ rich, Switzerland, Intermediary Metabolism. Research Monograph IX, Portland where he also received his Ph.D. After a postdoctoral stay in Prof. Press, London, GB. Andrew G. Szent-Gyo¨ rgyi’s laboratory at Brandeis University, Dolder, M., Walzel, B., Speer, O., Schlattner, U., and Wallimann, T. Boston, from 1975 to 1981, he returned to ETH-Zu¨ rich. His (2003). Inhibition of the mitochondrial permeability transition by research interests are in cellular energetics, especially creatine creatine kinase substrates. Requirement for microcompartmenta- kinases and AMP-activated protein kinases, as well as creatine tion. J. Biol. Chem. 278, 17760–17766. supplementation in health and diseases.