Murine diet-induced obesity remodels cardiac and liver mitochondrial phospholipid acyl chains with differential effects on respiratory enzyme activity

Cardiac phospholipids, notably cardiolipin, undergo acyl chain remodeling and/or loss of content in aging and cardiovascular diseases, which is postulated to mechanistically impair mitochondrial function. Less is known about how diet-induced obesity influences cardiac phospholipid acyl chain composition and thus mitochondrial responses. Here we first tested if a high fat diet remodeled murine cardiac mitochondrial phospholipid acyl chain composition and consequently disrupted membrane packing, supercomplex formation and respiratory enzyme activity. Mass spectrometry analyses revealed that mice consuming a high fat diet displayed 0.8–3.3 fold changes in cardiac acyl chain remodeling of cardiolipin, phosphatidylcholine, and phosphatidylethanolamine. Biophysical analysis of monolayers constructed from mitochondrial phospholipids of obese mice showed impairment in the packing properties of the membrane compared to lean mice. However, the high fat diet, relative to the lean controls, had no influence on cardiac mitochondrial supercomplex formation, respiratory enzyme activity, and even respiration. To determine if the effects were tissue specific, we subsequently conducted select studies with liver tissue. Compared to the control diet, the high fat diet remodeled liver mitochondrial phospholipid acyl chain composition by 0.6–5.3-fold with notable increases in n-6 and n-3 polyunsaturation. The remodeling in the liver was accompanied by diminished complex I to III respiratory enzyme activity by 3.5-fold. Finally, qRT-PCR analyses demonstrated an upregulation of liver mRNA levels of tafazzin, which contributes to cardiolipin remodeling. Altogether, these results demonstrate that diet-induced obesity remodels acyl chains in the mitochondrial phospholipidome and exerts tissue specific impairments of respiratory enzyme activity.     

Unremodeled and Remodeled Cardiolipin Are Functionally Indistinguishable in Yeast

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.     

The complexity of cardiolipin in health and disease

Cardiolipin, the signature phospholipid of mitochondria, is a lipid dimer that is important for a diverse range of mitochondrial activities beyond the process of ATP production. Thus not surprisingly, derangements in cardiolipin metabolism are now appreciated to contribute to an assortment of pathological conditions. A comprehensive inventory of enzymes involved in cardiolipin biosynthesis and remodeling was just recently obtained. Post-biosynthesis, the acyl chain composition of cardiolipin is modified by up to three distinct remodeling enzymes that produce either a homogeneous tissue-specific mature form of cardiolipin or alternatively 'bad' cardiolipin that has been linked to mitochondrial dysfunction. In this review, we initially focus on the newly identified players in cardiolipin metabolism and then shift our attention to how changes in cardiolipin metabolism contribute to human disease.     

心磷脂重构与心血管疾病

心磷脂（cardiolipin, CL）是线粒体内膜的特征性磷脂,参与线粒体嵴的形成。心磷脂在线粒体内的合成伴随着特殊的分子重构过程,从而使其自身的4条酰基链形成特定的组成,以发挥其特殊的生理功能。研究发现,心磷脂重构对维持线粒体的形态及功能至关重要,其重构异常是大多数心血管疾病（cardiovascular disease, CVD）共有的病理现象,相应的分子机制研究得到了广泛关注。本文主要对心磷脂的理化特性及其生物合成途径,以及心磷脂重构在巴氏综合征（Barth syndrome, BTHS）、糖尿病心肌病（diabetic cardiomyopathy, DCM）以及心力衰竭（heart failure, HF）等心血管疾病的病理生理过程研究中的进展进行综述,以期为与心磷脂重构相关的心血管疾病的病理生理基础研究和药物干预的分子机制研究提供参考。     

In yeast,cardiolipin unsaturation level plays a key role in mitochondrial function and inner membrane integrity

Cardiolipin is the signature phospholipid of the mitochondrial inner membrane. It participates in shaping the inner membrane as well as in modulating the activity of many membrane-bound proteins. The acyl chain composition of cardiolipin is finely tuned post-biosynthesis depending on the surrounding phospholipids to produce mature or unsaturated cardiolipin. However, experimental evidence showing that immature and mature cardiolipin are functionally equivalents for mitochondria poses doubts on the relevance of cardiolipin remodeling. In this work, we studied the role of cardiolipin acyl chain composition in mitochondrial bioenergetics, including a detailed bioenergetic profile of yeast mitochondria. Cardiolipin acyl chains were modified by genetic and nutritional manipulation. We found that both the bioenergetic efficiency and osmotic stability of mitochondria are dependent on the unsaturation level of cardiolipin acyl chains. It is proposed that cardiolipin remodeling and, consequently, mature cardiolipins play an important role in mitochondrial inner membrane integrity and functionality.     

Identification and characterization of a gene encoding human LPGAT1, an endoplasmic reticulum-associated lysophosphatidylglycerol acyltransferase

Phosphatidylglycerol (PG) is an important membrane polyglycerolphospholipid required for the activity of a variety of enzymes and is a precursor for synthesis of cardiolipin and bis(monoacylglycerol) phosphate. PG is subjected to remodeling subsequent to its de novo biosynthesis to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. The enzymes involved in the remodeling process have not yet been identified. We report here the identification and characterization of a human gene encoding an acyl-CoA: lysophosphatidylglycerol acyltransferase (LPGAT1). Expression of the LPGAT1 cDNA in Sf9 insect and COS-7 cells led to a significant increase in LPG acyltransferase activity. In contrast, no significant acyltransferase activities were detected against glycerol 3-phosphate or a variety of lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The recombinant human LPGAT1 enzyme recognized various acyl-CoAs and LPGs as substrates but demonstrated clear preference to long chain saturated fatty acyl-CoAs and oleoyl-CoA as acyl donors, which is consistent with the lipid composition of endogenous PGs identified from different tissues. Kinetic analyses of LPGAT1 expressed in COS-7 cells showed that oleoyl-LPG was preferred over palmitoyl-LPG as an acyl receptor, whereas oleoyl-CoA was preferred over lauroyl-CoA as an acyl donor. Consistent with its proposed microsomal origin, LPGAT1 was localized to the endoplasmic reticulum by subcellular fractionation and immunohistochemical analyses. Northern blot analysis indicated that the human LPGAT1 was widely distributed, suggesting a dynamic functional role of the enzyme in different tissues.     

The enigmatic role of tafazzin in cardiolipin metabolism

The mitochondrial phospholipid cardiolipin plays an important role in cellular metabolism as exemplified by its involvement in mitochondrial energy production and apoptosis. Following its biosynthesis, cardiolipin is actively remodeled to achieve its final acyl composition. An important cardiolipin remodeling enzyme is tafazzin, of which several mRNA splice variants exist. Mutations in the tafazzin gene cause the X-linked recessive disorder Barth syndrome. In addition to providing an overview of the current knowledge in literature about tafazzin, we present novel experimental data and use this to discuss the functional role of the different tafazzin variants in cardiolipin metabolism in relation to Barth syndrome. We developed and performed specific quantitative PCR analyses of different tafazzin mRNA splice variants in 16 human tissues and correlated this with the tissue cardiolipin profile. In BTHS fibroblasts we showed that mutations in the tafazzin gene affected both the level and distribution of tafazzin mRNA variants. Transient expression of selected human tafazzin variants in BTHS fibroblasts showed for the first time in a human cell system that tafazzin lacking exon5 indeed functions in cardiolipin remodeling.     

A novel function of the human CLS1 in phosphatidylglycerol synthesis and remodeling

Phosphatidylglycerol (PG) is a precursor for the biosynthesis of cardiolipin and a signaling molecule required for various cellular functions. PG is subjected to remodeling subsequent to its de novo biosynthesis in mitochondria to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. Yet, a gene encoding a mitochondrial LPG acyltransferase has not been identified. In this report, we identified a novel function of the human cardiolipin synthase (hCLS1) in regulating PG remodeling. In addition to the reported cardiolipin synthase activity, the recombinant hCLS1 protein expressed in COS-7 cells and Sf-9 insect cells exhibited a strong acyl-CoA-dependent LPG acyltransferase activity, which was further confirmed by purified hCLS1 protein overexpressed in Sf-9 cells. The recombinant hCLS1 displayed an acyl selectivity profile in the order of in the order of C18:1 > C18:2 > C18:0 > C16:0, which is similar to that of hCLS1 toward PGs in cardiolipin synthesis, suggesting that the PG remodeling by hCLS1 is an intrinsic property of the enzyme. In contrast, no significant acyltransferase activity was detected from the recombinant hCLS1 enzyme toward lysocardiolipin which shares a similar structure with LPG. In support of a key function of hCLS1 in PG remodeling, overexpression of hCLS1 in COS-7 cells significantly increased PG biosynthesis concurrent with elevated levels of cardiolipin without any significant effects on the biosynthesis of other phospholipids. These results demonstrate for the first time that hCLS1 catalyzes two consecutive steps in cardiolipin biosynthesis by acylating LPG to PG and then converting PG to cardiolipin.     

Formation of molecular species of mitochondrial cardiolipin. 1. A novel transacylation mechanism to shuttle fatty acids between sn-1 and sn-2 positions of multiple phospholipid species

Mitochondrial cardiolipin undergoes extensive remodeling of its acyl groups to generate uniformly substituted species, such as tetralinoleoyl-cardiolipin, but the mechanism of this remodeling has not been elucidated, except for the fact that it requires tafazzin. Here we show that purified recombinant Drosophila tafazzin exchanges acyl groups between cardiolipin and phosphatidylcholine by a combination of forward and reverse transacylations. The acyl exchange is possible in the absence of phospholipase A₂ because it requires only trace amounts of lysophospholipids. We show that purified tafazzin reacts with various phospholipid classes and with various acyl groups both in sn-1 and sn-2 position. Expression studies in Sf9 insect cells suggest that the effect of tafazzin on cardiolipin species is dependent on the cellular environment and not on enzymatic substrate specificity. Our data demonstrate that tafazzin catalyzes general acyl exchange between phospholipids, which raises the question whether pattern formation in cardiolipin is the result of the equilibrium distribution of acyl groups between multiple phospholipid species.     

A novel cardiolipin-remodeling pathway revealed by a gene encoding an endoplasmic reticulum-associated acyl-CoA:lysocardiolipin acyltransferase (ALCAT1) in mouse

Cardiolipin is a major membrane polyglycerophospholipid that is required for the reconstituted activity of a number of key mitochondrial enzymes involved in energy metabolism. Cardiolipin is subjected to remodeling subsequent to its de novo biosynthesis to attain appropriate acyl composition for its biological functions. Yet, the enzyme(s) involved in the remodeling process have not been identified. We report here the identification and characterization of a murine gene that encodes an acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1). Expression of the ALCAT1 cDNA in either insect or mammalian cells led to a significant increase in acyl-CoA:monolysocardiolipin acyltransferase and acyl-CoA: dilysocardiolipin acyltransferase activities that exhibited a dependence upon ALCAT1 enzyme levels. The recombinant ALCAT1 enzyme recognizes both monolysocardiolipin and dilysocardiolipin as substrates with a preference for linoleoyl-CoA and oleoyl-CoA as acyl donors. In contrast, no significant increases in acyltransferase activities by the recombinant ALCAT1 were detected against either glycerol-3-phosphate or a variety of other lysophospholipids as substrates, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine. Immunocytohistochemical analysis showed that the ALCAT1 enzyme is localized in the endoplasmic reticulum, which is supported by a significant ALCAT activity in isolated liver and heart microsomes. Northern blot analysis indicates that the mouse ALCAT1 is widely distributed, with the highest expression in heart and liver. In support of a role for ALCAT1 in maintaining heart function, the ALCAT1 gene is conserved among different species of vertebrates, but not in non-atrium organisms. ALCAT1 represents the first identified cardiolipin-remodeling enzyme from any living organism; its identification implies a novel role for the endoplasmic reticulum in cardiolipin metabolism.     

Formation of molecular species of mitochondrial cardiolipin: 2. A mathematical model of pattern formation by phospholipid transacylation

Formation of the unique molecular species of mitochondrial cardiolipin requires tafazzin, a transacylase that exchanges acyl groups between phospholipid molecular species without strict specificity for acyl groups, head groups, or carbon positions. However, it is not known whether phospholipid transacylations can cause the accumulation of specific fatty acids in cardiolipin. Here, a model is shown in linear algebra representation, in which acyl specificity emerges from the transacylation equilibrium of multiple molecular species, provided that different species have different free energies. The model defines the conditions and energy terms, under which transacylations may generate the characteristic composition of mitochondrial cardiolipin. It is concluded that acyl-specific cardiolipin patterns could arise from phospholipid transacylations in the tafazzin domain, even if tafazzin itself does not have substrate specificity.     

Distinct effects of tafazzin deletion in differentiated and undifferentiated mitochondria

Tafazzin is a conserved mitochondrial protein that is required to maintain normal content and composition of cardiolipin. We used electron tomography to investigate the effect of tafazzin deletion on mitochondrial structure and found that cellular differentiation plays a crucial role in the manifestation of abnormalities. This conclusion was reached by comparing differentiated cardiomyocytes with embryonic stem cells from mouse and by comparing different tissues from Drosophila melanogaster. The data suggest that tafazzin deficiency affects cardiolipin in all mitochondria, but significant alterations of the ultrastructure, such as remodeling and aggregation of inner membranes, will only occur after specific differentiation.     

Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes

In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease.     

Cardiolipin Remodeling in a Chinese Hamster Lung Fibroblast Cell Line Deficient in Oxidative Energy Production

The metabolism of cardiolipin was investigated in a Chinese hamster lung fibroblast cell line CCL16-B2 deficient in oxidative energy metabolism and its parental cell line CCL16-B1. Mitochondrial enzyme activities involved in de novo cardiolipin biosynthesis were elevated in CCL16-B2 cells compared with CCL16-B1 cells, indicating initially an elevation in cardiolipin biosynthesis. Content of all phospholipids, including cardiolipin and its precursors, and high energy nucleotides were unaltered in CCL 16-B2 cells compared to CCL 16-B1 cells. When cells were incubated with [1,3-³H]glycerol for up to 4 h radioactivity incorporated into cardiolipin in CCL16-B2 cells did not differ compared with CCL16-B1 cells. In contrast, radioactivity incorporated into phosphatidylglycerol, the immediate precursor of cardiolipin, was elevated over 2-fold in CCL16-B2 cells compared with CCL16-B1 cells. Analysis of the fatty acid molecular species in cardiolipin revealed alterations in the level of unsaturated but not saturated fatty acids in B2 compared with B1 cells. In vivo cardiolipin remodeling, that is, the deacylation of cardiolipin to monolysocardiolipin followed by reacylation back to cardiolipin, with [1-¹⁴C]palmitate and [l-¹⁴C]oleate and in vitro mitochondrial phospholipid remodeling with [1-¹⁴C]linoleate were altered in CCL16-B2 cells compared to CCL16-B1 cells. Since both the appropriate content and molecular composition of cardiolipin is required for optimum mitochondrial oxidative phosphorylation, we suggest that the difference in CL molecular species composition observed in CCL16-B2 cells, mediated by alterations in in vivo cardiolipin remodeling, may be one of the underlying mechanisms for the reduction in oxidative energy production in CCL16-B2 cells.     

Separation and characterization of cardiolipin molecular species by reverse-phase ion pair high-performance liquid chromatography-mass spectrometry

An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented. Reverse-phase ion pair chromatography with acidified triethylamine resulted in increased chromatographic retention and resolution when compared with chromatography without acidified triethylamine. Using a hybrid triple quadrupole linear ion trap mass spectrometer to generate MS/MS spectra revealed three regions within each spectrum that could be used to deduce the structure of the cardiolipin molecular species: the diacylglycerol phosphate region, the monoacylglycerol phosphate region, and the fatty acid region. Cardiolipin standards of known composition were analyzed and exhibited expected chromatographic and mass spectral results. Two minor components in commercial bovine heart cardiolipin, (with the same molecular weight but different chromatographic retention times), were shown to differ by fatty acid composition: (C18:2)₂(C18:1)₂ versus (C18:2)₃(C18:0)₁. These compounds were then analyzed by HPLC-MS³ to examine specific diac ylglycerol phosphate generated fatty acid fragmentation. Also, two commercial sources of bovine heart cardiolipin were shown to have minor differences in cardiolipin species content. Cardiolipin isolated from rat liver, mouse heart, and dog heart mitochondria were then characterized and the relative distributions of the major cardiolipin species were determined.     

Molecular symmetry in mitochondrial cardiolipins

Cardiolipin is a unique mitochondrial phospholipid with an atypical fatty acid profile, but the significance of its acyl specificity has not been understood. We explored the enormous combinatorial diversity among cardiolipin species, which results from the presence of four fatty acids in each molecule, by integrated use of high-performance liquid chromatography, mass spectrometry, diacylglycerol species analysis, fatty acid analysis, and selective cleavage of fatty acids by phospholipase A2. The most abundant cardiolipin species from various organisms and tissues (human heart, human lymphoblasts, rat liver, Drosophila, sea urchin sperm, yeast, mung bean hypocotyls) contained only one or two types of fatty acids, which generated a high degree of structural uniformity and molecular symmetry. However, an exception was found in patients with Barth syndrome, in whom an acyltransferase deficiency led to loss of acyl selectivity and formation of multiple molecular species. These results suggest that restriction of the number of fatty acid species, rather than the selection of a particular kind of fatty acid, is the common theme of eukaryotic cardiolipins. This limits the structural diversity of the cardiolipin species and creates molecular symmetry with implications for the stereochemistry of cardiolipin.     

Modulation of erythrocyte acetylcholinesterase by cardiolipin: Effect on subunit coupling revealed by irradiation inactivation

The functional molecular weights of two kinetically distinct forms of bovine erythrocyte acetylcholinesterase were determined by irradiation inactivation. Whereas both forms have similar molecular weights by hydrodynamic measurements and contain 33 molecules of cardiolipin, the functional molecular weight of form α (140,000) was found to be twice that of form β (73,000). As form β is derived from form α by treatment with high salt concentration in alkaline Ca²⁺-chelating conditions, a procedure which is considered to disrupt the functional association of a Ca²⁺-cardiolipin complex with the enzyme, it is suggested that cardiolipin mediates the energy transfer between enzyme subunits, thereby modulating the kinetic properties of the lipoprotein.     

Myocardial regulation of lipidomic flux by cardiolipin synthase: setting the beat for bioenergetic efficiency

Lipidomic regulation of mitochondrial cardiolipin content and molecular species composition is a prominent regulator of bioenergetic efficiency. However, the mechanisms controlling cardiolipin metabolism during health or disease progression have remained elusive. Herein, we demonstrate that cardiac myocyte-specific transgenic expression of cardiolipin synthase results in accelerated cardiolipin lipidomic flux that impacts multiple aspects of mitochondrial bioenergetics and signaling. During the postnatal period, cardiolipin synthase transgene expression results in marked changes in the temporal maturation of cardiolipin molecular species during development. In adult myocardium, cardiolipin synthase transgene expression leads to a marked increase in symmetric tetra-18:2 molecular species without a change in total cardiolipin content. Mechanistic analysis demonstrated that these alterations result from increased cardiolipin remodeling by sequential phospholipase and transacylase/acyltransferase activities in conjunction with a decrease in phosphatidylglycerol content. Moreover, cardiolipin synthase transgene expression results in alterations in signaling metabolites, including a marked increase in the cardioprotective eicosanoid 14,15-epoxyeicosatrienoic acid. Examination of mitochondrial bioenergetic function by high resolution respirometry demonstrated that cardiolipin synthase transgene expression resulted in improved mitochondrial bioenergetic efficiency as evidenced by enhanced electron transport chain coupling using multiple substrates as well as by salutary changes in Complex III and IV activities. Furthermore, transgenic expression of cardiolipin synthase attenuated maladaptive cardiolipin remodeling and bioenergetic inefficiency in myocardium rendered diabetic by streptozotocin treatment. Collectively, these results demonstrate the unanticipated role of cardiolipin synthase in maintaining physiologic membrane structure and function even under metabolic stress, thereby identifying cardiolipin synthase as a novel therapeutic target to attenuate mitochondrial dysfunction in diabetic myocardium.     

Quantitation of cardiolipin molecular species in spontaneously hypertensive heart failure rats using electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry has previously been used to probe qualitative changes in the phospholipid cardiolipin (CL), but it has rarely been used in a quantitative manner. We assessed changes in the amount of individual molecular species of cardiac CL present in a model of congestive heart failure using 1,1',2,2'-tetramyristoyl cardiolipin as an internal standard. There was a linear relationship between the ratio of the negative molecular ion ([M-H]-) current from four different CL reference standards and the [M-H]- from the internal standard, as a function of the concentration of CL molecular species. Therefore, this internal standard can be used to quantitate many naturally occurring CL molecular species over a wide range of CL concentrations. Using this method, changes to individual molecular species of CL in failing hearts from male spontaneously hypertensive heart failure rats were examined. CL isolated from cardiac mitochondria was compared with left ventricular tissue to demonstrate the feasibility of extracting and quantitating CL from either mitochondrial or tissue samples. The acyl chain composition of individual CL molecular species was identified using tandem mass spectrometry. In animals with heart failure, the major cardiac CL species (tetralinoloyl) decreased significantly, whereas other minor CL species were significantly increased.     

Protein denaturation during heat shock and related stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase inactivation in mouse cells

In an attempt to question the toxic effect of heat shock and related stress, we have studied the activity of reporter enzymes during stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase were synthesized in mouse and Drosophila cells after transfection of the corresponding genes. Both enzymes are rapidly inactivated during hyperthermia. The corresponding polypeptides are not degraded but become insoluble even in the presence of non-ionic detergents. The heat inactivation is more dramatic in vivo within the living cell than in vitro, in a detergent-free crude cell lysate. The extent of enzyme inactivation at a given temperature depends on the cell type in which the enzyme is expressed. Luciferase is inactivated at lower temperatures within Drosophila cells than within mouse cells, whereas beta-galactosidase is inactivated at higher temperatures in E. coli than in mouse cells. A "priming" heat shock confers a transient increased resistance (thermotolerance) of cells against a second "challenging" heat shock. Enzyme inactivation during heat shock or exposure of the cells to ethanol is attenuated in heat shock-primed cells. A comparable thermoprotection is raised by a priming heat shock for both luciferase activity and protein synthesis. Thus, the study of reporter enzyme inactivation is a promising tool for understanding the molecular basis of the toxicity of heat shock and related stress as well as the mechanisms leading to thermotolerance.