Naoshi Yamazaki

High expression of a novel carnitine palmitoyltransferase I like protein in rat brown adipose tissue and heart: isolation and characterization of its cDNA clone

To characterize energy metabolism in rat brown adipose tissue (BAT), we carried out differential screening of a cDNA library of BAT with a cDNA probe of white adipose tissue (WAT) and isolated one cDNA clone. It contained a single open reading frame of 2,316 bases which encodes a protein of 88.2 kDa. The predicted amino acid sequence showed the highest homology (62.6%) with that of rat carnitine palmitoyltransferase I (CPTI). The transcript corresponding to this cDNA was found to be abundantly expressed in BAT and heart. Therefore, the isolated clone is concluded to encode a CPTI like protein expressed in BAT and heart.

Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I

With a cDNA probe encoding rat muscle type carnitine palmitoyltransferase I (CPTI), we isolated cDNA and genomic clones encoding the human homologue and deduced the primary structure of human muscle type CPTI. By Northern analysis, we confirmed the dominant expression of this isoform in heart and skeletal muscle.

Structural features of the gene encoding human muscle type carnitine palmitoyltransferase I

We isolated a human muscle type of carnitine palmitoyltransferase I (CPTI‐M) genomic clone and determined its entire nucleotide sequence. By comparison of the nucleotide sequence of the genomic clone with that of cDNA, we determined the intron/exon junctions. For detection of the exon(s) in the 5′‐region of the CPTI‐M gene, we isolated cDNA clones corresponding to the 5′‐region of its transcript by 5′‐rapid amplification of cDNA ends (5′‐RACE method). Results showed two alternative exons, 1A and 1B, that do not encode amino acids in the 5′‐region of the human CPTI‐M gene. The gene encoding human CPTI‐M was found to consist of two 5′‐non‐coding exons, 18 coding exons and one 3′‐non‐coding exon spanning approximately 10 kbp. Furthermore, on analysis of the 5′‐flanking region, a putative gene encoding a ‘choline kinase homologue’ was found to be located only about 300 bp upstream from exon 1A of the human CPTI‐M gene. Comparison of the gene structure of human CPTI‐M with the reported partial gene structure of human liver type CPTI (CPTI‐L) showed that the intron insertion sites were completely conserved in these two genes.

Expression of the bovine heart mitochondrial ADP/ATP carrier in yeast mitochondria: significantly enhanced expression by replacement of the N-terminal region of the bovine carrier by the corresponding regions of the yeast carriers

To characterize the transport mechanism mediated by the mammalian mitochondrial ADP/ATP carrier (AAC), we tried to express bovine heart mitochondrial AAC (bhAAC) in Saccharomyces cerevisiae. The open reading frame of the bhAAC was introduced into the haploid strain WB-12, in which intrinsic AAC genes were disrupted. Growth of the transformant was very low in glycerol medium, and a little amount of bhAAC was detected in the mitochondrial membrane. For improvement of bhAAC expression in WB-12, we introduced DNA fragments encoding chimeric bhAACs, in which the N-terminal region of the bhAAC extending into the cytosol was replaced by the corresponding regions of the type 1 and type 2 yeast AAC isoforms (yAAC1 and yAAC2). These transformants grew well, and the amounts of the chimeric bhAACs in their mitochondria were as high as that of yAAC2. The carriers expressed showed essentially the same ADP transport activities as that of AAC in bovine heart mitochondria.

Dramatic enhancement of the specific expression of the heart-type fatty acid binding protein in rat brown adipose tissue by cold exposure.

To understand the difference in energy metabolisms in brown (BAT) and white (WAT) adipose tissues, we examined the steady‐state transcript levels of the heart‐type and adipose‐type fatty acid binding proteins (H‐FABP and A‐FABP, respectively) by Northern blot analysis. The transcript of H‐FABP in rat BAT was increased about 100‐fold by cold exposure, whereas that in WAT was negligible, and was increased only slightly by cold exposure. The transcript of A‐FABP was observed in both BAT and WAT, the level being slightly greater in WAT. However, its transcript level was not affected by cold exposure in either adipose tissue. In addition, on treatment with norepinephrine (NE), transcript level of H‐FABP was elevated markedly but that of A‐FABP was not changed in rat brown adipocytes. Therefore, the stimulatory effect of cold exposure on the transcript of H‐FABP in BAT was concluded to be mediated by NE, like that of the uncoupling protein (UCP). Thus, the expressions of H‐FABP and UCP may be controlled by the same mechanism.

Ca2+-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions

Yeast mitochondria have generally been believed not to undergo the permeability transition (PT) by the accumulation of Ca(2+) within the mitochondrial matrix, unlike mammalian mitochondria. However, the reason why the yeast PT is not induced by Ca(2+) has remained obscure. In this study, we examined in detail the effects of Ca(2+) on yeast mitochondria under various conditions. As a result, we discovered that the PT could be induced even in yeast mitochondria by externally added Ca(2+) under optimized experimental conditions. The 2 essential parameters for proper observation of the PT-inducing effects of Ca(2+) were the concentrations of the respiratory substrate and that of inorganic phosphate (Pi) in the incubation medium. The yeast mitochondrial PT induced by Ca(2+) was found to be insensitive to cyclosporin A and suppressed in the presence of a high concentration of Pi. Furthermore, when the PT was induced in yeast mitochondria by Ca(2+), the release of cytochrome c from mitochondria was also observed.

Isolation and characterization of cDNA clones and a genomic clone encoding rat mitochondrial adenine nucleotide translocator

Two cDNA clones encoding rat mitochondrial adenine nucleotide translocator were isolated from libraries constructed from mRNAs of heart and liver. These two clones corresponded to the heart-skeletal muscle type (ANT1) and fibroblast type (ANT2), respectively. A genomic clone encoding rat ANT1 was also isolated and characterized.

Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure

To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of beta-adrenergic receptor (beta-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of beta(1)-AR and C/EBPbeta in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.

Classification of FABP isoforms and tissues based on quantitative evaluation of transcript levels of these isoforms in various rat tissues.

In mammals, 10 isoforms of fatty acid-binding protein (FABP) are expressed in various tissues. To understand the role of multiple FABP isoforms, we have quantitatively examined the transcript levels of individual FABP isoforms in each of various tissues by Northern blotting using synthesized RNAs corresponding to the mRNA of each isoform as external standards. As a result, absolute transcript levels of individual FABP isoforms expressed in each tissue were successfully determined. The 10 FABP isoforms were classified into three categories: (1) isoforms FABP7 and 12 were not markedly expressed in any tissue examined; (2) isoforms showing certain transcript levels in multiple tissues; and (3) isoforms FABP6, 8, and 9, expressed at certain levels in one particular tissue. Based on the expression profiles of the isoforms, individual tissues were also classified into three groups: (1) tissues in which high-level expression of FABP isoforms was not observed, (2) tissues in which multiple FABP isoforms were expressed at certain levels, and (3) tissues in which a single FABP isoform was dominantly expressed at a certain level. These results give a better understanding of the meaning of the presence of multiple FABP isoforms in mammals.

Differential Permeabilization Effects of Ca2+ and Valinomycin on the Inner and Outer Mitochondrial Membranes as Revealed by Proteomics Analysis of Proteins Released from Mitochondria

It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca2+. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224–5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed.