Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase

Two fission yeast temperature-sensitive mutants, cut6 and lsd1, show a defect in nuclear division. The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter). Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei. EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei. The small nuclei lacked electron- dense nuclear materials and contained highly compacted chromatin. The cut6+ and lsd1+ genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis. Gene disruption of lsd1+ led to the lsd phenotype. Palmitate in medium fully suppressed the phenotypes of lsd1. Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type. The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation. A reduced level of fatty acid thus led to impaired separation of non- chromosomal nuclear components. We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters.     

Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA

In this review we examine the effects of the allosteric activator, acetyl CoA on both the structure and catalytic activities of pyruvate carboxylase. We describe how the binding of acetyl CoA produces gross changes to the quaternary and tertiary structures of the enzyme that are visible in the electron microscope. These changes serve to stabilize the tetrameric structure of the enzyme. The main locus of activation of the enzyme by acetyl CoA is the biotin carboxylation domain of the enzyme where ATP-cleavage and carboxylation of the biotin prosthetic group occur. As well as enhancing reaction rates, acetyl CoA also enhances the binding of some substrates, especially HCO3-, and there is also a complex interaction with the binding of the cofactor Mg2. The activation of pyruvate carboxylase by acetyl CoA is generally a cooperative processes, although there is a large degree of variability in the degree of cooperativity exhibited by the enzyme from different organisms. The X-ray crystallographic holoenzyme structures of pyruvate carboxylases from Rhizobium etli and Staphylococcus aureus have shown the allosteric acetyl CoA binding domain to be located at the interfaces of the biotin carboxylation and carboxyl transfer and the carboxyl transfer and biotin carboxyl carrier protein domains.     

Biotin subunits of acetyl CoA carboxylase and pyruvate carboxylase from a thermophilic Bacillus.

Two biotin-containing polypeptides of molecular weights 140,000 and 22,000 have been identified by gel electrophoresis in a sodium dodecyl sulfate-denatured extract of cells of a thermophilic Bacillus. These polypeptides can be separated from each other by either gel filtration through Sepharose 6B or affinity chromatography on a Sepharose-avidin column. The larger polypeptide is renatured under appropriate conditions to yield enzymically active pyruvate carboxylase. Enzyme reconstitution experiments show that the smaller polypeptide is a component of acetyl CoA carboxylase. The biotin subunits of these two carboxylases are thus distinct from, and dissimilar to, each other. The demonstration that a pyruvate carboxylase-deficient mutant of the Bacillus contains the smaller, but not the larger, polypeptide corroborates this conclusion.     

Effect of insulin on association of acetyl CoA carboxylase phosphatase and acetyl CoA carboxylase

Insulin promotes an association between acetyl CoA carboxylase and acetyl CoA carboxylase phosphatase. The association between rat epididymal fat tissue carboxylase and the phosphatase occurs in both a tissue culture system and in vivo and is accompanied by an increase in acetyl CoA carboxylase activity.     

Subcellular localization of fatty acid acetyl coa and malonyl CoA elongation systems in rat adipocytes

The distribution of fatty acid elongation systems in the subcellular fractions of isolated fat cells was studied. These systems were found to be located essentially in the particulate fractions, Acetyl CoA elongation was found to occur predominantly in the mitochondria (2.8 nmoles of acetyl CoA incorporated into fatty acids/mg of proteins per 30 min) while malonyl CoA elongation was predominant in the purified plasma membranes (10 nmoles of malonyl CoA incorporated into fatty acids/mg of proteins per 30 min). The acetyl CoA elongation activity in the mitochondria and the malonyl CoA elongation activity in the purified plasma membranes were found to be 6.4 and 2.4 times higher, respectively, than in the purified microsomes, the next most active fraction. In liver, on the other hand, greater malonyl CoA elongation activity was found in microsomes than in plasma membranes.     

Differential regulation of the yeast isozymes of pyruvate carboxylase and the locus of action of acetyl CoA

Unlike other eukaryotes studied to date, yeast has two genes for pyruvate carboxylase coding for very similar, but not identical, isozymes (Pyc1 and Pyc2), both of which are located in the cytoplasm. We have found that there are marked differences in the kinetic properties of the isozymes potentially leading to differential regulation of Pyc1 and Pyc2 activity by both activators and substrates. For example, Pyc2 is only activated 3.7-fold by acetyl CoA, and 9.6-fold by NH(4)(+), whilst the figures for Pyc1 are 16 and 14.6-fold, respectively. Pyc1 and Pyc2 display different allosteric properties with respect to acetyl CoA activation and aspartate inhibition, with Pyc1 showing a higher degree of cooperativity than Pyc2, even in the absence of aspartate. We have investigated the locus of action in the amino acid sequence of the isozymes of this activator by measuring its regulation of various chimeric constructs of the two isozymes. In this way, we conclude that the main locus of action of acetyl CoA lies in the N-terminal half of the enzyme, within the biotin-carboxylation domain, between amino acids 99 and 478 of Pyc1.     

Cloning of acetyl CoA carboxylase cDNA and the effects of spirodiclofen on the expression of acetyl CoA carboxylase mRNA in Panonychus citri

The citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), is an important pest worldwide. Previous studies showed that acetyl CoA carboxylase (ACCase) may be a critical target of spirodiclofen, a recently introduced insecticide. Therefore, we cloned the PcACCase gene and obtained a full‐length cDNA (Genbank accession number KJ675439 ). The open reading frame was 6 972 bp, coding for 2 323 amino acids. The PcACCase protein had a theoretical molecular weight of 262.82 kDa and an isoelectric point of 6.31, and it contained six conserved domains and one low‐complexity region. Quantitative real‐time PCR showed that spirodiclofen can up‐regulate the expression levels of ACCase mRNA during all four developmental stages.     

Role of pyruvate carboxylase in fatty acid synthesis: Alterations during preadipocyte differentiation

Transport of mitochondrial acetyl units to the cytoplasm for fatty acid synthesis via the citrate cleavage pathway requires replenishment of mitochondrial oxaloacetate. Pyruvate carboxylase is though to fulfill this role although compelling evidence has been lacking. During lipogenic differentiation of 3T3-L1 preadipocytes, pyruvate carboxylase activity rises 18-fold in close coordination with fat accumulation and the activity of ATP-citrate lyase, an established lipogenic enzyme. The activities of enzymes less directly related to lipogenesis rise only 3–5-fold while other unrelated enzymes do not increase significantly. These results indicate that pyruvate carboxylase is in fact a lipogenic enzyme.     

TR4 promotes fatty acid synthesis in 3T3-L1 adipocytes by activation of pyruvate carboxylase expression

We show that testicular orphan nuclear receptor 4 (TR4) increases the expression of pyruvate carboxylase (PC) gene in 3T3-L1 adipocytes by direct binding to a TR4 responsive element in the murine PC promoter. While TR4 overexpression increased PC activity, oxaloacetate (OAA) and glycerol levels with enhanced incorporation of ¹⁴C from ¹⁴C-pyruvate into fatty acids in 3T3-L1 adipocytes, PC knockdown by short interfering RNA (siRNA) or inhibition of PC activity by phenylacetic acid (PAA) abolished TR4-enhanced fatty acid synthesis. Moreover, TR4 microRNA reduced PC expression with decreased fatty acid synthesis in 3T3-L1 adipocytes, suggesting that TR4-mediated enhancement of fatty acid synthesis in adipocytes requires increased expression of PC gene.     

Expression profiling during adipocyte differentiation of 3T3-L1 fibroblasts

The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. Changes in gene expression were measured by DNA microarrays at three time points (24 h, 4 days, and 1 week) during the course of differentiation from preadipocytes to mature adipocytes. Several functional categories of genes were affected by adipocyte conversion. In addition, seven genes were found to be commonly altered by 5-fold or more by adipocyte conversion at all three time points. Lipocalin 2, haptoglobin, serum amyloid A3, stearoyl-CoA desaturase, and 11beta-hydroxysteroid dehydrogenase 1 were induced while actin alpha2 and procollagen VIII alpha1 were suppressed by adipocyte differentiation. Further study of the regulation of these genes and pathways will lead to an increased understanding of the biochemical pathways involved in adipocyte differentiation and possibly to the identification of new therapeutic targets for treatment of obesity and other metabolic diseases.     

Mammalian pyruvate carboxylase: effect of biotin on the synthesis and translocation of apo-enzyme into 3T3-L adipocyte mitochondria

The effect of biotin on the induction (and possible requirement for uptake into mitochondria) of apopyruvate carboxylase has been examined in 3T3-L adipocytes. Cells fed biotin-sufficient medium contained only holoenzyme in mitochondria and no apoenzyme was detected. The amount of apoenzyme elaborated in biotin-deficient 3T3-L adipocytes was comparable to the holopyruvate carboxylase protein found in cells maintained on biotin-sufficient medium. Like the holoenzyme, the apoenzyme was detected exclusively in the mitochondrial fraction of 3T3-L adipocytes. This indicates that the synthesis of apopyruvate carboxylase and its translocation into mitochondria occur independently of the cofactor, biotin.     

Hormonal regulation of acetyl CoA carboxylase effect of insulin and epinephrine

Acetyl CoA carboxylase in the isolated epididymal fat tissue is activated by insulin and inactivated by epinephrine and dibutyryl cAMP. Insulin activation of the enzyme occurs in the presence of inhibitors of protein synthesis and can be observed as early as 15 min. A time dependent inactivation of the enzyme occurs in the presence of ATP and Mg⁺⁺$\text{in vitro}$.