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At least 91 records · Page 5Linked to original sources
PubMed2000

Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis.

Ribosome biogenesis requires, in addition to rRNA molecules and ribosomal proteins, a multitude of trans-acting factors. Recently it has become clear that in the yeast Saccharomyces cerevisiae many RNA helicases of the DEAD-box and related families are involved in ribosome biogenesis. Here we show that the previously uncharacterised open reading frame YDL031w (renamed DBP10 for DEAD-box protein 10) encodes an essential putative RNA helicase that is required for accurate ribosome biogenesis. Genetic depletion of Dbp10p results in a deficit in 60S ribosomal subunits and an accumulation of half-mer polysomes. Furthermore, pulse-chase analyses of pre-rRNA processing reveal a strong delay in the maturation of 27SB pre-rRNA intermediates into 25S rRNA and 7S pre-rRNA. Northern blot analyses indicate that this delay leads to higher steady-state levels of 27SB species and reduced steady-state levels of 7S pre-rRNA and 25S/5.8S mature rRNAs, thus explaining the final deficit in 60S subunit and the formation of half-mer polysomes. Consistent with a direct role in ribosome biogenesis, Dbp10p was found to be located predominantly in the nucleolus.

Amino Acid Sequence↗
PubMed2003

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis.

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.

Acrosome↗
PubMed2003

Raising Ca2+ in L6 myotubes mimics effects of exercise on mitochondrial biogenesis in muscle.

Skeletal muscle adapts to endurance exercise with an increase in mitochondria. Muscle contractions generate numerous potential signals. To determine which of these stimulates mitochondrial biogenesis, we are using L6 myotubes. Using this model we have found that raising cytosolic Ca2+ induces an increase in mitochondria. In this study, we tested the hypothesis that raising cytosolic Ca2+ in L6 myotubes induces increased expression of PGC-1, NRF-1, NRF-2, and mtTFA, factors that have been implicated in mitochondrial biogenesis and in the adaptation of muscle to exercise. Raising cytosolic Ca2+ by exposing L6 myotubes to caffeine for 5 h induced significant increases in PGC-1 and mtTFA protein expression and in NRF-1 and NRF-2 binding to DNA. These adaptations were prevented by dantrolene, which blocks Ca2+ release from the SR. Exposure of L6 myotubes to caffeine for 5 h per day for 5 days induced significant increases in mitochondrial marker enzyme proteins. Our results show that the adaptive response of L6 myotubes to an increase in cytosolic Ca2+ mimics the stimulation of mitochondrial biogenesis by exercise. They support the hypothesis that an increase in cytosolic Ca2+ is one of the signals that mediate increased mitochondrial biogenesis in muscle.

Animals↗
PubMed1996

A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus.

Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50-70 MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasmid-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10 kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. coli strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The BFP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.

Bacterial Adhesion↗
PubMed2004

Has1p, a member of the DEAD-box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae.

The Has1 protein, a member of the DEAD-box family of ATP-dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre-60S ribosomal complexes. Here, we show that Has1p is an essential trans-acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature-sensitive has1-1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre-rRNA processing by pulse-chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre-rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre-rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high-molecular-weight complex sedimenting at positions equivalent to 60S and pre-60S ribosomal particles. A mutation in the ATP-binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.

Amino Acid Motifs↗
PubMed2005

Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occurring after pilus assembly, essential for fibre stability and function.

Type IV pili (Tfp) play a critical role in the pathogenic lifestyle of Neisseria meningitidis and N. gonorrhoeae, notably by facilitating bacterial attachment to human cells, but our understanding of their biogenesis, during which the fibres are assembled in the periplasm, then emerge onto the cell surface and are stabilized, remains fragmentary. We therefore sought to identify the genes required for Tfp formation in N. meningitidis by screening a genome-wide collection of mutants for those that were unable to form aggregates, another phenotype mediated by these organelles. Fifteen proteins, of which only seven were previously characterized, were found to be essential for Tfp biogenesis. One novel component, named PilW, was studied in more detail. We found that PilW is an outer-membrane protein necessary for the stabilization of the fibres but not for their assembly or surface localization, because Tfp could be restored on the surface in a pilW mutant by a mutation in the twitching motility gene pilT. However, Tfp-linked properties, including adherence to human cells, were not restored in a pilW/T mutant, which suggests that PilW is also essential for the functionality of the fibres. Together with the finding that PilW is important for the stability of PilQ multimers, our results extend the current model for Tfp biogenesis by suggesting that a multiprotein machinery in the outer-membrane is involved in the terminal stage of Tfp biogenesis during which growing fibres are not only stabilized, but also become perfectly functional.

Cells, Cultured↗
PubMed2004

How to roll an endothelial cigar: the biogenesis of Weibel-Palade bodies.

Weibel-Palade bodies (WPB) are the regulated secretory organelles of endothelial cells. These cigar-shaped membrane-bound structures function in both hemostasis and inflammation but their biogenesis is poorly understood. Here, we review what is currently known about their formation. The content of WPBs is dominated by the hemostatic factor von Willebrand factor (VWF), whose complex biogenesis ends in the formation of high molecular weight multimers. VWF is also organized into proteinaceous tubules which underlie the striated interior of WPBs as seen in the EM. VWF expression is necessary for formation of WPBs, and its heterologous expression can even lead to the specific recruitment of WPB membrane proteins, including the leukocyte receptor P-selectin, the tetraspanin CD63, and Rab27a. Unusually, the VWF propeptide is implicated in the biogenesis of WPBs, being essential for formation of the storage compartment. The elongation of the cigars and the formation of the tubules are determined by non-covalent interactions between pro- and mature VWF proteins. Surprisingly, high molecular weight multimers seem neither necessary nor sufficient to trigger formation of a storage compartment, and do not seem to have any role in WPB biogenesis. Von Willebrand's disease, usually caused by mutations within VWF, has provided many of the insights into the way in which VWF drives the formation of these organelles.

Animals↗
PubMed2005

Mitochondrial biogenesis in mtDNA-depleted cells involves a Ca2+-dependent pathway and a reduced mitochondrial protein import.

Alterations in mitochondrial activity resulting from defects in mitochondrial DNA (mtDNA) can modulate the biogenesis of mitochondria by mechanisms that are still poorly understood. In order to study mitochondrial biogenesis in cells with impaired mitochondrial activity, we used rho-L929 and rho(0)143 B cells (partially and totally depleted of mtDNA, respectively), that maintain and even up-regulate mitochondrial population, to characterize the activity of major transcriptional regulators (Sp1, YY1, MEF2, PPARgamma, NRF-1, NRF-2, CREB and PGC-1alpha) known to control the expression of numerous nuclear genes encoding mitochondrial proteins. Among these regulators, cyclic AMP-responsive element binding protein (CREB) activity was the only one to be increased in mtDNA-depleted cells. CREB activation mediated by a calcium-dependent pathway in these cells also regulates the expression of cytochrome c and the abundance of mitochondrial population as both are decreased in mtDNA-depleted cells that over-express CREB dominant negative mutants. Mitochondrial biogenesis in mtDNA-depleted cells is also dependent on intracellular calcium as its chelation reduces mitochondrial mass. Despite a slight increase in mitochondrial mass in mtDNA-depleted cells, the mitochondrial protein import activity was reduced as shown by a decrease in the import of radiolabeled matrix-targeted recombinant proteins into isolated mitochondria and by the reduced mitochondrial localization of ectopically expressed HA-apoaequorin targeted to the mitochondria. Decrease in ATP content, in mitochondrial membrane potential as well as reduction in mitochondrial Tim44 abundance could explain the lower mitochondrial protein import in mtDNA-depleted cells. Taken together, these results suggest that mitochondrial biogenesis is stimulated in mtDNA-depleted cells and involves a calcium-CREB signalling pathway but is associated with a reduced mitochondrial import for matrix proteins.

Adenosine Triphosphate↗
PubMed2002

Regulation of mitochondrial biogenesis in skeletal muscle by CaMK.

Endurance exercise training promotes mitochondrial biogenesis in skeletal muscle and enhances muscle oxidative capacity, but the signaling mechanisms involved are poorly understood. To investigate this adaptive process, we generated transgenic mice that selectively express in skeletal muscle a constitutively active form of calcium/calmodulin-dependent protein kinase IV (CaMKIV*). Skeletal muscles from these mice showed augmented mitochondrial DNA replication and mitochondrial biogenesis, up-regulation of mitochondrial enzymes involved in fatty acid metabolism and electron transport, and reduced susceptibility to fatigue during repetitive contractions. CaMK induced expression of peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), a master regulator of mitochondrial biogenesis in vivo, and activated the PGC-1 gene promoter in cultured myocytes. Thus, a calcium-regulated signaling pathway controls mitochondrial biogenesis in mammalian cells.

Animals↗
PubMed2003

Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide.

Nitric oxide was found to trigger mitochondrial biogenesis in cells as diverse as brown adipocytes and 3T3-L1, U937, and HeLa cells. This effect of nitric oxide was dependent on guanosine 3',5'-monophosphate (cGMP) and was mediated by the induction of peroxisome proliferator-activated receptor gamma coactivator 1alpha, a master regulator of mitochondrial biogenesis. Moreover, the mitochondrial biogenesis induced by exposure to cold was markedly reduced in brown adipose tissue of endothelial nitric oxide synthase null-mutant (eNOS-/-) mice, which had a reduced metabolic rate and accelerated weight gain as compared to wild-type mice. Thus, a nitric oxide-cGMP-dependent pathway controls mitochondrial biogenesis and body energy balance.

3T3 Cells↗
PubMed2006

Two gene determinants are differentially involved in the biogenesis of Fap1 precursors in Streptococcus parasanguis.

Mature Fap1, a 200-kDa fimbria-associated adhesin, is required for fimbrial biogenesis and biofilm formation in Streptococcus parasanguis. Fap1-like proteins are found in the genomes of many streptococcal and staphylococcal species. Fap1 is a serine-rich glycoprotein modified by O-linked glycan moieties. In this study, we identified a seven-gene cluster including secY2, orf1, orf2, orf3, secA2, gtf1, and gtf2 that is localized immediately downstream of fap1. The lower G+C contents and the presence of a putative transposase element suggest that this gene cluster was horizontally transferred from other bacteria and represents a genomic island. At least two genes in this island mediated Fap1 biogenesis. Mutation of a glucosyltransferase (Gtf1) gene led to accumulation of a Fap1 precursor, which had no detectable glycan moieties. Inactivation of a gene coding for an accessory Sec protein (SecY2) resulted in expression of a distinct Fap1 precursor, which reacted with one glycan-specific Fap1 antibody but not with another glycan-specific antibody. Furthermore, partially glycosylated Fap1 was detected on the cell surface and in the culture supernatant. These data suggest that SecY2 has a role in complete glycosylation of Fap1 and imply that SecY2 is not the only translocation channel for the Fap1 precursor and that alternative secretion machinery exists. Together, Gtf1 and SecY2 are involved in biogenesis of two distinct Fap1 precursors in S. parasanguis. Discovery of the effect of an accessory Sec protein on Fap1 glycosylation suggests that Fap1 secretion and glycosylation are coupled during Fap1 biogenesis.

Bacterial Proteins↗
PubMed2002

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis.

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.

Amino Acid Sequence↗
PubMed2006

Characterization of Saccharomyces cerevisiae Npa2p (Urb2p) reveals a low-molecular-mass complex containing Dbp6p, Npa1p (Urb1p), Nop8p, and Rsa3p involved in early steps of 60S ribosomal subunit biogenesis.

We report the characterization of the yeast Npa2p (Urb2p) protein, which is essential for 60S ribosomal subunit biogenesis. We identified this protein in a synthetic lethal screening with the rsa3 null allele. Rsa3p is a genetic partner of the putative RNA helicase Dbp6p. Mutation or depletion of Npa2p leads to a net deficit in 60S subunits and a decrease in the levels all 27S pre-rRNAs and mature 25S and 5.8S rRNAs. This is likely due to instability of early pre-60S particles. Consistent with a role of Npa2p in 60S subunit biogenesis, green fluorescent protein-tagged Npa2p localizes predominantly to the nucleolus and TAP-tagged Npa2p sediments with large complexes in sucrose gradients and is associated mainly with 27SA(2) pre-rRNA-containing preribosomal particles. In addition, we reveal a genetic synthetic interaction between Npa2p, several factors required for early steps of 60S subunit biogenesis (Dbp6p, Dbp7p, Dbp9p, Npa1p, Nop8p, and Rsa3p), and the 60S protein Rpl3p. Furthermore, coimmunoprecipitation and gel filtration analyses demonstrated that at least Npa2p, Dbp6p, Npa1p, Nop8p, and Rsa3p are present together in a subcomplex of low molecular mass whose integrity is independent of RNA. Our results support the idea that these five factors work in concert during the early steps of 60S subunit biogenesis.

Alleles↗
PubMed1997

Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.

Animals↗
PubMed2005

Myc stimulates nuclearly encoded mitochondrial genes and mitochondrial biogenesis.

Although several genes involved in mitochondrial function are direct Myc targets, the role of Myc in mitochondrial biogenesis has not been directly established. We determined the effects of ectopic Myc expression or the loss of Myc on mitochondrial biogenesis. Induction of Myc in P493-6 cells resulted in increased oxygen consumption and mitochondrial mass and function. Conversely, compared to wild-type Myc fibroblasts, Myc null rat fibroblasts have diminished mitochondrial mass and decreased number of normal mitochondria. Reconstitution of Myc expression in Myc null fibroblasts partially restored mitochondrial mass and function and normal-appearing mitochondria. Concordantly, we also observed in primary hepatocytes that acute deletion of floxed murine Myc by Cre recombinase resulted in diminished mitochondrial mass in primary hepatocytes. Our microarray analysis of genes responsive to Myc in human P493-6 B lymphocytes supports a role for Myc in mitochondrial biogenesis, since genes involved in mitochondrial structure and function are overrepresented among the Myc-induced genes. In addition to the known direct binding of Myc to many genes involved in mitochondrial structure and function, we found that Myc binds the TFAM gene, which encodes a key transcriptional regulator and mitochondrial DNA replication factor, both in P493-6 lymphocytes with high ectopic MYC expression and in serum-stimulated primary human 2091 fibroblasts with induced endogenous MYC. These observations support a pivotal role for Myc in regulating mitochondrial biogenesis.

Animals↗
PubMed2005

The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis.

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.

ATP-Binding Cassette Transporters↗
PubMed2005

Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels.

ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels.

ATP-Binding Cassette Transporters↗
PubMed2002

Intermittent increases in cytosolic Ca2+ stimulate mitochondrial biogenesis in muscle cells.

Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+ can stimulate mitochondrial biogenesis. Continuous exposure to 1 microM ionomycin, a Ca2+ ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an approximately 40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+ from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+ intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+ release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+ stimulates mitochondrial biogenesis in muscle cells.

5-Aminolevulinate Synthetase↗