Airway Resistance Caused by Sphingomyelin Synthase 2 Insufficiency in Response to Cigarette Smoke.

Sphingomyelin synthase is responsible for the production of sphingomyelin (SGM), the second most abundant phospholipid in mammalian plasma, from ceramide, a major sphingolipid. Knowledge of the effects of cigarette smoke on SGM production is limited. In the present study, we examined the effect of chronic cigarette smoke on sphingomyelin synthase (SGMS) activity and evaluated how the deficiency of Sgms2, one of the two isoforms of mammalian SGMS, impacts pulmonary function. Sgms2-knockout and wild-type control mice were exposed to cigarette smoke for 6 months, and pulmonary function testing was performed. SGMS2-dependent signaling was investigated in these mice and in human monocyte-derived macrophages of nonsmokers and human bronchial epithelial (HBE) cells isolated from healthy nonsmokers and subjects with chronic obstructive pulmonary disease (COPD). Chronic cigarette smoke reduces SGMS activity and Sgms2 gene expression in mouse lungs. Sgms2-deficient mice exhibited enhanced airway and tissue resistance after chronic cigarette smoke exposure, but had similar degrees of emphysema, compared with smoke-exposed wild-type mice. Sgms2-/- mice had greater AKT phosphorylation, peribronchial collagen deposition, and protease activity in their lungs after smoke inhalation. Similarly, we identified reduced SGMS2 expression and enhanced phosphorylation of AKT and protease production in HBE cells isolated from subjects with COPD. Selective inhibition of AKT activity or overexpression of SGMS2 reduced the production of several matrix metalloproteinases in HBE cells and monocyte-derived macrophages. Our study demonstrates that smoke-regulated Sgms2 gene expression influences key COPD features in mice, including airway resistance, AKT signaling, and protease production.

Computational study on human sphingomyelin synthase 1 (hSMS1).

Human sphingomyelin synthase 1 (hSMS1) is the last enzyme for sphingomyelin (SM) biosynthesis. It has been discovered that in different human tumor tissues the SM levels are lower compared to normal tissues and the activation of hSMS1, to restore the normal levels of SM, inhibits cell cycle proliferation of cancer cells. Since the importance of SM and other lipid metabolism genes in the malignant transformation, we decided to explore the hSMS1 mechanism of action. Enzymes capable to regulate the formation of lipids are therefore of paramount importance. Here we present a computational study on sphingomyelin synthases hSMS1. The full structure of the enzyme was obtained by means of homology and ab initio techniques. Further molecular dynamics and docking studies permitted to identify putative binding sites and to identify the key residues for binding. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Impact of sphingomyelin synthase 1 deficiency on sphingolipid metabolism and atherosclerosis in mice.

OBJECTIVE: Sphingomyelin synthase (SMS) catalyzes the conversion of ceramide to sphingomyelin and sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. Although they have the same SMS activity, they are different enzymes with distinguishable subcellular localizations and cell expression patterns. It is conceivable that these differences could yield different consequences, in terms of sphingolipid metabolism and its related atherogenesis. METHODS AND RESULTS: We created Sms1 gene knockout mice and found that Sms1 deficiency significantly decreased plasma, liver, and macrophage sphingomyelin (59%, 45%, and 54%, respectively), but only had a marginal effect on ceramide levels. Surprisingly, we found that Sms1 deficiency dramatically increased glucosylceramide and GM3 levels in plasma, liver, and macrophages (4- to 12-fold), whereas Sms2 deficiency had no such effect. We evaluated the total SMS activity in tissues and found that Sms1 deficiency causes 77% reduction in SMS activity in macrophages, indicating SMS1 is the major SMS in macrophages. Moreover, Sms1-deficient macrophages have a significantly higher glucosylceramide synthase activity. We also found that Sms1 deficiency significantly attenuated toll-like 4 receptor-mediated nuclear factor-κB and mitogen-activated protein kinase activation after lipopolysaccharide treatment. To evaluate atherogenicity, we transplanted Sms1 knockout mouse bone marrow into low-density lipoprotein receptor knockout mice (Sms1(-/-)→Ldlr(-/-)). After 3 months on a western diet, these animals showed a significant decrease of atherosclerotic lesions in the root and the entire aorta (35% and 44%, P<0.01, respectively) and macrophage content in lesions (51%, P<0.05), compared with wild-type→Ldlr(-/-) mice. CONCLUSIONS: Sms1 deficiency decreases sphingomyelin, but dramatically increases the levels of glycosphingolipids. Atherosclerosis in Sms1(-/-)→Ldlr(-/-) mice is significantly decreased.

Sphingomyelin synthases regulate protein trafficking and secretion.

Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.

Chemopreventive effects of silymarin against 1,2-dimethylhydrazine plus dextran sodium sulfate-induced inflammation-associated carcinogenicity and genotoxicity in the colon of gpt delta rats.

Silymarin, a natural flavonoid from the seeds of milk thistle, is used for chemoprevention against various cancers in clinical settings and in experimental models. To examine the chemopreventive mechanisms of silymarin against colon cancer, we investigated suppressive effects of silymarin against carcinogenicity and genotoxicity induced by 1,2-dimethylhydrazine (DMH) plus dextran sodium sulfate (DSS) in the colon of F344 gpt delta transgenic rats. Male gpt delta rats were given a single subcutaneous injection of 40 mg/kg DMH and followed by 1.5% DSS in drinking water for a week. They were fed diets containing silymarin for 4 weeks, starting 1 week before DMH injection and samples were collected at 4, 20 and 32 weeks after the DMH treatment. Silymarin at doses of 100 and 500 p.p.m. suppressed the tumor formation in a dose-dependent manner and the reduction was statistically significant. In the mutation assays, DMH plus DSS enhanced the gpt mutant frequency (MF) in the colon, and the silymarin treatments reduced the MFs by 20%. Silymarin also reduced the genotoxicity of DMH in a dose-dependent manner in bacterial mutation assay with Salmonella typhimurium YG7108, a sensitive strain to alkylating agents, and the maximum reduction was >80%. These results suggest that silymarin is chemopreventive against DMH/DSS-induced inflammation-associated colon carcinogenesis and silymarin might act as an antigenotoxic agent, in part.

Mitochondrial dysfunction and increased reactive oxygen species impair insulin secretion in sphingomyelin synthase 1-null mice.

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic ß-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic ß-cells.

Proteolytic processing of the gamma-subunit is associated with the failure to form GlcNAc-1-phosphotransferase complexes and mannose 6-phosphate residues on lysosomal enzymes in human macrophages.

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

E. coli cardiolipin synthase: function of N-terminal conserved residues.

The E. coli cls open reading frame (ORF) predicts a 54.8 kDa polypeptide, whereas mature cardiolipin (CL) synthase is 46 kDa. The N-terminal region extending to residue 60 contains several conserved residues but is not essential for enzyme activity. A deletion mutant that is missing residues 2-60 produces a fully active protein. These findings raise the question of why several residues in a region that is not required for enzyme activity are conserved. Recombinant DNA technology was used to introduce an EYMPE epitope (EE) tag into the interior of CL synthase. The EE tagged polypeptide retained the biological properties of wild type CL synthase, including full enzymatic activity. Site-directed mutagenesis was used to alter conserved residues in the N-terminal region. An EE tagged CL synthase in which Leu-7 and Val-8 were both replaced by Ser residues retains in vitro activity but loses most of its in vivo activity. Furthermore, the mutant protein has a higher apparent molecular mass than its parent protein. Taken together, these findings suggest that conserved residues L7 and V8 play a role in polypeptide processing, topology, or both.

Sphingomyelin synthase 2 deficiency attenuates NFkappaB activation.

BACKGROUND: NFkappaB has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. Metabolism of sphingomyelin (SM) has been suggested to affect NFkappaB activation, but the mechanism is largely unknown. SMS2 regulates SM levels in cell plasma membrane and lipid rafts and has a potential to regulate NFkappaB activation. METHODS AND RESULTS: To investigate the role of SMS2 in NFkappaB activation we used macrophages from SMS2 knockout (KO) mice and SMS2 siRNA-treated HEK 293 cells. We found that NFkappaB activation and its target gene expression are attenuated in macrophages from SMS2 KO mice in response to lipopolysaccharide (LPS) stimulation and in SMS2 siRNA- treated HEK 293 cells after tumor necrosis factor (TNF)-alpha simulation. In line with attenuated NFkappaB activation, we found that SMS2 deficiency substantially diminished the abundance of toll like receptor 4 (TLR4)-MD2 complex levels on the surface of macrophages after LPS stimulation, and SMS2 siRNA treatment reduced TNF-alpha-stimulated lipid raft recruitment of TNF receptor-1 (TNFR1) in HEK293 cells. SMS2 deficiency decreased the relative amounts of SM and diacylglycerol (DAG) and increased ceramide, suggesting multiple mechanisms for the decrease in NFkappaB activation. CONCLUSIONS: SMS2 is a modulator of NFkappaB activation, and thus it could play an important role in NFkappaB-mediated proatherogenic process.

TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway.

TAZ is a WW domain containing a transcription coactivator that modulates mesenchymal differentiation and development of multiple organs. In this study, we show that TAZ is phosphorylated by the Lats tumor suppressor kinase, a key component of the Hippo pathway, whose alterations result in organ and tissue hypertrophy in Drosophila and contribute to tumorigenesis in humans. Lats phosphorylates TAZ on several serine residues in the conserved HXRXXS motif and creates 14-3-3 binding sites, leading to cytoplasmic retention and functional inactivation of TAZ. Ectopic expression of TAZ stimulates cell proliferation, reduces cell contact inhibition, and promotes epithelial-mesenchymal transition (EMT). Elimination of the Lats phosphorylation sites results in a constitutively active TAZ, enhancing the activity of TAZ in promoting cell proliferation and EMT. Our results elucidate a molecular mechanism for TAZ regulation and indicate a potential function of TAZ as an important target of the Hippo pathway in regulating cell proliferation tumorigenesis.

Phosphatidylcholine synthesis is required for optimal function of Legionella pneumophila virulence determinants.

The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N-methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Second, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady-state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion.

SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis.

Sphingomyelin synthase (SMS), the last enzyme in the sphingomyelin (SM) biosynthetic pathway, uses ceramide and phosphatidylcholine as substrates to produce SM and diacylglycerol (DAG). To evaluate the role of SMS in apoptosis, we generated Chinese hamster ovary cells that stably express human SMS1 or SMS2. We found that SMS1 or SMS2 overexpression results in a significant increase in cellular levels of SM (24% or 20%) and DAG (35% or 31%), respectively, compared with controls. Cells overexpressing SMS1 or SMS2 were more likely to undergo lysis mediated by lysenin (a protein that causes lysis through its affinity with SM-rich microdomains in the plasma membrane) than were controls, indicating SM enrichment of the plasma membrane. SMS1 and SMS2 overexpression also led to higher retention of DiIC16 fluorescence compared with wild-type cells, indicating an increased number of detergent-insoluble microdomains and significantly increased tumor necrosis factor-alpha-mediated apoptosis. To further evaluate the relationship between SMS activity and cell apoptosis, we used SMS1 and SMS2 small interfering RNA (siRNA) to knock down their mRNA in THP-1-derived macrophages. We found that SMS1 or SMS2 siRNA significantly reduces intracellular SM (by 20% or 23%), plasma membrane SM (as indicated by the rate of lysenin-mediated cell lysis), and DAG levels (24% or 20%), respectively, while significantly reducing lipopolysaccharide-mediated apoptosis compared with controls. These results indicate that SMS1 and SMS2 are key factors in the control of SM and DAG levels within the cell and thus influence apoptosis.

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase.

The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h=1.46, K50=0.751 microM, Vmax=2.236 micromol mg(-1) min(-1)) and CoA (h=0.704, K50=5.627 microM, Vmax=0.557 micromol mg(-1) min(-1)), and biphasic kinetics towards adenosine 5'-triphosphate (Km1=3.149 microM, Vmax1=373.3 nmol mg(-1) min(-1), Km2=121.0 microM, and Vmax2=563.7 nmol mg(-1) min(-1)). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50=6.64 microM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA.

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.

Nuclear sphingomyelin-synthase and protein kinase C delta in melanoma cells.

The aim of this research is to study the influence of protein kinase C delta on the nuclear phospholipids metabolism. Murine and human melanoma cells, in which overexpression of protein kinase delta was induced, were used. After purification of the nuclei, the phosphatidylcholine-dependent phospholipase C, sphingomyelin-synthase, and sphingomyelinase activities were measured. The results showed that the nuclear sphingomyelin-synthase activity increased and sphingomyelinase activity decreased in the protein kinase C delta overexpressive cells with respect to the controls. As a consequence, the ceramide pool decreased and diacylglycerol pool increased; this effect was not due to the phosphatidylcholine-dependent phospholipase C activity that did not change. The inhibition of sphingomyelinase could be due to protein kinase C delta as well as to existence of a sort of nuclear self-regulation between sphingomyelin-synthase and sphingomyelinase. The possible role of nuclear sphingomyelin-synthase in cell proliferation is discussed.

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments.

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses. Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK), phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms. In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid). Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions. In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.

Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) causes a novel congenital disorder of Glycosylation Type Ij.

Defects in the assembly of dolichol-linked oligosaccharide or its transfer to proteins result in severe, multi-system human diseases called Type I congenital disorders of glycosylation. We have identified a novel CDG type, CDG-Ij, resulting from deficiency in UDP-GlcNAc: dolichol phosphate N-acetyl-glucosamine-1 phosphate transferase (GPT) activity encoded by DPAGT1. The patient presents with severe hypotonia, medically intractable seizures, mental retardation, microcephaly, and exotropia. Metabolic labeling of cultured dermal fibroblasts from the patient with [2-(3)H]-mannose revealed lowered incorporation of radiolabel into full-length dolichol-linked oligosaccharides and glycoproteins. In vitro enzymatic analysis of microsomal fractions from the cultured cells indicated that oligosaccharyltransferase activity is normal, but the GPT activity is reduced to approximately 10% of normal levels while parents have heterozygous levels. The patient's paternal DPAGT1 allele contains a point mutation (660A>G) that replaces a highly conserved tyrosine with a cysteine (Y170C). The paternal allele cDNA produces a full-length protein with almost no activity when over-expressed in CHO cells. The maternal allele makes only about 12% normal mature mRNA, while the remainder shows a complex exon skipping pattern that shifts the reading frame encoding a truncated non-functional GPT protein. Thus, we conclude that the DPAGT1 gene defects are responsible for the CDG symptoms in this patient. Hum Mutat 22:144-150, 2003.

Lack of mitochondrial anionic phospholipids causes an inhibition of translation of protein components of the electron transport chain. A yeast genetic model system for the study of anionic phospholipid function in mitochondria.

Reduction of mitochondrial cardiolipin (CL) levels has been postulated to compromise directly the function of several essential enzymes and processes of the mitochondria. There is limited genetic evidence for the critical roles with which CL and its precursor phosphatidylglycerol (PG) have been associated. A null allele of the PGS1 gene from Saccharomyces cerevisiae, which encodes the enzyme responsible for the synthesis of the CL precursor PG phosphate, was created in a yeast strain in which PGS1 expression is exogenously regulated by doxycycline. The addition of increasing concentrations of doxycycline to the growth medium causes a proportional decrease to undetectable levels of PGS1 transcript, PG phosphate synthase activity, and PG plus CL. The doubling time of this strain with increasing doxycycline increases to senescence in non-fermentable carbon sources or at high temperatures, conditions that do not support growth of the pgs1Delta strain. Doxycycline addition also causes mitochondrial abnormalities as observed by fluorescence microscopy. Products of four mitochondrial encoded genes (COX1, COX2, COX3, and COB) and one nuclear encoded gene (COX4) associated with the mitochondrial inner membrane are not present when PGS1 expression is fully repressed. No translation of these proteins can be detected in cells lacking the PGS1 gene product, although transcription and splicing appear unaffected. Protein import of other nuclear encoded proteins remains unaffected. The remaining proteins encoded by mitochondrial DNA are expressed and translated normally. Thus, the molecular basis for the lack of mitochondrial function in pgs1Delta cells is the failure to translate gene products essential to the electron transport chain.

Molecular dissection of the genetic targets of ALG7 in the serpentine receptor-mediated signal transduction pathway in yeast.

These initial studies show that deregulated expression of ALG7 affects diverse cellular functions crucial to development, including proliferation, differentiation, and morphogenesis. Furthermore, the data suggest multiple genetic targets for ALG7 and provide the basis for future dissection of these developmentally relevant pathways.