@article {1456, title = {Saccharomyces cerevisiae essential genes with an Opi- phenotype.}, journal = {G3 (Bethesda)}, volume = {4}, year = {2014}, month = {2014 Apr}, pages = {761-7}, abstract = {

The overproduction and secretion of inositol (i.e., Opi(-)) phenotype is associated with defects in regulation of phospholipid biosynthesis in yeast. Here we report a screen of the essential yeast gene set using a conditional-expression library. This screen identified novel functions previously unknown to affect phospholipid synthesis.

}, keywords = {Cluster Analysis, Genes, Essential, Histone Acetyltransferases, Inositol, Phenotype, Phospholipids, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {2160-1836}, doi = {10.1534/g3.113.010140}, author = {Salas-Santiago, Bryan and Lopes, John M} } @article {1458, title = {Transcription regulation of a yeast gene from a downstream location.}, journal = {J Mol Biol}, volume = {425}, year = {2013}, month = {2013 Feb 8}, pages = {457-65}, abstract = {

Mechanisms for coregulation of transcription of tandem genes in yeast remain largely speculative. This study focused on inositol-mediated regulation of the tandem gene pair SNA3-INO1. While the pattern of regulation of these two genes was similar, results showed that intermediate levels of inositol repressed INO1 and induced SNA3. Results also showed that inositol-mediated regulation of the SNA3 gene was not a function of its promoter but occurred from factors within the SNA3-INO1 intergenic region. The basic helix-loop-helix proteins, Ino2p and Ino4p, mediated this regulation through the upstream activation sequence (UAS)(INO) (E-box) sequences in the intergenic region. These results provide a model for studying coregulation of yeast tandem genes. This is especially significant given that many tandem gene pairs in yeast are coregulated even though context-specific UAS sequences are known only for one gene in the pair.

}, keywords = {Gene Expression Regulation, Fungal, Inositol, Membrane Proteins, Myo-Inositol-1-Phosphate Synthase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic}, issn = {1089-8638}, doi = {10.1016/j.jmb.2012.11.018}, author = {Shetty, Ameet and Swaminathan, Aishwarya and Lopes, John M} } @article {32, title = {Transcription regulation of the Saccharomyces cerevisiae PHO5 gene by the Ino2p and Ino4p basic helix-loop-helix proteins.}, journal = {Mol Microbiol}, volume = {83}, year = {2012}, month = {2012 Jan}, pages = {395-407}, abstract = {

The Saccharomyces cerevisiae PHO5 gene product accounts for a majority of the acid phosphatase activity. Its expression is induced by the basic helix-loop-helix (bHLH) protein, Pho4p, in response to phosphate depletion. Pho4p binds predominantly to two UAS elements (UASp1 at -356 and UASp2 at -247) in the PHO5 promoter. Previous studies from our lab have shown cross-regulation of different biological processes by bHLH proteins. This study tested the ability of all yeast bHLH proteins to regulate PHO5 expression and identified inositol-mediated regulation via the Ino2p/Ino4p bHLH proteins. Ino2p/Ino4p are known regulators of phospholipid biosynthetic genes. Genetic epistasis experiments showed that regulation by inositol required a third UAS site (UASp3 at -194). ChIP assays showed that Ino2p:Ino4p bind the PHO5 promoter and that this binding is dependent on Pho4p binding. These results demonstrate that phospholipid biosynthesis is co-ordinated with phosphate utilization via the bHLH proteins.

}, keywords = {Acid Phosphatase, Basic Helix-Loop-Helix Transcription Factors, Chromatin Immunoprecipitation, DNA, Fungal, Enhancer Elements, Genetic, Gene Expression Regulation, Fungal, Inositol, Models, Biological, Phospholipids, Promoter Regions, Genetic, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors, Transcription, Genetic}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2011.07941.x}, author = {He, Ying and Swaminathan, Aishwarya and Lopes, John M} } @article {687, title = {Inositol lipid metabolism in mycobacteria: biosynthesis and regulatory mechanisms.}, journal = {Biochim Biophys Acta}, volume = {1810}, year = {2011}, month = {2011 Jun}, pages = {630-41}, abstract = {BACKGROUND: The genus Mycobacterium includes a number of medically important pathogens. The cell walls of these bacteria have many unique features, including the abundance of various inositol lipids, such as phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). The biosynthesis of these lipids is believed to be prime drug targets, and has been clarified in detail over the past several years. SCOPE OF REVIEW: Here we summarize our current understanding of the inositol lipid metabolism in mycobacteria. We will highlight unsolved issues and future directions especially in the context of metabolic regulation. MAJOR CONCLUSIONS: Inositol is a building block of phosphatidylinositol (PI), which is further elaborated to become PIMs, LM and LAM. d-myo-inositol 3-phosphate is an intermediate of the de novo inositol synthesis, but it is also the starting substrate for mycothiol synthesis. Controlling the level of d-myo-inositol 3-phosphate appears to be important for maintaining the steady state levels of mycothiol and inositol lipids. Several additional control mechanisms must exist to control the complex biosynthetic pathways of PI, PIMs, LM and LAM. These may include regulatory proteins such as a lipoprotein LpqW, and spatial separation of enzymes, such as the amphipathic PimA mannosyltransferase and later enzymes in the PIMs/LM biosynthetic pathway. Finally, we discuss mechanisms that underlie control of LM/LAM glycan polymer elongation. GENERAL SIGNIFICANCE: Mycobacteria have evolved a complex network of inositol metabolism. Clarifying its metabolism will not only provide better understanding of bacterial pathogenesis, but also understanding of the evolution and general functions of inositol lipids in nature.}, keywords = {Inositol, Lipid Metabolism, Lipids, Models, Biological, Mycobacterium, Phosphatidylinositols}, issn = {0006-3002}, doi = {10.1016/j.bbagen.2011.03.017}, author = {Morita, Yasu S and Fukuda, Takeshi and Sena, Chubert B C and Yamaryo-Botte, Yoshiki and McConville, Malcolm J and Kinoshita, Taroh} } @article {33, title = {Multiple bHLH proteins regulate CIT2 expression in Saccharomyces cerevisiae.}, journal = {Yeast}, volume = {27}, year = {2010}, month = {2010 Jun}, pages = {345-59}, abstract = {

The basic helix-loop-helix (bHLH) proteins comprise a eukaryotic transcription factor family involved in multiple biological processes. They have the ability to form multiple dimer combinations and most of them also bind a 6 bp site (E-box) with limited specificity. These properties make them ideal for combinatorial regulation of gene expression. The Saccharomyces cerevisiae CIT2 gene, which encodes citrate synthase, was previously known to be induced by the bHLH proteins Rtg1p and Rtg3p in response to mitochondrial damage. Rtg1p-Rtg3p dimers bind two R-boxes (modified E-boxes) in the CIT2 promoter. The current study tested the ability of all nine S. cerevisiae bHLH proteins to regulate the CIT2 gene. The results showed that expression of CIT2-lacZ reporter was induced in a rho(0) strain by the presence of inositol via the Ino2p and Ino4p bHLH proteins, which are known regulators of phospholipid synthesis. Promoter mutations revealed that inositol induction required a distal E-box in the CIT2 promoter. Interestingly, deleting the INO2, INO4 genes or the cognate E-box revealed phosphate induction of CIT2 expression. This layer of expression required the two R-boxes and the Pho4p bHLH protein, which is known to be required for phosphate-specific regulation. Lastly, the data show that the Hms1p and Sgc1p bHLH proteins also play important roles in repression of CIT2-lacZ expression. Collectively, these results support the model that yeast bHLH proteins coordinate different biological pathways.

}, keywords = {Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, Basic Helix-Loop-Helix Transcription Factors, Citrate (si)-Synthase, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Fungal, Inositol, Regulatory Sequences, Nucleic Acid, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Species Specificity}, issn = {1097-0061}, doi = {10.1002/yea.1757}, author = {Chen, Linan and Lopes, John M} } @article {34, title = {Transcription regulation of the Saccharomyces cerevisiae PIS1 gene by inositol and the pleiotropic regulator, Ume6p.}, journal = {Mol Microbiol}, volume = {70}, year = {2008}, month = {2008 Dec}, pages = {1529-39}, abstract = {In Saccharomyces cerevisiae, transcription of most of the phospholipid biosynthetic genes (e.g. INO1, CHO1, CHO2 and OPI3) is repressed by growth in the presence of inositol and choline and derepressed in their absence. This regulation requires the Ino2p and Ino4p activators and the Opi1p repressor. The PIS1 structural gene is required for the synthesis of the essential lipid phosphatidylinositol. Previous reports show that PIS1 expression is uncoupled from inositol/choline regulation, but is regulated by carbon source, hypoxia and zinc. However, in this study we found that the expression of PIS1 is induced twofold by inositol. This regulation did not require Ino2p and Ino4p, although Ino4p was required for full expression. Ino4p is a basic helix-loop-helix protein that requires a binding partner. Curiously, none of the other basic helix-loop-helix proteins affected PIS1 expression. Inositol induction did require another general regulator of phospholipid biosynthesis, Ume6p. Ume6p was found to be a positive regulator of PIS1 gene expression. Ume6p, and several associated factors, were required for inositol-mediated induction and chromatin immunoprecipitation analysis showed that Ume6p directly regulates PIS1 expression. Thus, we demonstrate novel regulation of the PIS1 gene by Ume6p.}, keywords = {Chromatin Immunoprecipitation, DNA-Binding Proteins, Gene Expression Regulation, Fungal, Histone Deacetylases, Inositol, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Transferases (Other Substituted Phosphate Groups)}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2008.06506.x}, author = {Jani, Niketa M and Lopes, John M} } @article {37, title = {Genomic analysis of the Opi- phenotype.}, journal = {Genetics}, volume = {173}, year = {2006}, month = {2006 Jun}, pages = {621-34}, abstract = {Most of the phospholipid biosynthetic genes of Saccharomyces cerevisiae are coordinately regulated in response to inositol and choline. Inositol affects the intracellular levels of phosphatidic acid (PA). Opi1p is a repressor of the phospholipid biosynthetic genes and specifically binds PA in the endoplasmic reticulum. In the presence of inositol, PA levels decrease, releasing Opi1p into the nucleus where it represses transcription. The opi1 mutant overproduces and excretes inositol into the growth medium in the absence of inositol and choline (Opi(-) phenotype). To better understand the mechanism of Opi1p repression, the viable yeast deletion set was screened to identify Opi(-) mutants. In total, 89 Opi(-) mutants were identified, of which 7 were previously known to have the Opi(-) phenotype. The Opi(-) mutant collection included genes with roles in phospholipid biosynthesis, transcription, protein processing/synthesis, and protein trafficking. Included in this set were all nonessential components of the NuA4 HAT complex and six proteins in the Rpd3p-Sin3p HDAC complex. It has previously been shown that defects in phosphatidylcholine synthesis (cho2 and opi3) yield the Opi(-) phenotype because of a buildup of PA. However, in this case the Opi(-) phenotype is conditional because PA can be shuttled through a salvage pathway (Kennedy pathway) by adding choline to the growth medium. Seven new mutants present in the Opi(-) collection (fun26, kex1, nup84, tps1, mrpl38, mrpl49, and opi10/yol032w) were also suppressed by choline, suggesting that these affect PC synthesis. Regulation in response to inositol is also coordinated with the unfolded protein response (UPR). Consistent with this, several Opi(-) mutants were found to affect the UPR (yhi9, ede1, and vps74).}, keywords = {Gene Expression Regulation, Fungal, Genes, Fungal, Genes, Reporter, Genome, Fungal, Genomics, Inositol, Lac Operon, Models, Biological, Mutation, Phenotype, Phospholipids, Protein Folding, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {0016-6731}, doi = {10.1534/genetics.106.057489}, author = {Hancock, Leandria C and Behta, Ryan P and Lopes, John M} } @article {693, title = {Removal or maintenance of inositol-linked acyl chain in glycosylphosphatidylinositol is critical in trypanosome life cycle.}, journal = {J Biol Chem}, volume = {281}, year = {2006}, month = {2006 Apr 28}, pages = {11595-602}, abstract = {The protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol (GPI)-anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins in the bloodstream form, whereas it remains acylated in procyclins in the procyclic form. We have cloned a T. brucei GPI inositol deacylase (GPIdeAc2). In accordance with the acylation/deacylation profile, the level of GPIdeAc2 mRNA was 6-fold higher in the bloodstream form than in the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating that inositol-deacylation is essential for the growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI biosynthetic intermediates lacking inositol-linked acyl chain and decreased cell surface procyclins because of release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. Therefore, the GPI inositol deacylase activity must be tightly regulated in trypanosome life cycle.}, keywords = {Acylation, Animals, Cloning, Molecular, Glycosylphosphatidylinositols, Inositol, Life Cycle Stages, Membrane Glycoproteins, Phosphoric Monoester Hydrolases, Protozoan Proteins, RNA, Messenger, Trypanosoma brucei brucei}, issn = {0021-9258}, doi = {10.1074/jbc.M513061200}, author = {Hong, Yeonchul and Nagamune, Kisaburo and Morita, Yasu S and Nakatani, Fumiki and Ashida, Hisashi and Maeda, Yusuke and Kinoshita, Taroh} } @article {697, title = {Function of phosphatidylinositol in mycobacteria.}, journal = {J Biol Chem}, volume = {280}, year = {2005}, month = {2005 Mar 25}, pages = {10981-7}, abstract = {Phosphatidylinositol (PI) is an abundant phospholipid in the cytoplasmic membrane of mycobacteria and the precursor for more complex glycolipids, such as the PI mannosides (PIMs) and lipoarabinomannan (LAM). To investigate whether the large steady-state pools of PI and apolar PIMs are required for mycobacterial growth, we have generated a Mycobacterium smegmatis inositol auxotroph by disruption of the ino1 gene. The ino1 mutant displayed wild-type growth rates and steady-state levels of PI, PIM, and LAM when grown in the presence of 1 mM inositol. The non-dividing ino1 mutant was highly resistant to inositol starvation, reflecting the slow turnover of inositol lipids in this stage. In contrast, dilution of growing or stationary-phase ino1 mutant in inositol-free medium resulted in the rapid depletion of PI and apolar PIMs. Whereas depletion of these lipids was not associated with loss of viability, subsequent depletion of polar PIMs coincided with loss of major cell wall components and cell viability. Metabolic labeling experiments confirmed that the large pools of PI and apolar PIMs were used to sustain polar PIM and LAM biosynthesis during inositol limitation. They also showed that under non-limiting conditions, PI is catabolized via lyso-PI. These data suggest that large pools of PI and apolar PIMs are not essential for membrane integrity but are required to sustain polar PIM biosynthesis, which is essential for mycobacterial growth.}, keywords = {Inositol, Lipopolysaccharides, Mannose, Mycobacterium smegmatis, Phosphatidylinositols}, issn = {0021-9258}, doi = {10.1074/jbc.M413443200}, author = {Haites, Ruth E and Morita, Yasu S and McConville, Malcolm J and Billman-Jacobe, Helen} } @article {41, title = {Identification of novel dominant INO2c mutants with an Opi- phenotype.}, journal = {Mol Microbiol}, volume = {52}, year = {2004}, month = {2004 Jun}, pages = {1271-80}, abstract = {The INO2 gene of Saccharomyces cerevisiae is required for derepression of the phospholipid biosynthetic genes in response to inositol depletion. Conversely, the OPI1 gene is required for repression in response to inositol supplementation. Results of an in vitro assay have led to a model in which Opi1p interacts with Ino2p. However, there is no in vivo evidence to support this model. Additionally, most of the previously isolated ino2 mutants offer little insight into this model. Here, we report the isolation of a new class of dominant mutations in the INO2 gene, which yield constitutive expression of a target gene (i.e. an Opi(-) mutant phenotype). Two mutations reside in a region of the Ino2p required for interaction with Opi1p in vitro. Three other mutations are at the amino-terminus in a transcriptional activation domain.}, keywords = {Basic Helix-Loop-Helix Transcription Factors, Gene Expression Regulation, Fungal, Inositol, Mutation, Phenotype, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors}, issn = {0950-382X}, doi = {10.1111/j.1365-2958.2004.04069.x}, author = {Gardenour, Kyle R and Levy, Jamie and Lopes, John M} } @article {42, title = {Expression of the yeast PIS1 gene requires multiple regulatory elements including a Rox1p binding site.}, journal = {J Biol Chem}, volume = {278}, year = {2003}, month = {2003 Oct 3}, pages = {38646-52}, abstract = {The PIS1 gene is required for de novo synthesis of phosphatidylinositol (PI), an essential phospholipid in Saccharomyces cerevisiae. PIS1 gene expression is unusual because it is uncoupled from the other phospholipid biosynthetic genes, which are regulated in response to inositol and choline. Relatively little is known about regulation of transcription of the PIS1 gene. We reported previously that PIS1 transcription is sensitive to carbon source. To further our understanding of the regulation of PIS1 transcription, we carried out a promoter deletion analysis that identified three regions required for PIS1 gene expression (upstream activating sequence (UAS) elements 1-3). Deletion of either UAS1 or UAS2 resulted in an approximately 45\% reduction in expression, whereas removal of UAS3 yielded an 84\% decrease in expression. A comparison of promoters among several Saccharomyces species shows that these sequences are highly conserved. Curiously, the UAS3 element region (-149 to -138) includes a Rox1p binding site. Rox1p is a repressor of hypoxic genes under aerobic growth conditions. Consistent with this, we have found that expression of a PIS1-cat reporter was repressed under aerobic conditions, and this repression was dependent on both Rox1p and its binding site. Furthermore, PI levels were elevated under anaerobic conditions. This is the first evidence that PI levels are affected by regulation of PIS1 transcription.}, keywords = {Anoxia, Base Sequence, Binding Sites, Carbon, Chloramphenicol O-Acetyltransferase, Choline, Chromatography, Thin Layer, Conserved Sequence, DNA, DNA, Complementary, DNA-Binding Proteins, Gene Deletion, Gene Expression Regulation, Fungal, Genes, Reporter, Inositol, Lipid Metabolism, Models, Biological, Models, Genetic, Molecular Sequence Data, Oxygen, Phospholipids, Plasmids, Promoter Regions, Genetic, Protein Binding, Repressor Proteins, RNA, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Transferases (Other Substituted Phosphate Groups)}, issn = {0021-9258}, doi = {10.1074/jbc.M305251200}, author = {Gardocki, Mary Elizabeth and Lopes, John M} }