Phosphatidylinositol 4-kinase: Gene structure and requirement for yeast cell viability
Department of Molecular and Cell Biology, University of California, Berkeley 94720. Science
(Impact Factor: 33.61).
12/1993; 262(5138):1444-8. DOI: 10.1126/science.8248783
Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first step in the biosynthesis of PtdIns-4,5-bisphosphate (PtdIns[4,5]P2).
Hydrolysis of PtdIns[4,5]P2 in response to extracellular stimuli is thought to initiate intracellular signaling cascades that
modulate cell proliferation and differentiation. The PIK1 gene encoding a PtdIns 4-kinase from the yeast Saccharomyces cerevisiae
was isolated by polymerase chain reaction (PCR) with oligonucleotides based on the sequence of peptides derived from the purified
enzyme. The sequence of the PIK1 gene product bears similarities to that of PtdIns 3-kinases from mammals (p110) and yeast
(Vps34p). Expression of PIK1 from a multicopy plasmid elevated PtdIns 4-kinase activity and enhanced the response to mating
pheromone. A pik1 null mutant was inviable, indicating that PtdIns4P and presumably PtdIns[4,5]P2 are indispensable phospholipids.
Available from: Isaac Corcoles-Saez
- "Likewise, ectopic expression of MSS4, the PI(4)P 5-kinase-encoding gene, stimulated the growth of wild-type yeast cells at 15 °C (Fig. 2B). However , only a subtle improvement on cold growth could be observed by a high-copy number expression of PIK1, a PI 4-kinase, with nuclear and Golgi localization, whereas overexpression of LSB6 impaired growth at both 30 and 15 °C (Fig. 2B). Unlike Pik1 and Stt4, Lsb6, the third PI 4-kinase in S. cerevisiae, which is found at the plasma and vacuolar membranes, is non-essential and its contribution to total cellular PI(4)P appears to be scarce[3,116]. "
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ABSTRACT: Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its derivatives diphosphoinositol phosphates (DPIPs) play key signaling and regulatory roles. However, a direct function of these molecules in lipid and membrane homeostasis remains obscure. Here, we have studied the cold tolerance phenotype of yeast cells lacking the Inp51-mediated phosphoinositide-5-phosphatase. Genetic and biochemical approaches showed that increased metabolism of PI(4,5)P2 reduces the activity of the Pho85 kinase by increasing the levels of the DPIP isomer 1-IP7. This effect was key in the cold tolerance phenotype. Indeed, pho85 mutant cells grew better than the wild-type at 15 °C, and lack of this kinase abolished the inp51-mediated cold phenotype. Remarkably, reduced Pho85 function by loss of Inp51 affected the activity of the Pho85-regulated target Pah1, the yeast phosphatidate phosphatase. Cells lacking Inp51 showed reduced Pah1 abundance, derepression of an INO1-lacZ reporter, decreased content of triacylglycerides and elevated levels of phosphatidate, hallmarks of the pah1 mutant. However, the inp51 phenotype was not associated to low Pah1 activity since deletion of PAH1 caused cold sensitivity. In addition, the inp51 mutant exhibited features not shared by pah1, including a 40%-reduction in total lipid content and decreased membrane fluidity. These changes may influence the activity of membrane-anchored and/or associated proteins since deletion of INP51 slows down the transit to the vacuole of the fluorescent dye FM4-64. In conclusion, our work supports a model in which changes in the PI(4,5)P2 pool affect the 1-IP7 levels modulating the activity of Pho85, Pah1 and likely additional Pho85-controlled targets, and regulate lipid composition and membrane properties.
Available from: George M Carman
- "The inositol used in PI synthesis is either synthesized de novo (discussed below) or obtained from the growth medium via the ITR1-and ITR2-encoded inositol transporters (Table 2) (Nikawa et al. 1991). Once formed, PI may be converted to PI 3-P by the VPS34-encoded PI 3 kinase (Herman and Emr 1990; Schu et al. 1993) or to PI 4-P by the PI 4 kinases encoded by LSB6 (Han G-S et al. 2002; Shelton et al. 2003), STT4 (Yoshida et al. 1994a), and PIK1 (Flanagan et al. 1993; Garcia-Bustos et al. 1994). PI 4-P may be further phosphorylated to PI 4,5-P 2 by the MSS4- encoded PI 4-P 5 kinase (Yoshida et al. 1994b), whereas PI 3-P may be phosphorylated to PI 3,5-P 2 by the FAB1- encoded PI 3-P 5 kinase (Yamamoto et al. 1995). "
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ABSTRACT: Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces cerevisiae is a model system of choice for the study of the genetics, biochemistry, and cell biology of eukaryotic lipid metabolism. Glycerolipids (e.g., phospholipids and triacylglycerol) and their precursors are synthesized and metabolized by enzymes associated with the cytosol and membranous organelles, including endoplasmic reticulum, mitochondria, and lipid droplets. Genetic and biochemical analyses have revealed that glycerolipids play important roles in cell signaling, membrane trafficking, and anchoring of membrane proteins in addition to membrane structure. The expression of glycerolipid enzymes is controlled by a variety of conditions including growth stage and nutrient availability. Much of this regulation occurs at the transcriptional level and involves the Ino2-Ino4 activation complex and the Opi1 repressor, which interacts with Ino2 to attenuate transcriptional activation of UAS(INO)-containing glycerolipid biosynthetic genes. Cellular levels of phosphatidic acid, precursor to all membrane phospholipids and the storage lipid triacylglycerol, regulates transcription of UAS(INO)-containing genes by tethering Opi1 to the nuclear/endoplasmic reticulum membrane and controlling its translocation into the nucleus, a mechanism largely controlled by inositol availability. The transcriptional activator Zap1 controls the expression of some phospholipid synthesis genes in response to zinc availability. Regulatory mechanisms also include control of catalytic activity of glycerolipid enzymes by water-soluble precursors, products and lipids, and covalent modification of phosphorylation, while in vivo function of some enzymes is governed by their subcellular location. Genome-wide genetic analysis indicates coordinate regulation between glycerolipid metabolism and a broad spectrum of metabolic pathways.
Available from: Kerry Kornfeld
- "This nonconservative missense change substitutes a basic amino acid for an acidic residue. Furthermore, Glu 725 is a highly conserved residue in the PIK domain of the AGE-1 protein, which is a homolog of the vertebrate PI3 kinase (Flanagan et al. 1993; Morris et al. 1996; Domin and Waterfield 1997) (Figure 3). This mutation segregated with the lifespan extension phenotype after four rounds of backcrossing (data not shown), supporting the conclusion that the missense change resulting in the E725K substitution is the am88 mutation, and am88 is a novel allele of age-1. "
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ABSTRACT: Aging is an important feature of animal biology characterized by progressive, degenerative changes in somatic and reproductive tissues. The rate of age-related degeneration is genetically controlled, since genes that influence lifespan have been identified. However, little is known about genes that affect reproductive aging or aging of specific somatic tissues. To identify genes that are important for controlling these degenerative changes, we used chemical mutagenesis to perform forward genetic screens in Caenorhabditis elegans. By conducting a screen focused on somatic aging, we identified mutant hermaphrodites that displayed extended periods of pharyngeal pumping, body movement, or survival. One of these mutations is a novel allele of the age-1 gene. age-1 encodes a phosphatidylinositol-3-kinase (PI3K) that functions in the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway. age-1(am88) creates a missense change in the conserved PIK domain and causes dramatic extensions of the pharyngeal pumping and body movement spans, as well as a twofold extension of the lifespan. By conducting screens focused on reproductive aging in mated hermaphrodites, we identified mutants that displayed increased progeny production late in life. To characterize these mutations, we developed quantitative measurements of age-related morphological changes in the gonad. The am117 mutation delayed age-related declines in progeny production and morphological changes in the gonad. These studies provide new insights into the genetic regulation of age-related degenerative changes in somatic and reproductive tissues.
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