Home LiteratureArticle Details
PMID: 8395015 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.

Molecular and cellular biology ·Vol. 13 ·No. 9 ·1993-09-00 ·Pages 5861-76

Flick JS, Thorner J

Abstract

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.

Related Genes
PLC1
MeSH Terms
Amino Acid Sequence Base Sequence Cell Division Cloning, Molecular DNA Mutational Analysis Gene Expression Genes, Fungal Molecular Sequence Data Oligodeoxyribonucleotides/chemistry Phosphatidylinositol Diacylglycerol-Lyase Phosphoinositide Phospholipase C Phosphoric Diester Hydrolases/genetics,metabolism RNA, Messenger/genetics Restriction Mapping Saccharomyces cerevisiae/enzymology,genetics Sequence Alignment Sequence Deletion Structure-Activity Relationship Type C Phospholipases/genetics,metabolism
Chemicals
Oligodeoxyribonucleotides RNA, Messenger Phosphoric Diester Hydrolases Type C Phospholipases Phosphoinositide Phospholipase C Phosphatidylinositol Diacylglycerol-Lyase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Flick J S
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Thorner J
References (68)
68 references, click to expand
  1. Separation of large DNA molecules by contour-clamped homogeneous electric fields.
    Science. 1986 Dec 19;234(4783):1582-5 PMID: 3538420
  2. Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.
    Mol Cell Biol. 1987 May;7(5):1906-16 PMID: 3037338
  3. Purification and characterization of two immunologically distinct phosphoinositide-specific phospholipases C from bovine brain.
    J Biol Chem. 1987 Sep 15;262(26):12511-8 PMID: 3040753
  4. Bovine brain cytosol contains three immunologically distinct forms of inositolphospholipid-specific phospholipase C.
    Proc Natl Acad Sci U S A. 1987 Oct;84(19):6649-53 PMID: 3477795
  5. Identification of critical regions on phospholipase C-beta 1 required for activation by G-proteins.
    J Biol Chem. 1993 Feb 15;268(5):3704-9 PMID: 8381437
  6. The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth.
    Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1804-8 PMID: 8383328
  7. An osmosensing signal transduction pathway in yeast.
    Science. 1993 Mar 19;259(5102):1760-3 PMID: 7681220
  8. A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation.
    Mol Cell Biol. 1993 Jul;13(7):4351-64 PMID: 8391635
  9. Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae.
    Cell. 1991 Aug 9;66(3):497-505 PMID: 1868547
  10. Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase.
    Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7376-80 PMID: 1651503
  11. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier.
    Yeast. 1991 Apr;7(3):253-63 PMID: 1882550
  12. GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats.
    Mol Cell Biol. 1991 Oct;11(10):5101-12 PMID: 1922034
  13. Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression.
    Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8597-601 PMID: 1924319
  14. The occurrence of diphosphoinositide and triphosphoinositide in Saccharomyces cerevisiae.
    J Biol Chem. 1968 Sep 25;243(18):4889-93 PMID: 4301226
  15. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  16. Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells.
    J Physiol. 1973 Dec;235(2):551-69 PMID: 4271735
  17. Identification and properties of two distinct phosphatidylinositol-specific phospholipase C enzymes from sheep seminal vesicular glands.
    J Biol Chem. 1982 Jun 10;257(11):6461-9 PMID: 7076678
  18. RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA.
    Proc Natl Acad Sci U S A. 1983 May;80(9):2432-6 PMID: 6189122
  19. One-step gene disruption in yeast.
    Methods Enzymol. 1983;101:202-11 PMID: 6310324
  20. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.
    Gene. 1984 Jun;28(3):351-9 PMID: 6235151
  21. Hydrolysis of polyphosphoinositides by purified sheep seminal vesicle phospholipase C enzymes.
    J Biol Chem. 1984 Oct 10;259(19):11718-24 PMID: 6090445
  22. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.
    Mol Cell Biol. 1984 Aug;4(8):1440-8 PMID: 6092912
  23. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA.
    DNA. 1985 Apr;4(2):165-70 PMID: 3996185
  24. Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae.
    Mol Cell Biol. 1988 Jan;8(1):210-25 PMID: 3275867
  25. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
    Science. 1988 Jan 29;239(4839):487-91 PMID: 2448875
  26. Sequence similarity of phospholipase C with the non-catalytic region of src.
    Nature. 1988 Mar 17;332(6161):269-72 PMID: 2831461
  27. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector.
    Gene. 1987;60(2-3):237-43 PMID: 3327750
  28. Improved tools for biological sequence comparison.
    Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8 PMID: 3162770
  29. Saturation mutagenesis of a yeast his3 "TATA element": genetic evidence for a specific TATA-binding protein.
    Proc Natl Acad Sci U S A. 1988 Apr;85(8):2691-5 PMID: 3282236
  30. Cloning and sequence of multiple forms of phospholipase C.
    Cell. 1988 Jul 15;54(2):161-9 PMID: 3390863
  31. Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities.
    Cell. 1988 Jul 15;54(2):171-7 PMID: 2455601
  32. Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products.
    Proc Natl Acad Sci U S A. 1988 Aug;85(15):5419-23 PMID: 2840660
  33. Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction.
    Cell. 1988 Aug 26;54(5):723-33 PMID: 2457447
  34. Yeast/E. coli shuttle vectors with multiple unique restriction sites.
    Yeast. 1986 Sep;2(3):163-7 PMID: 3333305
  35. Elevated recombination rates in transcriptionally active DNA.
    Cell. 1989 Feb 24;56(4):619-30 PMID: 2645056
  36. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
    Genetics. 1989 May;122(1):19-27 PMID: 2659436
  37. A novel inositol-phospholipid-specific phospholipase C. Rapid purification and characterization.
    Eur J Biochem. 1989 Jul 1;182(3):673-7 PMID: 2753038
  38. A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity.
    J Biol Chem. 1989 Dec 25;264(36):21885-90 PMID: 2557343
  39. A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle.
    Cell. 1990 Jul 27;62(2):213-24 PMID: 2196995
  40. Phospholipase C isozymes: structural and functional similarities.
    Ciba Found Symp. 1990;150:112-23; discussion 124-7 PMID: 2373024
  41. Essential role for induced Ca2+ influx followed by [Ca2+]i rise in maintaining viability of yeast cells late in the mating pheromone response pathway. A study of [Ca2+]i in single Saccharomyces cerevisiae cells with imaging of fura-2.
    J Biol Chem. 1990 Aug 5;265(22):13391-9 PMID: 2198292
  42. Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae.
    Mol Cell Biol. 1990 Sep;10(9):4757-69 PMID: 2201902
  43. EF-hand motifs in inositol phospholipid-specific phospholipase C.
    FEBS Lett. 1990 Sep 3;269(2):454-6 PMID: 2401372
  44. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers.
    Yeast. 1990 Sep-Oct;6(5):363-6 PMID: 2220072
  45. A second gene product of the inositol-phospholipid-specific phospholipase C delta subclass.
    Eur J Biochem. 1991 Feb 26;196(1):159-65 PMID: 1848183
  46. Tackling the protease problem in Saccharomyces cerevisiae.
    Methods Enzymol. 1991;194:428-53 PMID: 2005802
  47. Immunofluorescence methods for yeast.
    Methods Enzymol. 1991;194:565-602 PMID: 2005809
  48. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation.
    Science. 1991 Mar 8;251(4998):1231-3 PMID: 1848725
  49. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.
    Science. 1991 May 3;252(5006):668-74 PMID: 1708916
  50. Multiple Ca2+/calmodulin-dependent protein kinase genes in a unicellular eukaryote.
    EMBO J. 1991 Jun;10(6):1511-22 PMID: 2026147
  51. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.
    Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8972-6 PMID: 1924358
  52. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect.
    J Cell Biol. 1992 Mar;116(5):1221-9 PMID: 1740473
  53. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast.
    FEBS Lett. 1992 Feb 24;298(2-3):149-53 PMID: 1544438
  54. Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone.
    Mol Cell Biol. 1992 Aug;12(8):3460-9 PMID: 1321337
  55. CDC25-dependent induction of inositol 1,4,5-trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen.
    FEBS Lett. 1992 Aug 3;307(3):249-52 PMID: 1322832
  56. Cloning, sequencing, expression, and Gq-independent activation of phospholipase C-beta 2.
    J Biol Chem. 1992 Aug 15;267(23):16048-55 PMID: 1644792
  57. Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis.
    Nature. 1992 Aug 20;358(6388):678-81 PMID: 1379697
  58. Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis.
    Nature. 1992 Aug 20;358(6388):681-4 PMID: 1379698
  59. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.
    Science. 1992 Oct 23;258(5082):607-14 PMID: 1411571
  60. Multiple forms of phospholipase C isozymes and their activation mechanisms.
    Adv Second Messenger Phosphoprotein Res. 1992;26:35-61 PMID: 1419362
  61. Purification and characterization of a soluble phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae.
    J Biol Chem. 1992 Nov 25;267(33):24117-25 PMID: 1331109
  62. Inositol trisphosphate and calcium signalling.
    Nature. 1993 Jan 28;361(6410):315-25 PMID: 8381210
  63. Analysis of inositol metabolites produced by Saccharomyces cerevisiae in response to glucose stimulation.
    J Biol Chem. 1993 Feb 15;268(5):3374-83 PMID: 8429013
  64. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
    Mol Cell Biol. 1985 Dec;5(12):3610-6 PMID: 3915782
  65. Random-clone strategy for genomic restriction mapping in yeast.
    Proc Natl Acad Sci U S A. 1986 Oct;83(20):7826-30 PMID: 3463999
  66. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis.
    Science. 1986 Oct 17;234(4774):364-8 PMID: 2876518
  67. Isolation of the yeast calmodulin gene: calmodulin is an essential protein.
    Cell. 1986 Nov 7;47(3):423-31 PMID: 3533275
  68. Possible involvement of RAS-encoded proteins in glucose-induced inositolphospholipid turnover in Saccharomyces cerevisiae.
    Proc Natl Acad Sci U S A. 1986 Nov;83(21):8172-6 PMID: 3022283
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.13.9.5861-5876.1993
Published
1993-09-00
Pages
5861-76
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC360334
Subset
IM
Grants
NIGMS NIH HHS · GM21841 · United States
Databases
GENBANK
L13036
External Links
PubMed source DOI: 10.1128/mcb.13.9.5861-5876.1993 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2