REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae.

Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae. The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase. Previously, we characterized a mutation, glc7‐T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism. We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism. Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit. We show that REG1 is physically associated with GLC7. REG1 interacts with GLC7 strongly and specifically in the two‐hybrid system, and REG1 and GLC7 fusion proteins co‐immunoprecipitate from cell extracts. Moreover, overexpression of a REG1 fusion protein suppresses the glc7‐T152K mutant defect in glucose repression. This and other genetic evidence indicate that the two proteins function together in regulating glucose repression. These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway.

[1]  M. Yanagida,et al.  Mitotic regulation of protein phosphatases by the fission yeast sds22 protein , 1993, Current Biology.

[2]  M. Yanagida,et al.  Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle , 1990, Cell.

[3]  K. Matsumoto,et al.  Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae , 1983, Journal of bacteriology.

[4]  P. Cohen,et al.  On target with a new mechanism for the regulation of protein phosphorylation. , 1993, Trends in biochemical sciences.

[5]  Gerald R. Fink,et al.  Methods in Yeast Genetics: A Laboratory Course Manual , 1987 .

[6]  P. Cohen The structure and regulation of protein phosphatases. , 1989, Annual review of biochemistry.

[7]  J. Jaehning,et al.  Glucose repression of yeast mitochondrial transcription: kinetics of derepression and role of nuclear genes , 1994, Molecular and cellular biology.

[8]  T. Hunter A thousand and one protein kinases , 1987, Cell.

[9]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[10]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .

[11]  M. Yanagida,et al.  S. pombe gene sds22 + essential for a midmitotic transition encodes a leucine-rich repeat protein that positively modulates protein phosphatase-1 , 1991, Cell.

[12]  J. Doonan,et al.  The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1 , 1989, Cell.

[13]  S. Fields,et al.  The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Clarence S. M. Chan,et al.  Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation , 1994, Molecular and cellular biology.

[15]  H. Ronne,et al.  Yeast MIG1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. , 1990, The EMBO journal.

[16]  S. Elledge,et al.  The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. , 1993, Genes & development.

[17]  M. Stark,et al.  The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase , 1995, Molecular and cellular biology.

[18]  M. Carlson,et al.  A yeast gene that is essential for release from glucose repression encodes a protein kinase. , 1986, Science.

[19]  M. Carlson,et al.  Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations. , 1987, Genetics.

[20]  K. Matsumoto,et al.  The Glc7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M , 1994, Molecular and cellular biology.

[21]  Fred Winston,et al.  Methods in Yeast Genetics: A Laboratory Course Manual , 1990 .

[22]  Alexander D. Johnson,et al.  Ssn6-Tup1 is a general repressor of transcription in yeast , 1992, Cell.

[23]  Mark Johnston,et al.  5 Regulation of Carbon and Phosphate Utilization , 1992 .

[24]  K. Matsumoto,et al.  The EGP1 gene may be a positive regulator of protein phosphatase type 1 in the growth control of Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[25]  M. Carlson,et al.  Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Carlson,et al.  A protein kinase substrate identified by the two-hybrid system. , 1992, Science.

[27]  J. E. Mermoud,et al.  Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. , 1994, The EMBO journal.

[28]  M. Johnston,et al.  Genetic and molecular characterization of GAL83: its interaction and similarities with other genes involved in glucose repression in Saccharomyces cerevisiae. , 1993, Genetics.

[29]  R. Trumbly,et al.  The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase. , 1991, The Journal of biological chemistry.

[30]  K. Tatchell,et al.  The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit , 1994, Molecular and cellular biology.

[31]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[32]  Yi Li,et al.  Generating yeast transcriptional activators containing no yeast protein sequences , 1991, Nature.

[33]  M. Carlson,et al.  A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex. , 1994, The EMBO journal.

[34]  F. Bischoff,et al.  Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[35]  P. Greengard,et al.  Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Johnston,et al.  Three different regulatory mechanisms enable yeast hexose transporter (HXT) genes to be induced by different levels of glucose , 1995, Molecular and cellular biology.

[37]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[38]  K. Entian,et al.  Characterization of Hex2 protein, a negative regulatory element necessary for glucose repression in yeast. , 1991, European journal of biochemistry.

[39]  R. Rothstein One-step gene disruption in yeast. , 1983, Methods in enzymology.

[40]  A. Hopper,et al.  SRN1, a yeast gene involved in RNA processing, is identical to HEX2/REG1, a negative regulator in glucose repression , 1992, Molecular and cellular biology.

[41]  Jeremy Luban,et al.  Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B , 1993, Cell.

[42]  K. Arndt,et al.  The SIT4 protein phosphatase is required in late G1 for progression into S phase. , 1991, Cold Spring Harbor symposia on quantitative biology.

[43]  D. Beach,et al.  Involvement of a type 1 protein phosphatase encoded by bws1 + in fission yeast mitotic control , 1989, Cell.

[44]  V. Chester Heritable glycogen-storage deficiency in yeast and its induction by ultra-violet light. , 1968, Journal of general microbiology.

[45]  P. Cohen,et al.  Signal integration at the level of protein kinases, protein phosphatases and their substrates. , 1992, Trends in biochemical sciences.

[46]  D. Glover,et al.  One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis , 1990, Cell.

[47]  J. Pringle,et al.  Characterization of glycogen-deficient glc mutants of Saccharomyces cerevisiae. , 1994, Genetics.

[48]  A. Hinnebusch,et al.  Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2 , 1992, Molecular and cellular biology.