Genomewide Screen Reveals a Wide Regulatory Network for Di/Tripeptide Utilization in Saccharomyces cerevisiae

Small peptides of two to six residues serve as important sources of amino acids and nitrogen required for growth by a variety of organisms. In the yeast Saccharomyces cerevisiae, the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tripeptides. To identify genes involved in regulation of dipeptide utilization, we performed a systematic, functional examination of this process in a haploid, nonessential, single-gene deletion mutant library. We have identified 103 candidate genes: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. On the basis of Ptr2p-GFP expression studies, together with PTR2 expression analysis and dipeptide uptake assays, 42 genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes did not apparently affect Ptr2p-GFP expression or localization. The 103 genes regulating dipeptide utilization were distributed among most of the Gene Ontology functional categories, indicating a very wide regulatory network involved in transport and utilization of dipeptides in yeast. It is anticipated that further characterization of how these genes affect peptide utilization should add new insights into the global mechanisms of regulation of transport systems in general and peptide utilization in particular.

[1]  D. Wolf,et al.  Proteinase yscD (oligopeptidase yscD). Structure, function and relationship of the yeast enzyme with mammalian thimet oligopeptidase (metalloendopeptidase, EP 24.15). , 1994, European journal of biochemistry.

[2]  P. Ljungdahl,et al.  Genetic and Biochemical Analysis of the Yeast Plasma Membrane Ssy1p-Ptr3p-Ssy5p Sensor of Extracellular Amino Acids , 2001, Molecular and Cellular Biology.

[3]  A. Varshavsky,et al.  The E2–E3 interaction in the N‐end rule pathway: the RING‐H2 finger of E3 is required for the synthesis of multiubiquitin chain , 1999, The EMBO journal.

[4]  Angelika Amon,et al.  A Genome-Wide Screen Identifies Genes Required for Centromeric Cohesion , 2004, Science.

[5]  Ronald W. Davis,et al.  Systematic screen for human disease genes in yeast , 2002, Nature Genetics.

[6]  T. Stevens,et al.  Protein–Protein Interactions of ESCRT Complexes in the Yeast Saccharomyces cerevisiae , 2004, Traffic.

[7]  H. Dohlman,et al.  Analysis of RGS proteins in Saccharomyces cerevisiae. , 2002, Methods in enzymology.

[8]  G. Marzluf,et al.  Genetic regulation of nitrogen metabolism in the fungi , 1997, Microbiology and molecular biology reviews : MMBR.

[9]  M. Peter,et al.  A Genome-Wide Screen in Saccharomyces cerevisiae Reveals Altered Transport As a Mechanism of Resistance to the Anticancer Drug Bleomycin , 2004, Cancer Research.

[10]  Carl Wu,et al.  Involvement of actin-related proteins in ATP-dependent chromatin remodeling. , 2003, Molecular cell.

[11]  J. Becker,et al.  Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene , 1994, Molecular and cellular biology.

[12]  R. Planta,et al.  Transcriptional regulation of the Saccharomyces cerevisiae amino acid permease gene BAP2 , 2001, Molecular and General Genetics MGG.

[13]  M. Carlson,et al.  A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II holoenzyme. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Andrew J. Link,et al.  Proteomics of the Eukaryotic Transcription Machinery: Identification of Proteins Associated with Components of Yeast TFIID by Multidimensional Mass Spectrometry , 2002, Molecular and Cellular Biology.

[15]  P. Ljungdahl,et al.  The role of the yeast plasma membrane SPS nutrient sensor in the metabolic response to extracellular amino acids , 2001, Molecular microbiology.

[16]  J. Becker,et al.  The PTR family: a new group of peptide transporters , 1995, Molecular microbiology.

[17]  M. Gustin,et al.  MAP Kinase Pathways in the YeastSaccharomyces cerevisiae , 1998, Microbiology and Molecular Biology Reviews.

[18]  T. Baker,et al.  On the Unique Structural Organization of the Saccharomyces cerevisiae Pyruvate Dehydrogenase Complex* , 1997, The Journal of Biological Chemistry.

[19]  H. Stenmark,et al.  Protein sorting into multivesicular endosomes. , 2003, Current opinion in cell biology.

[20]  V. Lee.  Membrane transporters. , 2000, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[21]  Hannelore Daniel,et al.  Molecular and integrative physiology of intestinal peptide transport. , 2004, Annual review of physiology.

[22]  T. Miyake,et al.  A novel cis-acting cysteine-responsive regulatory element of the gene for the high-affinity glutathione transporter of Saccharomyces cerevisiae , 2002, Molecular Genetics and Genomics.

[23]  B. Lapeyre,et al.  The Two Proteins Pat1p (Mrt1p) and Spb8p Interact In Vivo, Are Required for mRNA Decay, and Are Functionally Linked to Pab1p , 2000, Molecular and Cellular Biology.

[24]  A. Varshavsky,et al.  Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[25]  H. Bussey,et al.  A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin. , 2003, Genetics.

[26]  Bertrand Séraphin,et al.  A Sm‐like protein complex that participates in mRNA degradation , 2000, The EMBO journal.

[27]  R. Young,et al.  RNA Polymerase II Holoenzymes and Subcomplexes* , 1998, The Journal of Biological Chemistry.

[28]  M. Romero,et al.  Expression cloning of a mammalian proton-coupled oligopeptide transporter , 1994, Nature.

[29]  P. Ljungdahl,et al.  The N-Terminal Regulatory Domain of Stp1p Is Modular and, Fused to an Artificial Transcription Factor, Confers Full Ssy1p-Ptr3p-Ssy5p Sensor Control , 2004, Molecular and Cellular Biology.

[30]  Karsten Weis,et al.  Exportin 1 (Crm1p) Is an Essential Nuclear Export Factor , 1997, Cell.

[31]  J. Becker,et al.  PTR3, a novel gene mediating amino acid‐inducible regulation of peptide transport in Saccharomyces cerevisiae , 1998, Molecular microbiology.

[32]  X. Mao,et al.  Rpn4 Is a Physiological Substrate of the Ubr2 Ubiquitin Ligase* , 2004, Journal of Biological Chemistry.

[33]  J. Becker,et al.  Isolation and characterization of S. cerevisiae mutants deficient in amino acid-inducible peptide transport , 1991, Current Genetics.

[34]  Ronald W. Davis,et al.  Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. , 1999, Science.

[35]  J. Becker,et al.  Regulation of dipeptide transport in Saccharomyces cerevisiae by micromolar amino acid concentrations , 1987, Journal of bacteriology.

[36]  A. Varshavsky,et al.  Peptides accelerate their uptake by activating a ubiquitin-dependent proteolytic pathway , 2000, Nature.

[37]  C. Brown,et al.  The Type 1 Phosphatase Reg1p-Glc7p Is Required for the Glucose-induced Degradation of Fructose-1,6-bisphosphatase in the Vacuole* , 2004, Journal of Biological Chemistry.

[38]  B. André,et al.  Amino Acid Signaling in Saccharomyces cerevisiae: a Permease-Like Sensor of External Amino Acids and F-Box Protein Grr1p Are Required for Transcriptional Induction of the AGP1 Gene, Which Encodes a Broad-Specificity Amino Acid Permease , 1999, Molecular and Cellular Biology.

[39]  Roger D Kornberg,et al.  Mediator and the mechanism of transcriptional activation. , 2005, Trends in biochemical sciences.

[40]  A. Varshavsky,et al.  The recognition component of the N‐end rule pathway. , 1990, The EMBO journal.

[41]  A. Moliner,et al.  Suppressors of ssy1 and ptr3 null mutations define novel amino acid sensor-independent genes in Saccharomyces cerevisiae. , 2001, Genetics.

[42]  A. Varshavsky,et al.  The N‐end rule pathway controls the import of peptides through degradation of a transcriptional repressor , 1998, The EMBO journal.

[43]  Tamás Fischer,et al.  The mRNA export machinery requires the novel Sac3p–Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores , 2002, The EMBO journal.

[44]  Michael Hampsey,et al.  A Review of Phenotypes in Saccharomyces cerevisiae , 1997, Yeast.

[45]  T. Coolbear,et al.  The physiology and biochemistry of the proteolytic system in lactic acid bacteria. , 1993, FEMS microbiology reviews.

[46]  P. Ljungdahl,et al.  Receptor-mediated endoproteolytic activation of two transcription factors in yeast. , 2002, Genes & development.

[47]  B. André,et al.  The Npr1 Kinase Controls Biosynthetic and Endocytic Sorting of the Yeast Gap1 Permease* , 2001, The Journal of Biological Chemistry.

[48]  Nicola J. Rinaldi,et al.  Transcriptional Regulatory Networks in Saccharomyces cerevisiae , 2002, Science.

[49]  C. Kaiser,et al.  Control of amino acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST8. , 1997, Genetics.

[50]  M H Saier,et al.  Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae , 1998, FEBS letters.

[51]  J. Aitchison,et al.  Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p, Nup59p, and Nup170p , 1998, The Journal of cell biology.

[52]  James I. Garrels,et al.  The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data , 1999, Nucleic Acids Res..

[53]  D. Balciunas,et al.  Three subunits of the RNA polymerase II mediator complex are involved in glucose repression. , 1995, Nucleic acids research.

[54]  G. Knipp,et al.  Current perspectives on established and putative mammalian oligopeptide transporters. , 2003, Journal of pharmaceutical sciences.

[55]  Michel Roberge,et al.  Yeast genome-wide drug-induced haploinsufficiency screen to determine drug mode of action. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[56]  R. Planta,et al.  Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae. , 2000, Nucleic acids research.

[57]  Martin Kupiec,et al.  A genome-wide screen for Saccharomyces cerevisiae deletion mutants that affect telomere length. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[58]  K. Struhl,et al.  Eaf3 Regulates the Global Pattern of Histone Acetylation in Saccharomyces cerevisiae , 2004, Molecular and Cellular Biology.

[59]  Steven P Gygi,et al.  A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[60]  M. Hediger,et al.  Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney. , 1995, Biochimica et biophysica acta.

[61]  B. André,et al.  Ubiquitin and the SCFGrr1 ubiquitin ligase complex are involved in the signalling pathway activated by external amino acids in Saccharomyces cerevisiae , 2001, FEBS Letters.

[62]  Kara Dolinski,et al.  Saccharomyces Genome Database (SGD) provides tools to identify and analyze sequences from Saccharomyces cerevisiae and related sequences from other organisms , 2004, Nucleic Acids Res..

[63]  B. Wu,et al.  Amino acid sensing by Ssy1. , 2005, Biochemical Society transactions.

[64]  I. Arias,et al.  A Yeast ATP-binding Cassette-type Protein Mediating ATP-dependent Bile Acid Transport* , 1997, The Journal of Biological Chemistry.

[65]  S. Hohmann.  Osmotic Stress Signaling and Osmoadaptation in Yeasts , 2002, Microbiology and Molecular Biology Reviews.

[66]  B. Brors,et al.  Two genes of the putative mitochondrial fatty acid synthase in the genome of Saccharomyces cerevisiae , 1997, Current Genetics.

[67]  J. Becker,et al.  An oligopeptide transport gene from Candida albicans. , 1997, Microbiology.

[68]  T. Hughes,et al.  Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[69]  P. Ljungdahl,et al.  Sensors of extracellular nutrients in Saccharomyces cerevisiae , 2001, Current Genetics.

[70]  M. Hauser,et al.  Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae , 2001, Molecular membrane biology.

[71]  H. Feldmann,et al.  Rpn4p acts as a transcription factor by binding to PACE, a nonamer box found upstream of 26S proteasomal and other genes in yeast , 1999, FEBS letters.

[72]  R. Storms,et al.  Molecular Characterization of GCV3, the Saccharomyces cerevisiae Gene Coding for the Glycine Cleavage System Hydrogen Carrier Protein* , 1997, The Journal of Biological Chemistry.

[73]  A. Goffeau,et al.  The uses of genome-wide yeast mutant collections , 2004, Genome Biology.

[74]  B. André,et al.  The external amino acid signaling pathway promotes activation of Stp1 and Uga35/Dal81 transcription factors for induction of the AGP1 gene in Saccharomyces cerevisiae. , 2004, Genetics.

[75]  M. Carlson,et al.  Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[76]  J. Bonifacino,et al.  Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. , 2002, Molecular biology of the cell.

[77]  J. Robert‐Baudouy,et al.  Bacterial aminopeptidases: properties and functions. , 1996, FEMS microbiology reviews.