TY - JOUR

T1 - Guanine nucleotide pool imbalance impairs multiple steps of protein synthesis and disrupts GCN4 translational control in Saccharomyces cerevisiae

AU - Iglesias Gato, Diego

AU - Martín-Marcos, Pilar

AU - Santos, María A

AU - Hinnebusch, Alan G

AU - Tamame, Mercedes

PY - 2011/1

Y1 - 2011/1

N2 - Purine nucleotides are structural components of the genetic material, function as phosphate donors, participate in cellular signaling, are cofactors in enzymatic reactions, and constitute the main carriers of cellular energy. Thus, imbalances in A/G nucleotide biosynthesis affect nearly the whole cellular metabolism and must be tightly regulated. We have identified a substitution mutation (G388D) that reduces the activity of the GMP synthase Gua1 in budding yeast and the total G-nucleotide pool, leading to precipitous reductions in the GDP/GTP ratio and ATP level in vivo. gua1-G388D strongly reduces the rate of growth, impairs general protein synthesis, and derepresses translation of GCN4 mRNA, encoding a transcriptional activator of diverse amino acid biosynthetic enzymes. Although processing of pre-tRNA(i)(Met) and other tRNA precursors, and the aminoacylation of tRNA(i)(Met) are also strongly impaired in gua1-G388D cells, tRNA(i)(Met)-containing complexes with the macromolecular composition of the eIF2·tRNA(i)(Met.)GTP complex (TC) and the multifactor complex (MFC) required for translation initiation accumulate ∼10-fold in gua1-G388D cells and, to a lesser extent, in wild-type (WT) cells treated with 6-azauracil (6AU). Consistently, addition of an external supply of guanine reverts all the phenotypes of gua1-G388D cells, but not those of gua1-G388D Δhpt1 mutants unable to refill the internal GMP pool through the salvage pathway. These and other findings suggest that a defect in guanine nucleotide biosynthesis evokes a reduction in the rate of general protein synthesis by impairing multiple steps of the process, disrupts the gene-specific reinitiation mechanism for translation of GCN4 mRNA and has far-reaching effects in cell biology and metabolism.

AB - Purine nucleotides are structural components of the genetic material, function as phosphate donors, participate in cellular signaling, are cofactors in enzymatic reactions, and constitute the main carriers of cellular energy. Thus, imbalances in A/G nucleotide biosynthesis affect nearly the whole cellular metabolism and must be tightly regulated. We have identified a substitution mutation (G388D) that reduces the activity of the GMP synthase Gua1 in budding yeast and the total G-nucleotide pool, leading to precipitous reductions in the GDP/GTP ratio and ATP level in vivo. gua1-G388D strongly reduces the rate of growth, impairs general protein synthesis, and derepresses translation of GCN4 mRNA, encoding a transcriptional activator of diverse amino acid biosynthetic enzymes. Although processing of pre-tRNA(i)(Met) and other tRNA precursors, and the aminoacylation of tRNA(i)(Met) are also strongly impaired in gua1-G388D cells, tRNA(i)(Met)-containing complexes with the macromolecular composition of the eIF2·tRNA(i)(Met.)GTP complex (TC) and the multifactor complex (MFC) required for translation initiation accumulate ∼10-fold in gua1-G388D cells and, to a lesser extent, in wild-type (WT) cells treated with 6-azauracil (6AU). Consistently, addition of an external supply of guanine reverts all the phenotypes of gua1-G388D cells, but not those of gua1-G388D Δhpt1 mutants unable to refill the internal GMP pool through the salvage pathway. These and other findings suggest that a defect in guanine nucleotide biosynthesis evokes a reduction in the rate of general protein synthesis by impairing multiple steps of the process, disrupts the gene-specific reinitiation mechanism for translation of GCN4 mRNA and has far-reaching effects in cell biology and metabolism.

KW - Alleles

KW - Amino Acid Sequence

KW - Animals

KW - Base Sequence

KW - Basic-Leucine Zipper Transcription Factors

KW - Carbon-Nitrogen Ligases

KW - Cloning, Molecular

KW - Eukaryotic Initiation Factor-2

KW - Guanine Nucleotides

KW - Guanosine Monophosphate

KW - Guanosine Triphosphate

KW - Humans

KW - Molecular Sequence Data

KW - Mutation

KW - Protein Biosynthesis

KW - RNA, Transfer

KW - Ribosome Subunits

KW - Saccharomyces cerevisiae

KW - Saccharomyces cerevisiae Proteins

KW - Transfer RNA Aminoacylation

U2 - 10.1534/genetics.110.122135

DO - 10.1534/genetics.110.122135

M3 - Journal article

C2 - 20980241

VL - 187

SP - 105

EP - 122

JO - Genetics

JF - Genetics

SN - 1943-2631

IS - 1

ER -