The essential function of not1 lies within the Ccr4-not complex

Our previous work suggests that the Nhp6 HMGB protein stimulates RNA polymerase II transcription via the TATA-binding protein TBP and that Nhp6 functions in the same functional pathway as the Gcn5 histone acetyltransferase. In this report we examine the genetic relationship between Nhp6 and Gcn5 with the Mot1 and Ccr4-Not complexes, both of which have been implicated in regulating DNA binding by TBP. We find that combining either a nhp6ab or a gcn5 mutation with mot1, ccr4, not4, or not5 mutations results in lethality. Combining spt15 point mutations (in TBP) with either mot1 or ccr4 also results in either a growth defect or lethality. Several of these synthetic lethalities can be suppressed by overexpression of TFIIA, TBP, or Nhp6, suggesting that these genes facilitate formation of the TBP-TFIIA-DNA complex. The growth defect of a not5 mutant can be suppressed by a mot1 mutant. HO gene expression is reduced by nhp6ab, gcn5, or mot1 mutations, and the additive decreases in HO mRNA levels in nhp6ab mot1 and gcn5 mot1 strains suggest different modes of action. Chromatin immunoprecipitation experiments show decreased binding of TBP to promoters in mot1 mutants and a further decrease when combined with either nhp6ab or gcn5 mutations.

Section: Genetic Interactions Of Nhp6 and Gcn5 With Mot1mentioning

confidence: 68%

Section: Genetic Interactions Of Nhp6 and Gcn5 With Mot1mentioning

confidence: 99%

Genetic Interactions Between Nhp6 and Gcn5 With Mot1 and the Ccr4–Not Complex That Regulate Binding of TATA-Binding Protein in Saccharomyces cerevisiae

Biswas

Mitra

et al. 2006

“…The Ccr4-Not complex is a global regulator of transcription consisting of five Not proteins (Not1-Not5), Pop2 (also known as Caf1), Caf40, Caf130, and Ccr4 Tucker et al 2001;Collart 2003). Among them, Not1 is the scaffold of the complex and is essential for viability (Maillet et al 2000). We found that deletion of CCR4, POP2, or NOT4 (also known as MOT2) resulted in substantial growth defects at high pressure and low temperature (Table 1 and Figure 2G).…”

Section: Resultsmentioning

confidence: 99%

Global Screening of Genes Essential for Growth in High-Pressure and Cold Environments: Searching for Basic Adaptive Strategies Using a Yeast Deletion Library

Abe

Minegishi

2008

Microorganisms display an optimal temperature and hydrostatic pressure for growth. To establish the molecular basis of piezo-and psychroadaptation, we elucidated global genetic defects that give rise to susceptibility to high pressure and low temperature in Saccharomyces cerevisiae. Here we present 80 genes including 71 genes responsible for high-pressure growth and 56 responsible for low-temperature growth with a significant overlap of 47 genes. Numerous previously known cold-sensitive mutants exhibit marked high-pressure sensitivity. We identified critically important cellular functions: (i) amino acid biosynthesis, (ii) microautophagy and sorting of amino acid permease established by the exit from rapamycin-induced growth arrest/Gap1 sorting in the endosome (EGO/GSE) complex, (iii) mitochondrial functions, (iv) membrane trafficking, (v) actin organization mediated by Drs2-Cdc50, and (vi) transcription regulated by the Ccr4-Not complex. The loss of EGO/GSE complex resulted in a marked defect in amino acid uptake following high-pressure and low-temperature incubation, suggesting its role in surface delivery of amino acid permeases. Microautophagy and mitochondrial functions converge on glutamine homeostasis in the target of rapamycin (TOR) signaling pathway. The localization of actin requires numerous associated proteins to be properly delivered by membrane trafficking. In this study, we offer a novel route to gaining insights into cellular functions and the genetic network from growth properties of deletion mutants under high pressure and low temperature.

“…Several genetic interactions within the Ccr4-Not complex are described (Maillet et al 2000), including synthetic lethality between deletions of NOT4 and NOT5. We hypothesized that if this function of Not4p depends on its ubiquitylation activity, the not4Dnot5D synthetic lethality would not be complemented by introduction of E2-interaction-deficient mutants.…”

Section: Resultsmentioning

confidence: 99%

Modulation of Ubc4p/Ubc5p-Mediated Stress Responses by the RING-Finger-Dependent Ubiquitin-Protein Ligase Not4p in Saccharomyces cerevisiae

Mulder

Inagaki

Cameroni³

et al. 2007

Self Cite

The Ccr4-Not complex consists of nine subunits and acts as a regulator of mRNA biogenesis in Saccharomyces cerevisiae. The human ortholog of yeast NOT4, CNOT4, displays UbcH5B-dependent ubiquitinprotein ligase (E3 ligase) activity in a reconstituted in vitro system. However, an in vivo role for this enzymatic activity has not been identified. Site-directed mutagenesis of the RING finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p-mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. To investigate the in vivo relevance of this activity, we performed synthetic genetic array (SGA) analyses using not4D and not4L35A alleles. This indicates involvement of the RING finger of Not4p in transcription, ubiquitylation, and DNA damage responses. In addition, we found a phenotypic overlap between deletions of UBC4 and mutants encoding single-aminoacid substitutions of the RING finger of Not4p. Together, our results show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p-mediated stress responses in vivo.