Academic journal article Genetics

The Inactivation of KlNOT4, a Kluyveromyces Lactis Gene Encoding a Component of the CCR4-NOT Complex, Reveals New Regulatory Functions

Academic journal article Genetics

The Inactivation of KlNOT4, a Kluyveromyces Lactis Gene Encoding a Component of the CCR4-NOT Complex, Reveals New Regulatory Functions

Article excerpt

ABSTRACT

We have isolated the KlNOT4 gene of the yeast Kluyveromyces lactis, which encodes a component of the evolutionarily conserved CCR4-NOT complex. We show that inactivation of the gene leads to pleiotropic defects that were differentially suppressed by the NOT4 gene of S. cerevisiae, indicating that these genes have overlapping, but not identical, functions. K. lactis strains lacking Not4p are defective in fermentation and show reduced transcription of glucose transporter and glycolytic genes, which are phenotypes that are not found in the corresponding mutant of S. cerevisiae. We also show that Not4 proteins control the respiratory pathway in both yeasts, although with some differences. They activate transcription of KlACS2 and KlCYC1, but repress KlICL1, ScICL1, ScACS1, and ScCYC1. Altogether, our results indicate that Not4p is a pivotal factor involved in the regulation of carbon metabolism in yeast.

THE CCR4-NOT complex is an evolutionarily conserved protein complex mostly involved in positive and negative regulation of gene transcription (CoLLART and STRUHL 1994; LIU et al. 2001). In Saccharomyces cerevisiae, this complex exists in two forms, one that is 1.9 MDa and one that is 1 MDa in size. The smaller form represents the core complex. As shown by mass spectrometric analysis, it contains Ccr4p, Caflp, Caf40p, Caf13Op, five Not proteins (Notl-5p), and other unidentified components, each with a specific function (DRAPER et al 1994; LIU et al 1998; CHEN et al 2001). In past years, the CCR4-NOT complex was shown to play roles in multiple cellular processes, including mRNA deadenylation (DAUGERON et al. 2001; TUCKER et al. 2001; CHEN et al. 2002), transcriptional repression, and, through contacts with TFIID, the initiation and elongation of mRNA (BADARINARAYANA et al. 2000; LEMAIRE and COLLART 2000; DENIS et al 2001). The isolation and characterization of each component of the yeast CCR4-NOT complex has been recently reviewed (CoLLART 2003).

In S. cerevisiae, NOT4 was originally isolated as a gene coding for a transcription factor involved in the repression of the TATA-less promoter of the HIS3 gene (CoLLART and STRUHL 1994). It was isolated independently as a negative regulator of several groups of genes, including the mating-pheromone-responsive genes (CADE and ERREDE 1994; IRIE et al. 1994; LEBERER et al. 1994), and was assigned different names (MOT2 and SIG1).

Not4p is a putative RNA-binding protein, and genetic interaction studies also revealed a link between the CCR4-NOT complex and the DEAD-RNA-helicase Dhhlp, a protein that promotes mRNA decapping (HATA et al. 1998; ALBERT et al. 2000; MAILLET and COLLART 2002). Recently it was demonstrated that the human Not4 protein has a ubiquitin-ligase activity in vitro, indicating a new function of this protein that suggests that it could also act as an E3 ligase enzyme (ALBERT et al. 2002).

Kluyveromyces lactis is a yeast that differs mainly from the related Saccharomyces cerevisiae in that respiration is dominant over fermentation. Although most structural and regulatory genes have been evolutionarily conserved, some factors show marked differences in the regulation of cellular pathways in the two organisms. One important difference is that, unlike in S. cerevisiae, in K. lactis cells glucose repression on respiratory enzymes is absent or less severe (MULDER et al. 1995; WESOLOWSKi-LouvEL et al. 1996) and, as a consequence, respiration and fermentation can coexist (FUKUHARA 2003). Moreover, the activation of gluconeogenesis in K. lactis is not dependent on KlCatSp, a regulatory transcription factor that in S. cerevisiae is required for derepression of the gluconeogenetic genes FBPl (fructose 1,6 biphosphatase), PCKl (pyruvate carboxykinase), and ICLl (isocitrate lyase) (GEORiS et al. 2000). Another difference between the two organisms is that the HAP complex is necessary for cell growth on respiratory carbon sources in S. cerevisiae, but not in K. …

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