Multiple, Nutrient Sensing Kinases Converge to Phosphorylate an Element of cdc34 That Increases Saccharomyces Cerevisiae Lifespan

Indiana University-Purdue University Indianapolis (IUPUI)Growth and division are tightly coordinated with available nutrient conditions. Cells of the budding yeast, Saccharomyces cerevisiae, grow to a larger size prior to budding and DNA replication when preferred carbon sources such as glucose, as opposed to less preferred sources like ethanol and acetate, are available. A culture’s doubling time is also significantly reduced when the available carbon and nitrogen sources are more favorable. These physiological phenomena are well documented but the precise molecular mechanisms relaying nutrient conditions to the growth and division machinery are not well defined. I demonstrate here that Cdc34, the ubiquitin conjugating enzyme that promotes S phase entry, is phosphorylated upon a highly conserved serine residue which is part of a motif that defines the family of Cdc34/Ubc7 ubiquitin conjugating enzymes. This phosphorylation is regulated by multiple, nutrient sensing kinases including Protein Kinase A, Sch9 and TOR. Furthermore, this phosphorylation event is regulated through the cell cycle with the sole induction occurring in the G1 phase which is when nutrients are sensed and cells commit to another round of division. This phosphorylation likely activates Cdc34 and in turn propagates a signal to the cell division cycle machinery that nutrient conditions are favorable for commitment to a new round of division. This phosphorylation is critical for normal cell cycle progression but must be carefully controlled when cells are deprived of nutrients. Crippling the activity of Protein Kinase A, SCH9 or TOR increases the proportion of cells that survive stationary phase conditions, which because of the metabolic conditions that must be maintained and the similarity to post-mitotic mammalian cells, is referred to as a yeast culture’s chronological lifespan. Yeast cells expressing Cdc34 mutants that are no longer subject to this regulation by phosphorylation have a reduced chronological lifespan. A precise molecular mechanism describing the change in Cdc34 activity after phosphorylation of this serine residue is discussed

Spatial and Functional Relationships Among Pol V-Associated Loci, Pol IV-Dependent siRNAs, and Cytosine Methylation in the \u3cem\u3eArabidopsis\u3c/em\u3e Epigenome

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%–60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites

Complementation of <i>cdc34-2</i> and <i>cdc34Δ</i> strains by Cdc34 S97 Mutants.

<p>A and B) YL10-1, a <i>cdc34-2</i> temperature sensitive strain, bearing 2 µm plasmids encoding the indicated <i>CDC34</i> mutant under control of the <i>GAL10</i> promoter were spotted in tenfold serial dilutions on the indicated media and grown at the indicated temperatures for 3 days. C) YL18, a <i>cdc34Δ</i> strain harboring a <i>URA3</i> marked plasmid encoding wild type <i>CDC34</i> and a <i>LEU2</i>-marked plasmid encoding the indicated <i>CDC34</i> allele under control of the <i>GAL10</i> promoter were spotted in tenfold serial dilution on the indicated media and grown at 30°C for 3 days.</p

Strains used in this study.

<p>Strains used in this study.</p

Model of Cdc34 S97 phosphorylation.

<p>Model of Cdc34 S97 phosphorylation.</p

The Cdc34 S73/S97/loop motif increases chronological lifespan and is required for rapamycin resistance.

<p>A) Prototrophic strains BL2 (WT) and RRC85 (CDC34^tm) were grown in synthetic defined liquid media lacking all amino acids for the indicated number of days at 32 C and the percent viability was determined by spotting a fraction of the culture on a YPD plate and counting colonies after incubation for 3 days at 32 C. B) Strains BL2 and RRC85 were grown overnight in SD lacking all amino acids, diluted to 5×10⁷ cells/ml and spotted in ten-fold serial dilution on YPD and YPD+25 nM rapamycin plates. Plates were incubated at 32°C for 3 days and then photographed.</p

A screen for kinases whose overexpression alters the level of Cdc34-S97 phosphorylation.

<p>A) Total soluble protein from an array of strains each overexpressing the indicated kinase was analyzed by western blot using the α-pS97 phospho-specific and α-Cdc34 antibodies.</p