Pkh1 interacts with and phosphorylates components of the yeast Gcn2/eIF2α system.

The yeast Saccharomyces cerevisiae responds to amino acid deprivation by increasing translation of the transcription factor Gcn4, which enhances expression of amino acid biosynthetic genes. Accumulation of uncharged tRNAs activates the Gcn2 protein kinase, which phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF2α). The resulting downregulation of eIF2 activity causes reduction of general translation and stimulation of GCN4 translation. S. cerevisiae contains three PDK1 orthologs (encoded by PKH1, PKH2 and PKH3) that have been implicated in nutrient signaling. Using heterologously expressed proteins, we demonstrate physical interaction between Pkh1 and all three subunits of eIF2 as well as Gcn2. We confirm the interaction between Pkh1 and Gcn2 by co-immunoprecipitation in yeast cell extracts and show that Pkh1 can phosphorylate Gcn2 in vitro. However, Pkh1 inactivation did not affect eIF2α-S51 phosphorylation in vivo or GCN4 translation in response to amino acid deprivation. Hence, the physiological importance of the close interactions between Pkh1 and Gcn2 or eIF2 could depend on other conditions and/or other targets of the Gcn2/eIF2 system.

Nutrient sensing systems for rapid activation of the protein kinase A pathway in yeast.

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae controls a variety of properties that depend on the nutrient composition of the medium. High activity of the pathway occurs in the presence of rapidly fermented sugars like glucose or sucrose, but only as long as growth is maintained. Growth arrest of fermenting cells or growth on a respiratory carbon source, like glycerol or ethanol, is associated with low activity of the PKA pathway. We have studied how different nutrients trigger rapid activation of the pathway. Glucose and sucrose activate cAMP synthesis through a G-protein-coupled receptor system, consisting of the GPCR Gpr1, the Galpha protein Gpa2 and its RGS protein Rgs2. Glucose is also sensed intracellularly through its phosphorylation. Specific mutations in Gpr1 abolish glucose but not sucrose signalling. Activation of the PKA pathway by addition of a nitrogen source or phosphate to nitrogen- or phosphate-starved cells, respectively, is not mediated by an increase in cAMP. Activation by amino acids is triggered by the general amino acid permease Gap1, which functions as a transporter/receptor. Short truncation of the C-terminus results in constitutively activating alleles. Activation by ammonium uses the ammonium permeases Mep1 and Mep2 as receptor. Specific point mutations in Mep2 uncouple signalling from transport. Activation by phosphate is triggered a.o. by the Pho84 phosphate permease. Several mutations in Pho84 separating transport and signalling or triggering constitutive activation have been obtained.