Some assembly required: yeast septins provide the instruction manual

Septins are a family of conserved proteins that form hetero-oligomeric complexes that assemble into filaments. The filaments can be organized into linear arrays, coils, rings and gauzes. They serve as membrane-associated scaffolds and as barriers to demarcate local compartments, especially for the establishment of the septation site for cytokinesis. Studies in budding and fission yeast have revealed many of the protein-protein interactions that govern the formation of multi-septin complexes. GTP binding and phosphorylation direct the polymerization of filaments that is required for septin-collar assembly in budding yeast, whereas a homolog of anillin instructs timely formation of the ring of septin filaments at the medial cortex in fission yeast. These insights should aid understanding of the organization and function of the diverse septin structures in animal cells.     

Novel mechanisms in nutrient activation of the yeast protein kinase A pathway

In yeast the Protein Kinase A (PKA) pathway can be activated by a variety of nutrients. Fermentable sugars, like glucose and sucrose, trigger a spike in the cAMP level, followed by activation of PKA and phosphorylation of target proteins causing a.o. mobilization of reserve carbohydrates, repression of stress-related genes and induction of growth-related genes. Glucose and sucrose are sensed by a G-protein coupled receptor system that activates adenylate cyclase and also activates a bypass pathway causing direct activation of PKA. Addition of other essential nutrients, like nitrogen sources or phosphate, to glucose-repressed nitrogen- or phosphate-starved cells, also triggers rapid activation of the PKA pathway. In these cases cAMP is not involved as a second messenger. Amino acids are sensed by the Gap1 transceptor, previously considered only as an amino acid transporter. Recent results indicate that the amino acid ligand has to induce a specific conformational change for signaling. The same amino acid binding site is involved in transport and signaling. Similar results have been obtained for Pho84 which acts as a transceptor for phosphate activation of the PKA pathway. Ammonium activation of the PKA pathway in nitrogen-starved cells is mediated mainly by the Mep2 transceptor, which belongs to a different class of transporter proteins. Hence, different types of sensing systems are involved in control of the yeast PKA pathway by nutrients.     

Sex and sugar in yeast: two distinct GPCR systems

Although eukaryotic G-protein coupled receptor (GPCR) systems are well known for their ability to detect and mediate rapid responses to extracellular signals, the full range of stimuli to which they respond may not yet have been identified. Activation of GPCRs by hormones, pheromones, odorants, neurotransmitters, light and different taste compounds is well established. However, the recent discovery of a glucose-sensing GPCR system in Saccharomyces cerevisiae has unexpectedly added common nutrients to this list of stimuli. This GPCR system mediates glucose activation of adenylate cyclase during the switch from respirative/gluconeogenic metabolism to fermentation. The GPCR system involved in pheromone signalling in S. cerevisiae has already served as an important model and tool for the study of GPCR systems in higher eukaryotic cell types. Here, we highlight the similarities and differences between these two signalling systems. We also indicate how the new glucose-sensing system can serve as a model for GPCR function and as a tool with which to screen for heterologous components of signalling pathways as well as for novel ligands in high-throughput assays.     

Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9

Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.     

Proliferation activity of stromal stem cells (CFU-f) from hemopoietic organs of pre- and postnatal mice

Stromal stem cells (CFU-f assay) from hemopoietic organs of fetuses, in contrast to adult animals, exhibit a high proliferation activity. This implies that these CFU-f are radiosensitive and potential target cells after radioactive contamination of fetuses. Furthermore, the percentage of CFU-f in DNA synthesis is correlated with the hemopoietic activity in liver, spleen, and bone marrow. As hemopoiesis starts, high numbers of CFU-f are in S phase. In fetal liver, spleen, and bone marrow, values of 70, 43, and 58%, respectively, are reached. As hemopoietic activity decreases in liver and stabilizes in spleen and bone marrow, mitotic activity of these stromal stem cells becomes undetectable.     

A baker's yeast mutant (fil1) with a specific, partially inactivating mutation in adenylate cyclase maintains a high stress resistance during active fermentation and growth

The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in stress resistance. This is disadvantageous for several biotechnological applications, e.g. the preparation of freeze doughs. We have isolated mutants in a laboratory strain which are deficient in fermentation-induced loss of stress resistance ('fil' mutants) using a heat shock selection protocol. We show that the fil1 mutant contains a mutation in the CYR1 gene which encodes adenylate cyclase. It causes a change at position 1682 of glutamate into lysine and results in a tenfold drop in adenylate cyclase activity. The fil1 mutant displays a reduction in the glucose-induced cAMP increase, trehalase activation and loss of heat resistance. Interestingly, the fil1 mutant shows the same growth and fermentation rate as the wild type strain, as opposed to other mutants with reduced activity of the cAMP pathway. Introduction of the fil1 mutation in the vigorous Y55 strain and cultivation of the mutant under pilot scale conditions resulted in a yeast that displayed a higher freeze and drought resistance during active fermentation compared to the wild type Y55 strain. These results show that high stress resistance and high fermentation activity are compatible biological properties. Isolation of fil-type mutations appears a promising avenue for development of industrial yeast strains with improved stress resistance during active fermentation.     

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.     

A novel regulator of G protein signalling in yeast, Rgs2, downregulates glucose-activation of the cAMP pathway through direct inhibition of Gpa2.

We have characterized a novel member of the recently identified family of regulators of heterotrimeric G protein signalling (RGS) in the yeast Saccharomyces cerevisiae. The YOR107w/RGS2 gene was isolated as a multi-copy suppressor of glucose-induced loss of heat resistance in stationary phase cells. The N-terminal half of the Rgs2 protein consists of a typical RGS domain. Deletion and overexpression of Rgs2, respectively, enhances and reduces glucose-induced accumulation of cAMP. Overexpression of RGS2 generates phenotypes consistent with low activity of cAMP-dependent protein kinase A (PKA), such as enhanced accumulation of trehalose and glycogen, enhanced heat resistance and elevated expression of STRE-controlled genes. Deletion of RGS2 causes opposite phenotypes. We demonstrate that Rgs2 functions as a negative regulator of glucose-induced cAMP signalling through direct GTPase activation of the Gs-alpha protein Gpa2. Rgs2 and Gpa2 constitute the second cognate RGS-G-alpha protein pair identified in yeast, in addition to the mating pheromone pathway regulators Sst2 and Gpa1. Moreover, Rgs2 and Sst2 exert specific, non-overlapping functions, and deletion mutants in Rgs2 and Sst2 are complemented to some extent by different mammalian RGS proteins.