Josep Clotet

The Yeast Ser/Thr Phosphatases Sit4 and Ppz1 Play Opposite Roles in Regulation of the Cell Cycle

ABSTRACT Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from α-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 onsit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression ofPPZ1 is intensified in strains with low G1cyclin levels (such as bck2Δ or cln3Δ mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.

Hypothalamic Ceramide Levels Regulated by CPT1C Mediate the Orexigenic Effect of Ghrelin

Recent data suggest that ghrelin exerts its orexigenic action through regulation of hypothalamic AMP-activated protein kinase pathway, leading to a decline in malonyl-CoA levels and desinhibition of carnitine palmitoyltransferase 1A (CPT1A), which increases mitochondrial fatty acid oxidation and ultimately enhances the expression of the orexigenic neuropeptides agouti-related protein (AgRP) and neuropeptide Y (NPY). However, it is unclear whether the brain-specific isoform CPT1C, which is located in the endoplasmic reticulum of neurons, may play a role in this action. Here, we demonstrate that the orexigenic action of ghrelin is totally blunted in CPT1C knockout (KO) mice, despite having the canonical ghrelin signaling pathway activated. We also demonstrate that ghrelin elicits a marked upregulation of hypothalamic C18:0 ceramide levels mediated by CPT1C. Notably, central inhibition of ceramide synthesis with myriocin negated the orexigenic action of ghrelin and normalized the levels of AgRP and NPY, as well as their key transcription factors phosphorylated cAMP-response element–binding protein and forkhead box O1. Finally, central treatment with ceramide induced food intake and orexigenic neuropeptides expression in CPT1C KO mice. Overall, these data indicate that, in addition to formerly reported mechanisms, ghrelin also induces food intake through regulation of hypothalamic CPT1C and ceramide metabolism, a finding of potential importance for the understanding and treatment of obesity.

Control of Cell Cycle in Response to Osmostress: Lessons from Yeast

To maximize the probability of survival and proliferation, cells coordinate various intracellular activities in response to changes in the extracellular environment. Eukaryotic cells transduce diverse cellular stimuli by multiple mitogen-activated protein kinase (MAPK) cascades. Exposure of cells to stress results in rapid activation of a highly conserved family of MAPKs, known as stress-activated protein kinases (SAPKs). Activation of SAPKs results in the generation of a set of adaptive responses that leads to the modulation of several aspects of cell physiology essential for cell survival, such as gene expression, translation, and morphogenesis. This chapter proposes that regulation of cell cycle progression is another general stress response critical for cell survival. Studies from yeast, both Schizosaccharomyces pombe and Saccharomyces cerevisiae, have served to start understanding how SAPKs control cell cycle progression in response to stress.

Structural model of a malonyl-CoA-binding site of carnitine octanoyltransferase and carnitine palmitoyltransferase I: Mutational analysis of a malonyl-CoA affinity domain

Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an “in silico” macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001–45008). The putative malonyl-CoA domain contained His340, implicated together with His131 in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubı́, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183–186). When we mutated COT His131 the IC50increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala332, present in the domain 8 amino acids away from His340, decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His277, His483, and Ala478 were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro479, which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.

Identification of multicopy suppressors of cell cycle arrest at the G1–S transition in Saccharomyces cerevisiae

Inactivation of HAL3 in the absence of SIT4 function leads to cell cycle arrest at the G1–S transition. To identify genes potentially involved in the control of this phase of the cell cycle, a screening for multicopy suppressors of a conditional sit4 hal3 mutant (strain JC002) has been developed. The screening yielded several genes known to perform key roles in cell cycle events, such as CLN3, BCK2 or SWI4, thus proving its usefulness as a tool for this type of studies. In addition, this approach allowed the identification of additional genes, most of them not previously related to the regulation of G1–S transition or even without known function (named here as VHS1‐3, for viable in a hal3 sit4 background). Several of these gene products are involved in phospho‐dephosphorylation processes, including members of the protein phosphatase 2A and protein phosphatases 2C families, as well as components of the Hal5 protein kinase family. The ability of different genes to suppress sit4 phenotypes (such as temperature sensitivity and growth on non‐fermentable carbon sources) or to mimic the functions of Hal3 was evaluated. The possible relationship between the known functions of these suppressor genes and the progress through the G1–S transition is discussed. Copyright

The gene DIS2S1 is essential in Saccharomyces cerevisiae and is involved in glycogen phosphorylase activation

SummaryS. cerevisiae gene DIS2S1, which codes for a protein very similar to the catalytic subunit of mammalian protein phosphatase 1, was disrupted “in vitro”. Diploid yeast cells were transformed and sporulated. Tetrad analysis demonstrated that disruption of DIS2S1 is lethal for the cell. Glycogen phosphorylase a and glycogen synthase activity ratio were measured in diploids carrying a disrupted allele of the gene. Phosphorylase was dramatically activated in mutant cells but, under the same conditions, glycogen synthase activity was essentially identical in both mutant and wild-type cells.

Saccharomyces cerevisiae gene SIT4 is involved in the control of glycogen metabolism.

The gene SIT4 of S. cerevisiae, which codes for a protein structurally related to the catalytic subunit of mammalian protein phosphatase 2A, was disrupted in vitro. Analysis of glycogen synthase activity ratio in mutant haploid cells indicated that the enzyme was less active than in wild‐type cells. On the contrary, glycogen phosphorylase a activity was much higher. The activation of glycogen synthase observed in wild‐type cells after incubation with lithium ions was not detected in mutant cells. These results suggest that the product of gene SIT4, a putative protein phosphatase, could be involved in the control of glycogen metabolism in yeast cells.

Identification of the two histidine residues responsible for the inhibition by malonyl‐CoA in peroxisomal carnitine octanoyltransferase from rat liver

Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl‐CoA. cDNAs encoding full‐length wild‐type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild‐type‐expressed COT was inhibited by malonyl‐CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl‐CoA in the range assayed (2–200 μM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl‐CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.

Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast

The gene pzl‐1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL‐1 protein, as well as its carboxyl‐terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL‐1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL‐1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL‐1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen‐activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL‐1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Δ strain in high potassium, but only partially complemented the sodium‐hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL‐1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH2‐terminal moieties. Copyright

Glycogen metabolism in a Saccharomyces cerevisiae phosphoglucose isomerase (pgi1) disruption mutant

Disruption of the gene pgi1 of Saccharomyces cerevisiae, which codes for phosphoglucose isomerase, results in a dramatic increase in the amount of intracellular glycogen in early exponential cultures. The level or glucose 6‐phosphate was much higher in mutant than in wild‐type cells. Phosphorylase a activity and the state of activation of glycogen synthase were also investigated. Phosphorylase a activity was rather low along the culture in wild‐type cells, whereas it was consistently higher in mutants. Glycogen synthase was mostly in the active form in early‐medium exponential cultures in wild‐type cells whereas the activation state of this enzyme in mutant cells, although lower at the earlier steps of the culture, did not differ from wild‐type cells at later stages. The fact that the intracellular levels of UDP‐glucose are markedly increased in mutant cells suggest that the observed accumulation of glycogen results from a rise in substrate availability rather than from the activation of the enzyme responsible for the synthesis of the polysaccharide.