The HOG1-like MAP kinase Sak1 of Botrytis cinerea is negatively regulated by the upstream histidine kinase Bos1 and is not involved in dicarboximide- and phenylpyrrole-resistance

In filamentous ascomycetes, HOG-like signal transduction cascades are involved in the resistance to hyper-osmotic conditions and to dicarboximides and phenylpyrroles. The histidine kinase (HK) Bos1 and the mitogen-activated protein kinase (MAPK) Sak1 are important for the adaptation to hyper-osmotic and oxidative stress, development, and pathogenicity in the phytopathogenic fungus Botrytis cinerea. However, bos1Δ and sak1Δ mutants created previously, also presented different phenotypes, especially the sak1Δ mutants were not resistant to high fungicide concentrations. Since both single mutants were constructed in different parental strains, phenotypic variations due to the genetic background might be suspected. In order to establish the relationship between both protein kinases, we analyzed Sak1 phosphorylation under the control of the Bos1 HK and we realized epistasis analysis between bos1Δ and sak1Δ mutations through the construction of isogenic single and double mutants. Our results show that Bos1 negatively regulates Sak1 phosphorylation and that Bos1 regulates certain phenotypes independently of Sak1. They include fungicide susceptibility, adaptation and conidiation on high neutral osmolarity.     

Regulation of snf1 protein kinase in response to environmental stress

The Saccharomyces cerevisiae Snf1 protein kinase, a member of the Snf1/AMPK (AMP-activated protein kinase) family, has important roles in metabolic control, particularly in response to nutrient stress. Here we have addressed the role of Snf1 in responses to other environmental stresses. Exposure of cells to sodium ion stress, alkaline pH, or oxidative stress caused an increase in Snf1 catalytic activity and phosphorylation of Thr-210 in the activation loop, whereas treatment with sorbitol or heat shock did not. Inhibition of respiratory metabolism by addition of antimycin A to cells also increased Snf1 activity. Analysis of mutants indicated that the kinases Sak1, Tos3, and Elm1, which activate Snf1 in response to glucose limitation, are also required under other stress conditions. Each kinase sufficed for activation in response to stress, but Sak1 had the major role. In sak1Delta tos3Delta elm1Delta cells expressing mammalian Ca(2+)/calmodulin-dependent protein kinase kinase alpha, Snf1 was activated by both sodium ion and alkaline stress, suggesting that stress signals regulate Snf1 activity by a mechanism that is independent of the upstream kinase. Finally, we showed that Snf1 protein kinase is regulated differently during adaptation of cells to NaCl and alkaline pH with respect to both temporal regulation of activation and subcellular localization. Snf1 protein kinase becomes enriched in the nucleus in response to alkaline pH but not salt stress. Such differences could contribute to specificity of the stress responses.     

Interaction of SNF1 protein kinase with its activating kinase Sak1

The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.     

Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro

The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation and is highly conserved from yeast to mammals. The upstream kinases are also functionally conserved, and the AMPK kinases LKB1 and Ca2+/calmodulin-dependent protein kinase kinase activate Snf1 in mutant yeast cells lacking the native Snf1-activating kinases, Sak1, Tos3, and Elm1. Here, we exploited the yeast genetic system to identify members of the mammalian AMPK kinase family by their function as Snf1-activating kinases. A mouse embryo cDNA library in a yeast expression vector was used to transform sak1Delta tos3Delta elm1Delta yeast cells. Selection for a Snf+ growth phenotype yielded cDNA plasmids expressing LKB1, Ca2+/calmodulin-dependent protein kinase kinase, and transforming growth factor-beta-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase family. We present genetic and biochemical evidence that TAK1 activates Snf1 protein kinase in vivo and in vitro. We further show that recombinant TAK1, fused to the activation domain of its binding partner TAB1, phosphorylates Thr-172 in the activation loop of the AMPK catalytic domain. Finally, expression of TAK1 and TAB1 in HeLa cells or treatment of cells with cytokines stimulated phosphorylation of Thr-172 of AMPK. These findings indicate that TAK1 is a functional member of the Snf1/AMPK kinase family and support TAK1 as a candidate for an authentic AMPK kinase in mammalian cells.     

Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae

Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.     

Regulatory domains of Snf1-activating kinases determine pathway specificity

In Saccharomyces cerevisiae, the Snf1 kinase can be activated by any one of three upstream kinases, Sak1, Tos3, or Elm1. All three Snf1-activating kinases contain serine/threonine kinase domains near their N termini and large C-terminal domains with little sequence conservation and previously unknown function. Deletion of the C-terminal domains of Sak1 and Tos3 greatly reduces their ability to activate the Snf1 pathway. In contrast, deletion of the Elm1 C-terminal domain has no effect on Snf1 signaling but abrogates the ability of Elm1 to participate in the morphogenetic-checkpoint signaling pathway. Thus, the C-terminal domains of Sak1, Tos3, and Elm1 help to determine pathway specificity. Additional deletion mutants of the Sak1 kinase revealed that the N terminus of the protein is essential for Snf1 signaling. The deletion of 43 amino acids from within the N terminus of Sak1 (residues 87 to 129) completely blocks Snf1 signaling and activation loop phosphorylation in vivo. The Sak1 kinase domain (lacking both N-terminal and C-terminal domains) is catalytically active and specific in vitro but is unable to promote Snf1 signaling in vivo when expressed at normal levels. Our studies indicate that the kinase domains of the Snf1-activating kinases are not sufficient by themselves for their proper function and that the nonconserved N-terminal and C-terminal domains are critical for the biological activities of these kinases.     

Roles of the Snf1-Activating Kinases during Nitrogen Limitation and Pseudohyphal Differentiation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Snf1 protein kinase is important for growth on carbon sources that are less preferred than glucose. When glucose becomes limiting, Snf1 undergoes catalytic activation, which requires phosphorylation of its T-loop threonine (Thr210). Thr210 phosphorylation can be performed by any of three Snf1-activating kinases: Sak1, Tos3, and Elm1. These kinases are redundant in that all three must be eliminated to confer snf1Δ-like growth defects on nonpreferred carbon sources. We previously showed that in addition to glucose signaling, Snf1 also participates in nitrogen signaling and is required for diploid pseudohyphal differentiation, a filamentous-growth response to nitrogen limitation. Here, we addressed the roles of the Snf1-activating kinases in this process. Loss of Sak1 caused a defect in pseudohyphal differentiation, whereas Tos3 and Elm1 were dispensable. Sak1 was also required for increased Thr210 phosphorylation of Snf1 under nitrogen-limiting conditions. Expression of a catalytically hyperactive version of Snf1 restored pseudohyphal differentiation in the sak1Δ/sak1Δ mutant. Thus, while the Snf1-activating kinases exhibit redundancy for growth on nonpreferred carbon sources, the loss of Sak1 alone produced a significant defect in a nitrogen-regulated phenotype, and this defect resulted from deficient Snf1 activation rather than from disruption of another pathway. Our results suggest that Sak1 is involved in nitrogen signaling upstream of Snf1.Snf1 protein kinase of the yeast Saccharomyces cerevisiae belongs to the conserved Snf1/AMP-activated protein kinase (AMPK) family; members of this family play central roles in responses to metabolic stress in eukaryotes (reviewed in references 17 and 18). Interest in Snf1/AMPK pathways is high due to their important functions. Deregulation of AMPK signaling in humans has been linked to type 2 diabetes, heart disease, and cancer (for a review, see reference 16). Snf1 homologs of pathogenic fungi have been implicated in virulence and drug resistance (23, 63, 64).Yeast Snf1 (Cat1, Ccr1) was first identified by its requirement for growth on carbon sources that are less preferred than glucose (5, 7, 65). Subsequent evidence indicated that Snf1 protein kinase (6) is directly involved in glucose signaling, since its activity is stimulated in response to glucose limitation (62). Catalytic activation of Snf1 occurs through phosphorylation of its conserved T-loop threonine (Thr210) (12) by upstream kinases (40, 62). Three protein kinases—Sak1, Tos3, and Elm1—have been identified that can phosphorylate Thr210 of Snf1 (22, 41, 55). These kinases are related to the mammalian kinases that activate AMPK by phosphorylating the equivalent T-loop threonine (Thr172) (reviewed in references 17 and 18). We recently presented evidence that Snf1 homologs of two pathogenic Candida species, Candida albicans and C. glabrata, also undergo T-loop phosphorylation (42).It is not entirely clear why S. cerevisiae has three different kinases that can activate Snf1. Judging by assays of Snf1 kinase activity, Sak1 makes the largest individual contribution to Snf1 activation in the cell (19, 22). However, deletion of SAK1 alone does not result in growth defects on alternative carbon sources, and all three Snf1-activating kinases must be eliminated to produce a phenotypic defect comparable to that of the snf1Δ mutant (22, 39, 55). Deletion of TOS3 was reported to moderately affect growth on nonfermentable carbon sources; this correlated with a reduction in Snf1 activity, although effects on another pathway(s) cannot be excluded (25). Mutation of ELM1 affects cell cycle progression and cell morphology, but this effect is unrelated to Elm1''s role as a Snf1-activating kinase and pertains to its role in the activation of Nim1-related protein kinases involved in morphogenesis checkpoint control (1, 56).While showing significant redundancy for growth on nonpreferred carbon sources, the Snf1-activating kinases could exhibit specialization in Snf1 signaling in response to stresses other than carbon stress. Evidence indicates that Snf1 is important for adaptation to a number of stress conditions (reviewed in reference 18). In some cases, such as genotoxic stress or exposure to hygromycin B, weak activity of unphosphorylated Snf1 appears to be sufficient for resistance (10, 48). In others, such as sodium ion stress and alkaline stress, Thr210 phosphorylation of Snf1 is required for adaptation, and Snf1 becomes activated upon stress exposure (21, 40). As with glucose limitation, however, in these latter cases Sak1 makes the largest contribution to Snf1 activation judging by biochemical assays, and yet it remains dispensable for wild-type levels of stress-resistant growth in phenotypic tests; loss of all three Snf1-activating kinases results in growth defects comparable to those of cells lacking Snf1 (21). Thus, investigation of these stresses provided no evidence for phenotypically relevant specialization of Sak1, Tos3, or Elm1 in Snf1 signaling.Diploid pseudohyphal differentiation is a developmental response to nitrogen limitation (15). When nitrogen becomes limiting, diploid cells adopt elongated morphology, alter their budding pattern, and generate filaments (pseudohyphae) consisting of chains of cells attached to one another. One of the key events in this process is activation of the FLO11 (MUC1) gene, which encodes a cell surface glycoprotein involved in cell-cell adhesion (29, 33, 34). Following up on an observation that Snf1 is important for FLO11 expression on low glucose, we previously found that diploids lacking Snf1 fail to undergo pseudohyphal differentiation on low nitrogen (27, 28). The requirement of Snf1 for a nitrogen-regulated process raised the possibility that Snf1 is directly involved in nitrogen signaling. In support of this notion, we subsequently showed that weak activity of nonphosphorylatable Snf1-T210A is not sufficient for pseudohyphal differentiation and that Thr210 phosphorylation of Snf1 increases in response to nitrogen limitation (43).Here, we have examined the roles of Sak1, Tos3, and Elm1 in pseudohyphal differentiation and Snf1 activation on low nitrogen. We show that elimination of Sak1 leads to a significant defect in nitrogen-regulated pseudohyphal differentiation, whereas Tos3 and Elm1 are dispensable. Sak1 is also required for normal Thr210 phosphorylation of Snf1 under nitrogen-limiting conditions. Our data strongly suggest that the loss of Sak1 affects pseudohyphal differentiation by affecting Snf1 activation and not by disruption of another pathway. Collectively, our findings implicate Sak1 in nitrogen signaling upstream of Snf1.     

Roles of Cch1 and Mid1 in Morphogenesis, Oxidative Stress Response and Virulence in Candida albicans

Ca²⁺ channel Cch1, and its subunit Mid1, has been suggested as the protein complex responsible for mediating Ca²⁺ influx, which is often employed by fungal cells to maintain cell survival. The abilities of morphological switch and response to stress conditions are closely related to pathogenicity in Candida albicans. Cch1 and Mid1 activity are required for virulence of Cryptococcus neoformans and Claviceps purpurea, respectively. To investigate whether Cch1 and Mid1 also play a role in the virulence of C. albicans, we constructed cch1Δ/Δ and mid1Δ/Δ mutant strains for functional analysis of CCH1 and MID1. Although both of the mutants displayed the ability of yeast-to-hypha transition, they were defective in hyphae maintenance and invasive growth. Interestingly, deletion of CCH1 or MID1 in C. albicans led to an obvious defect phenotype in oxidative stress response. Moreover, the virulence of the mutants was reduced in a mouse model. Our results demonstrated that Cch1 and Mid1 activity are related to the virulence of C. albicans and may provide a new antifungal target.     

Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

Highly conserved among eukaryotic cells, the AMP‐activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Δsnf1, Δsnf4, and Δsnf1Δsnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub‐network analysis, we showed the benefits of three‐level ome‐data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low‐energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases.     

Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.     

Sphingolipid signalling mediates mitochondrial dysfunctions and reduced chronological lifespan in the yeast model of Niemann‐Pick type C1

The Niemann‐Pick type C is a rare metabolic disease with a severe neurodegenerative phenotype characterized by an accumulation of high amounts of lipids (cholesterol and sphingolipids) in the late endosomal/lysosomal network. It is caused by loss‐of‐function point mutations in either NPC1 or NPC2, which seem to mediate proper intracellular lipid transport through endocytic pathway. In this study, we show that yeast cells lacking Ncr1p, an orthologue of mammalian NPC1, exhibited a higher sensitivity to hydrogen peroxide and a shortened chronological lifespan. These phenotypes were associated with increased levels of oxidative stress markers, decreased levels of antioxidant defences and mitochondrial dysfunctions. Moreover, we report that Ncr1p‐deficient cells displayed high levels of long chain bases (LCB), and that Sch9p‐phospho‐T570 and Sch9p levels increased in ncr1Δ cells through a mechanism regulated by Pkh1p, a LCB‐activated protein kinase. Notably, deletion of PKH1 or SCH9 suppressed ncr1Δ phenotypes but downregulation of de novo sphingolipid biosynthesis had no protective effect, suggesting that LCBs accumulation may result from an increased turnover of complex sphingolipids. These results suggest that sphingolipid signalling through Pkh1p‐Sch9p mediate mitochondrial dysfunction, oxidative stress sensitivity and shortened chronological lifespan in the yeast model of Niemann‐Pick type C disease.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell‐based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild‐type than rad30Δ cells. In contrast, higher number of Polη‐deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild‐type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild‐type C. albicans. Despite the morphological differences, both wild‐type and rad30? C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.     

Swi2/Snf2-like protein Uls1 functions in the Sgs1-dependent pathway of maintenance of rDNA stability and alleviation of replication stress

The Saccharomyces cerevisiae Uls1 belongs to the Swi2/Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here we show that Uls1 is implicated in DNA repair independently of the replication stress response pathways mediated by the endonucleases Mus81 and Yen1 and the helicases Mph1 and Srs2. Uls1 works together with Sgs1 and we demonstrate that the attenuation of replication stress-related defects in sgs1Δ by deletion of ULS1 depends on a functional of Rad51 recombinase and post-replication repair pathway mediated by Rad18 and Rad5, but not on the translesion polymerase, Rev3. The higher resistance of sgs1Δ uls1Δ mutants to genotoxic stress compared to single sgs1Δ cells is not the result of decreased formation or accelerated resolution of recombination-dependent DNA structures. Instead, deletion of ULS1 restores stability of the rDNA region in sgs1Δ cells. Our data suggest that Uls1 may contribute to genomic stability during DNA synthesis and channel the repair of replication lesions into the Sgs1-dependent pathway, with DNA translocase and SUMO binding activities of Uls1 as well as a RING domain being essential for its functions in replication stress response.     

The ceramide-activated protein phosphatase Sit4p controls lifespan,mitochondrial function and cell cycle progression by regulating hexokinase 2 phosphorylation

Sit4p is the catalytic subunit of a ceramide-activated PP2A-like phosphatase that regulates cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan in yeast. In this study, we show that hexokinase 2 (Hxk2p) is hyperphosphorylated in sit4Δ mutants grown in glucose medium by a Snf1p-independent mechanism and Hxk2p-S15A mutation suppresses phenotypes associated with SIT4 deletion, namely growth arrest at G1 phase, derepression of mitochondrial respiration, H₂O₂ resistance and lifespan extension. Consistently, the activation of Sit4p in isc1Δ mutants, which has been associated with premature aging, leads to Hxk2p hypophosphorylation, and the expression of Hxk2p-S15E increases the lifespan of isc1Δ cells. The overall results suggest that Hxk2p functions downstream of Sit4p in the control of cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan.