Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

Mathematical model for the role of multiple pericentromeric repeats on heterochromatin assembly.

Although the length and constituting sequences for pericentromeric repeats are highly variable across eukaryotes, the presence of multiple pericentromeric repeats is one of the conserved features of the eukaryotic chromosomes. Pericentromeric heterochromatin is often misregulated in human diseases, with the expansion of pericentromeric repeats in human solid cancers. In this article, we have developed a mathematical model of the RNAi-dependent methylation of H3K9 in the pericentromeric region of fission yeast. Our model, which takes copy number as an explicit parameter, predicts that the pericentromere is silenced only if there are many copies of repeats. It becomes bistable or desilenced if the copy number of repeats is reduced. This suggests that the copy number of pericentromeric repeats alone can determine the fate of heterochromatin silencing in fission yeast. Through sensitivity analysis, we identified parameters that favor bistability and desilencing. Stochastic simulation shows that faster cell division and noise favor the desilenced state. These results show the unexpected role of pericentromeric repeat copy number in gene silencing and provide a quantitative basis for how the copy number allows or protects repetitive and unique parts of the genome from heterochromatin silencing, respectively.

Bqt4 affects relative movement between SPB and nucleolus in fission yeast.

Movement dynamics in the nucleus involve various biological processes, including DNA repair, which is crucial for cancer prevention. Changes in the movement of the components of the nucleus indicate the changes in movement dynamics in the nucleus. In Schizosaccharomyces pombe, the inner nuclear membrane protein Bqt4 plays an essential role in attaching telomeres to the nuclear envelope. We observed that the deletion of bqt4+ caused a significant decrease in the mean square displacement (MSD) calculated from the distance between the nucleolar center and spindle pole body (SPB), hereafter referred to as MSD(SPB-Nucleolus). The MSD(SPB-Nucleolus) decrease in bqt4Δ was microtubule-dependent. The Rap1-binding ability loss mutant, bqt4F46A, and nonspecific DNA-binding ability mutants, bqt43E-A, did not exhibit an MSD(SPB-Nucleolus) decrease compared to the WT. Moreover, the bqt43E-Arap1Δ double mutant and 1-262 amino acids truncated mutant bqt4ΔN (263-432), which does not have either Rap1-binding or nonspecific DNA-binding abilities, did not exhibit the MSD(SPB-Nucleolus) decrease to the same extent as bqt4Δ. These results suggest that the unknown function of Bqt4 in the C-terminal domain is essential for the maintenance of the pattern of relative movement between SPB and the nucleolus.

Live-cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeast Schizosaccharomyces pombe.

The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.

Cdc48 and its co-factor Ufd1 extract CENP-A from centromeric chromatin and can induce chromosome elimination in the fission yeast Schizosaccharomyces pombe.

CENP-A determines the identity of the centromere. Because the position and size of the centromere and its number per chromosome must be maintained, the distribution of CENP-A is strictly regulated. In this study, we have aimed to understand mechanisms to regulate the distribution of CENP-A (Cnp1SP) in fission yeast. A mutant of the ufd1+ gene (ufd1-73) encoding a cofactor of Cdc48 ATPase is sensitive to Cnp1 expressed at a high level and allows mislocalization of Cnp1. The level of Cnp1 in centromeric chromatin is increased in the ufd1-73 mutant even when Cnp1 is expressed at a normal level. A preexisting mutant of the cdc48+ gene (cdc48-353) phenocopies the ufd1-73 mutant. We have also shown that Cdc48 and Ufd1 proteins interact physically with centromeric chromatin. Finally, Cdc48 ATPase with Ufd1 artificially recruited to the centromere of a mini-chromosome (Ch16) induce a loss of Cnp1 from Ch16, leading to an increased rate of chromosome loss. It appears that Cdc48 ATPase, together with its cofactor Ufd1 remove excess Cnp1 from chromatin, likely in a direct manner. This mechanism may play a role in centromere disassembly, a process to eliminate Cnp1 to inactivate the kinetochore function during development, differentiation, and stress response.

Regulation of sexual differentiation initiation in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an excellent model organism to explore cellular events owing to rich tools in genetics, molecular biology, cellular biology, and biochemistry. Schizosaccharomyces pombe proliferates continuously when nutrients are abundant but arrests in G1 phase upon depletion of nutrients such as nitrogen and glucose. When cells of opposite mating types are present, cells conjugate, fuse, undergo meiosis, and finally form 4 spores. This sexual differentiation process in S. pombe has been studied extensively. To execute sexual differentiation, the glucose-sensing cAMP-PKA (cyclic adenosine monophosphate-protein kinase A) pathway, nitrogen-sensing TOR (target of rapamycin) pathway, and SAPK (stress-activating protein kinase) pathway are crucial, and the MAPK (mitogen-activating protein kinase) cascade is essential for pheromone sensing. These signals regulate ste11 at the transcriptional and translational levels, and Ste11 is modified in multiple ways. This review summarizes the initiation of sexual differentiation in S. pombe based on results I have helped to obtain, including the work of many excellent researchers.

The deletion of ppr2 interferes iron sensing and leads to oxidative stress response in Schizosaccharomyces pombe.

Pentatricopeptide repeat proteins are involved in mitochondrial both transcriptional and posttranscriptional regulation. Schizosaccharomyces pombe Ppr2 is a general mitochondrial translation factor that plays a critical role in the synthesis of all mitochondrial DNA-encoded oxidative phosphorylation subunits, which are essential for mitochondrial respiration. Our previous analysis showed that ppr2 deletion resulted in increased expression of iron uptake genes and caused ferroptosis-like cell death in S. pombe. In the present work, we showed that deletion of ppr2 reduced viability on glycerol- and galactose-containing media.Php4 is a transcription repressor that regulates iron homeostasis in fission yeast. We found that in the ppr2 deletion strain, Php4 was constitutively active and accumulated in the nucleus in the stationary phase. We also found that deletion of ppr2 decreased the ferroptosis-related protein Gpx1 in the mitochondria. Overexpression of Gpx1 improves the viability of Δppr2 cells. We showed that the deletion of ppr2 increased the production of ROS, downregulated heme synthesis and iron-sulfur cluster proteins, and induced stress proteins. Finally, we observed the nuclear accumulation of Pap1-GFP and Sty1-GFP, suggesting that Sty1 and Pap1 in response to cellular stress in the ppr2 deletion strain. These results suggest thatppr2 deletion may cause mitochondrial dysfunction, which is likely to lead to iron-sensing defect and iron starvation response, resulting in perturbation of iron homeostasis and increased hydroxyl radical production. The increased hydroxyl radical production triggers cellular responses in theppr2 deletion strain.

RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.

Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.

LAMMER Kinase Governs the Expression and Cellular Localization of Gas2, a Key Regulator of Flocculation in Schizosaccharomyces pombe.

It was reported that LAMMER kinase in Schizosaccharomyces pombe plays an important role in cation-dependent and galactose-specific flocculation. Analogous to other flocculating yeasts, when cell wall extracts of the Δlkh1 strain were treated to the wild-type strain, it displayed flocculation. Gas2, a 1,3-ß-glucanosyl transferase, was isolated from the EDTA-extracted cell-surface proteins in the Δlkh1 strain. While disruption of the gas2+ gene was not lethal and reduced the flocculation activity of the ∆lkh1 strain, the expression of a secreted form of Gas2, in which the GPI anchor addition sequences had been removed, conferred the ability to flocculate upon the WT strain. The Gas2-mediated flocculation was strongly inhibited by galactose but not by glucose. Immunostaining analysis showed that the cell surface localization of Gas2 was crucial for the flocculation of fission yeast. In addition, we identified the regulation of mbx2+ expression by Lkh1 using RT-qPCR. Taken together, we found that Lkh1 induces asexual flocculation by regulating not only the localization of Gas2 but also the transcription of gas2+ through Mbx2.

A role for the carbon source of the cell and protein kinase A in regulating the S. pombe septation initiation network.

The septation initiation network (SIN) is a conserved signal transduction network, which is important for cytokinesis in Schizosaccharomyces pombe. The SIN component Etd1p is required for association of some SIN proteins with the spindle pole body (SPB) during anaphase and for contractile ring formation. We show that tethering of Cdc7p or Sid1p to the SIN scaffold Cdc11p at the SPB, rescues etd1-Δ. Analysis of a suppressor of the mutant etd1-M9 revealed that SIN signalling is influenced by the carbon source of the cell. Growth on a non-fermentable carbon source glycerol reduces the requirement for SIN signalling but does not bypass it. The decreased need for SIN signalling is mediated largely by reduction of protein kinase A activity, and it is phenocopied by deletion of pka1 on glucose medium. We conclude that protein kinase A is an important regulator of the SIN, and that SIN signalling is regulated by the carbon source of the cell.

Fission yeast Wee1 is required for stable kinetochore-microtubule attachment.

Wee1 is a cell cycle regulator that phosphorylates Cdk1/Cdc2 and inhibits G2/M transition. Loss of Wee1 in fission yeast results in an early onset of mitosis. Interestingly, we found that cells lacking Wee1 require the functional spindle checkpoint for their viability. Genetic analysis indicated that the requirement is not attributable to the early onset of mitosis. Live-cell imaging revealed that some kinetochores are not attached or bioriented in the wee1 mutant. Furthermore, Mad2, a component of the spindle checkpoint known to recognize unattached kinetochores, accumulates in the vicinity of the spindle, representing activation of the spindle checkpoint in the mutant. It appears that the wee1 mutant cannot maintain stable kinetochore-microtubule attachment, and relies on the delay imposed by the spindle checkpoint for establishing biorientation of kinetochores. This study revealed a role of Wee1 in ensuring accurate segregation of chromosomes during mitosis, and thus provided a basis for a new principle of cancer treatment with Wee1 inhibitors.

Heterologous expression of rice OsEXO70FX1 confers tolerance to cadmium in Arabidopsis thaliana and fission yeast.

Cadmium (Cd) is an environmental toxicant that accumulates in grains, which greatly increases the risk of human exposure to Cd via food chain. The exocytosis of Cd is one of the essential detoxification mechanisms in plants. OsEXO70s, which facilitate the fusion of secretory vesicles and target membranes, has undergone significant expansion in rice. Here, we uncovered 40 OsEXO70 genes characterized by genome-wide profiling and focused on the potential functions of OsEXO70s, especially OsEXO70FX1, in Cd stress. Overexpression of OsEXO70FX1 enhanced both diamide and Cd tolerances in Schizosaccharomyces pombe (S. pombe), and in Arabidopsis resulted in 11% more seedlings survival rate and about 70% longer primary roots under Cd treatment compared with WT (empty vector). Meanwhile, Cd treatment upregulated the expression levels of some exocyst subunits in overexpression lines. Trichomes isolated from overexpression lines were observed to accumulate more Cd. Also, reactive oxygen species (ROS) induced by Cd stress reflected less sensitivity of OsEXO70FX1 overexpression lines to Cd stress, which was evidenced in the Cd determination assay. These results provide the fundament to future research on rice EXO70 family and suggest that it may have evolved a specialized role in response to Cd stress.

The cysteine-rich domain in CENP-A chaperone Scm3HJURP ensures centromere targeting and kinetochore integrity.

Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.

Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP6 kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast.

The inositol pyrophosphate signaling molecule 1,5-IP8 is an agonist of RNA 3'-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1, pho84, and tgp1. IP8 is synthesized from 5-IP7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1+ cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP6 kinase Kcs1 that generates 5-IP7, the immediate precursor to IP8. Transcription profiling of the kcs1 mutants in an asp1+ background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP3, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP8 governs repression of fission yeast phosphate homeostasis genes pho1, pho84, and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP8 agonism of transcription termination. It was assumed that IP8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP3 and the essential Kcs1 kinase that converts IP6 to 5-IP7, the immediate precursor of IP8.

Phosphate uptake restriction, phosphate export, and polyphosphate synthesis contribute synergistically to cellular proliferation and survival.

Phosphate (Pi) is a macronutrient, and Pi homeostasis is essential for life. Pi homeostasis has been intensively studied; however, many questions remain, even at the cellular level. Using Schizosaccharomyces pombe, we sought to better understand cellular Pi homeostasis and showed that three Pi regulators with SPX domains, Xpr1/Spx2, Pqr1, and the VTC complex synergistically contribute to Pi homeostasis to support cell proliferation and survival. SPX domains bind to inositol pyrophosphate and modulate activities of Pi-related proteins. Xpr1 is a plasma membrane protein and its Pi-exporting activity has been demonstrated in metazoan orthologs, but not in fungi. We first found that S. pombe Xpr1 is a Pi exporter, activity of which is regulated and accelerated in the mutants of Pqr1 and the VTC complex. Pqr1 is the ubiquitin ligase downregulating the Pi importers, Pho84 and Pho842. The VTC complex synthesizes polyphosphate in vacuoles. Triple deletion of Xpr1, Pqr1, and Vtc4, the catalytic core of the VTC complex, was nearly lethal in normal medium but survivable at lower [Pi]. All double-deletion mutants of the three genes were viable at normal Pi, but Δpqr1Δxpr1 showed severe viability loss at high [Pi], accompanied by hyper-elevation of cellular total Pi and free Pi. This study suggests that the three cellular processes, restriction of Pi uptake, Pi export, and polyP synthesis, contribute synergistically to cell proliferation through maintenance of Pi homeostasis, leading to the hypothesis that cooperation between Pqr1, Xpr1, and the VTC complex protects the cytoplasm and/or the nucleus from lethal elevation of free Pi.

Activities, substrate specificity, and genetic interactions of fission yeast Siw14, a cysteinyl-phosphatase-type inositol pyrophosphatase.

IMPORTANCE: The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular 1,5-IP8 levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize Schizosaccharomyces pombe Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8. Genetic analyses implicate SpSiw14 in 1,5-IP8 catabolism in vivo, insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and siw14∆ aps1∆ lethality depends on synthesis of 1,5-IP8 by the Asp1 kinase. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of 3'-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP8 toxicosis.

Pkd2, mutations linking to autosomal dominant polycystic kidney disease, localizes to the endoplasmic reticulum and regulates calcium signaling in fission yeast.

Autosomal dominant polycystic kidney disease (ADPKD) is a renal disorder caused by mutations in the PKD2 gene, which encodes polycystin-2/Pkd2, a transient receptor potential channel. The precise role of Pkd2 in cyst formation remains unclear. The fission yeast Schizosaccharomyces pombe has a putative transient receptor potential channel, Pkd2, which shares similarities with human Pkd2. In this study, truncation analyses of fission yeast Pkd2 were conducted to investigate its localization and function. The results revealed that Pkd2 localizes not only to the plasma membrane but also to the endoplasmic reticulum (ER) in fission yeast. Furthermore, Pkd2 regulates calcium signaling in fission yeast, with the transmembrane domains of Pkd2 being sufficient for these processes. Specifically, the C-terminal region of Pkd2 plays a crucial role in the regulation of calcium signaling. Interestingly, human Pkd2 also localized to the ER and had some impact on calcium signaling in fission yeast. However, human Pkd2 failed to suppress the loss of fission yeast Pkd2. These findings indicate that hPkd2 may not completely substitute for cellular physiology of fission yeast Pkd2. This study provides insights into the localization and functional characteristics of Pkd2 in fission yeast, contributing to our understanding of the pathogenesis of ADPKD.

[Inactivation of Ras1 in Fission Yeast Aggravates the Oxidative Stress Response Induced by Tert Butyl Hydroperoxide (tBHP)].

Ras proteins are small GTPases and function as molecular switches to regulate cellular homeostasis. Ras-dependent signalling pathways regulate several essential processes such as cell cycle progression, growth, migration, apoptosis, and senescence. The dysregulation of Ras signaling pathway has been linked to several pathological outcomes. A potential role of RAS in regulating the redox signalling pathway has been established that includes the manipulation of ROS levels to provide a redox milieu that might be conducive to carcinogenesis. Reactive oxygen species (ROS) and mitochondrial impairment have been proposed as major factors affecting the physiology of cells and implicated in several pathologies. The present study was conducted to evaluate the role of Ras1, tert Butyl hydroperoxide (tBHP), and antimycin A in oxidative stress response in Schizosaccharomyces pombe cells. We observed decreased cell survival, higher levels of ROS, and mitochondrial dysfunctionality in ras1Δ cells and tBHP as well as respiratory inhibitor, antimycin A treated wild type cells. Furthermore, these defects were more profound in ras1Δ cells treated with tBHP or antimycin A. Additionally, Ras1 also has been shown to regulate the expression and activity of several antioxidant enzymes like glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), and catalase. Together, these results suggest the potential role of S. pombe Ras1 in mitigating oxidative stress response.

The AAA-ATPase Yta4/ATAD1 interacts with the mitochondrial divisome to inhibit mitochondrial fission.

Mitochondria are in a constant balance of fusion and fission. Excessive fission or deficient fusion leads to mitochondrial fragmentation, causing mitochondrial dysfunction and physiological disorders. How the cell prevents excessive fission of mitochondria is not well understood. Here, we report that the fission yeast AAA-ATPase Yta4, which is the homolog of budding yeast Msp1 responsible for clearing mistargeted tail-anchored (TA) proteins on mitochondria, plays a critical role in preventing excessive mitochondrial fission. The absence of Yta4 leads to mild mitochondrial fragmentation in a Dnm1-dependent manner but severe mitochondrial fragmentation upon induction of mitochondrial depolarization. Overexpression of Yta4 delocalizes the receptor proteins of Dnm1, i.e., Fis1 (a TA protein) and Mdv1 (the bridging protein between Fis1 and Dnm1), from mitochondria and reduces the localization of Dnm1 to mitochondria. The effect of Yta4 overexpression on Fis1 and Mdv1, but not Dnm1, depends on the ATPase and translocase activities of Yta4. Moreover, Yta4 interacts with Dnm1, Mdv1, and Fis1. In addition, Yta4 competes with Dnm1 for binding Mdv1 and decreases the affinity of Dnm1 for GTP and inhibits Dnm1 assembly in vitro. These findings suggest a model, in which Yta4 inhibits mitochondrial fission by inhibiting the function of the mitochondrial divisome composed of Fis1, Mdv1, and Dnm1. Therefore, the present work reveals an uncharacterized molecular mechanism underlying the inhibition of mitochondrial fission.

The Product of the Fission Yeast fhl1 Gene Binds to the HomolE Box and Activates In Vitro Transcription of Ribosomal Protein Genes.

Fission yeast ribosomal protein genes (RPGs) contain a HomolD box as a core promoter element required for transcription. Some of the RPGs also contain a consensus sequence named HomolE, located upstream of the HomolD box. The HomolE box acts as an upstream activating sequence (UAS), and it is able to activate transcription in RPG promoters containing a HomolD box. In this work, we identified a HomolE-binding protein (HEBP) as a polypeptide of 100 kDa, which was able to bind to the HomolE box in a Southwestern blot assay. The features of this polypeptide were similar to the product of the fhl1 gene of fission yeast. The Fhl1 protein is the homolog of the FHL1 protein of budding yeast and possesses fork-head-associated (FHA) and fork-head (FH) domains. The product of the fhl1 gene was expressed and purified from bacteria, and it was demonstrated that is able to bind the HomolE box in an electrophoretic mobility assay (EMSA), as well as being able to activate in vitro transcription from an RPG gene promoter containing HomolE boxes upstream of the HomolD box. These results indicate that the product of the fhl1 gene of fission yeast can bind to the HomolE box, and it activates the transcription of RPGs.