Nutrient-dependent control of RNA polymerase II elongation rate regulates specific gene expression programs by alternative polyadenylation.

Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.

The flavohemoglobin Yhb1 is a new interacting partner of the heme transporter Str3.

Nitric oxide (˙NO) is a free radical that induces nitrosative stress, which can jeopardize cell viability. Yeasts have evolved diverse detoxification mechanisms to effectively counteract ˙NO-mediated cytotoxicity. One mechanism relies on the flavohemoglobin Yhb1, whereas a second one requires the S-nitrosoglutathione reductase Fmd2. To investigate heme-dependent activation of Yhb1 in response to ˙NO, we use hem1Δ-derivative Schizosaccharomyces pombe strains lacking the initial enzyme in heme biosynthesis, forcing cells to assimilate heme from external sources. Under these conditions, yhb1+ mRNA levels are repressed in the presence of iron through a mechanism involving the GATA-type transcriptional repressor Fep1. In contrast, when iron levels are low, the transcription of yhb1+ is derepressed and further induced in the presence of the ˙NO donor DETANONOate. Cells lacking Yhb1 or expressing inactive forms of Yhb1 fail to grow in a hemin-dependent manner when exposed to DETANONOate. Similarly, the loss of function of the heme transporter Str3 phenocopies the effects of Yhb1 disruption by causing hypersensitivity to DETANONOate under hemin-dependent culture conditions. Coimmunoprecipitation and bimolecular fluorescence complementation assays demonstrate the interaction between Yhb1 and the heme transporter Str3. Collectively, our findings unveil a novel pathway for activating Yhb1, fortifying yeast cells against nitrosative stress.

Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal.

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH. In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using CD spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.

A novel role of the fission yeast sulfiredoxin Srx1 in heme acquisition.

The transporter Str3 promotes heme import in Schizosaccharomyces pombe cells that lack the heme receptor Shu1 and are deficient in heme biosynthesis. Under microaerobic conditions, the peroxiredoxin Tpx1 acts as a heme scavenger within the Str3-dependent pathway. Here, we show that Srx1, a sulfiredoxin known to interact with Tpx1, is essential for optimal growth in the presence of hemin. The expression of Srx1 is induced in response to low iron and repressed under iron repletion. Coimmunoprecipitation and bimolecular fluorescence complementation experiments show that Srx1 interacts with Str3. Although the interaction between Srx1 and Str3 is weakened, it is still observed in tpx1Δ mutant cells or when Str3 is coexpressed with a mutant form of Srx1 (mutD) that cannot bind Tpx1. Further analysis by absorbance spectroscopy and hemin-agarose pull-down assays confirms the binding of Srx1 to hemin, with an equilibrium constant value of 2.56 µM. To validate the Srx1-hemin association, we utilize a Srx1 mutant (mutH) that fails to interact with hemin. Notably, when Srx1 binds to hemin, it partially shields hemin from degradation caused by hydrogen peroxide. Collectively, these findings elucidate an additional function of the sulfiredoxin Srx1, beyond its conventional role in oxidative stress defense.

Fission yeast RNA-binding proteins Puf2 and Puf4 are involved in repression of ferrireductase Frp1 expression in response to iron.

This study identifies a post-transcriptional mechanism of iron uptake regulation by Puf2 and Puf4 of the Pumilio and FBF (Puf) family of RNA-binding proteins in Schizosaccharomyces pombe. Cells expressing Puf2 and Puf4 stimulate decay of the frp1+ mRNA encoding a key enzyme of the reductive iron uptake pathway. Results consistently showed that frp1+ mRNA is stabilized in puf2Δ puf4Δ mutant cells under iron-replete conditions. As a result, puf2Δ puf4Δ cells exhibit an increased sensitivity to iron accompanied by enhanced ferrireductase activity. A pool of GFP-frp1+ 3'UTR RNAs was generated using a reporter gene containing the 3' untranslated region (UTR) of frp1+ that was under the control of a regulatable promoter. Results showed that Puf2 and Puf4 accelerate the destabilization of mRNAs containing the frp1+ 3'UTR which harbors two Pumilio response elements (PREs). Binding studies revealed that the PUM-homology RNA-binding domain of Puf2 and Puf4 expressed in Escherichia coli specifically interacts with PREs in the frp1+ 3'UTR. Using RNA immunoprecipitation in combination with reverse transcription qPCR assays, results showed that Puf2 and Puf4 interact preferentially with frp1+ mRNA under basal and iron-replete conditions, thereby contributing to inhibit Frp1 production and protecting cells against toxic levels of iron.

Hemeprotein Tpx1 interacts with cell-surface heme transporter Str3 in Schizosaccharomyces pombe.

Str3 is a transmembrane protein that mediates low-affinity heme uptake in Schizosaccharomyces pombe. Under iron-limiting conditions, Str3 remains at the cell surface in the presence of increasing hemin concentrations. Using a proximity-dependent biotinylation approach coupled to mass spectrometry and coimmunoprecipitation assays, we report that the peroxiredoxin Tpx1 is a binding partner of Str3. Under microaerobic conditions, cells deficient in heme biosynthesis and lacking the heme receptor Shu1 exhibit poor hemin-dependent growth in the absence of Tpx1. Analysis of membrane protein preparations from iron-starved hem1Δ shu1Δ str3Δ tpx1Δ cells coexpressing Str3-GFP and TAP-Tpx1 showed that TAP-Tpx1 is enriched in membrane protein fractions in response to hemin. Bimolecular fluorescence complementation assays brought additional evidence that an interaction between Tpx1 and Str3 occurs at the plasma membrane. Results showed that Tpx1 exhibits an equilibrium constant value of 0.26 µM for hemin. The association of Tpx1 with hemin protects hemin from degradation by H2 O2 . The peroxidase activity of hemin is lowered when it is bound to Tpx1. Taken together, these results revealed that Tpx1 is a novel interacting partner of Str3. Our data are the first example of an interaction between a cytoplasmic heme-binding protein and a cell-surface heme transporter.

Iron deficiency leads to repression of a non-canonical methionine salvage pathway in Schizosaccharomyces pombe.

The methionine salvage pathway (MSP) regenerates methionine from 5'-methylthioadenosine (MTA). Aerobic MSP consists of six enzymatic steps. The mug14+ and adi1+ genes that are involved in the third and fifth steps of the pathway are repressed when Schizosaccharomyces pombe undergoes a transition from high- to low-iron conditions. Results consistently show that methionine auxotrophic cells (met6Δ) require iron for growth in the presence of MTA as the sole source of methionine. Inactivation of the iron-using protein Adi1 leads to defects in the utilization of MTA. In the case of the third step of the pathway, co-expression of two distinct proteins, Mta3 and Mde1, is required. These proteins are interdependent to rescue MTA-dependent growth deficit of met6Δ cells. Coimmunoprecipitation experiments showed that Mta3 is a binding partner of Mde1. Meiotic met6Δ cells co-expressing mta3+ and mde1+ or mta3+ and mug14+ produce comparable levels of spores in the presence of MTA, revealing that Mde1 and Mug14 share a common function when co-expressed with Mta3 in sporulating cells. In sum, our findings unveil several novel features of MSP, especially with respect to its regulation by iron and the discovery of a non-canonical third enzymatic step in the fission yeast.

Heme acquisition by Shu1 requires Nbr1 and proteins of the ESCRT complex in Schizosaccharomyces pombe.

Assimilation of heme is mediated by the cell surface protein Shu1 in Schizosaccharomyces pombe. Shu1 undergoes internalization from the cell surface to the vacuole in response to high concentrations of hemin. Here, we have identified cellular components that are involved in mediating vacuolar targeting of Shu1. Cells deficient in heme biosynthesis and lacking the polyubiquitin gene ubi4+ exhibit poor growth in the presence of exogenous hemin as a sole source of heme. Microscopic analyses of hem1Δ shu1Δ ubi4Δ cells expressing a functional HA4 -tagged Shu1 show that Shu1 localizes to the cell surface. Ubiquitinated Nbr1 functions as a receptor for the endosomal sorting complexes required for transport (ESCRT) that delivers cargos to the vacuole. Inactivation of nbr1+ , ESCRT-0 hse1+ or ESCRT-I sst6+ results in hem1Δ cells being unable to use exogenous hemin for the growth. Using lysate preparations from hemin-treated cells, Shu1-Nbr1 and Shu1-Hse1 complexes are detected by coimmunoprecipitation experiments. Further analysis by immunofluorescence microscopy shows that Shu1 is unable to reach vacuoles of hemin-treated cells harboring a deletion for one of the following genes: ubi4+ , nbr1+ , hse1+ and sst6+ . Together, these results reveal that hemin-mediated vacuolar targeting of Shu1 requires Ubi4-dependent ubiquitination, the receptor Nbr1 and the ESCRT proteins Hse1 and Sst6.

Machinery for fungal heme acquisition.

Iron is essential for nearly all aerobic organisms. One source of iron in nature is in the form of heme. Due to its critical physiological importance as a cofactor for several enzymes, organisms have evolved various means to secure heme for their needs. In the case of heme prototrophs, these organisms possess a highly conserved eight-step biosynthetic pathway. Another means used by many organisms is to acquire heme from external sources. As opposed to the knowledge of enzymes responsible for heme biosynthesis, the nature of the players and mechanisms involved in the acquisition of exogenous heme is limited. This review focuses on a description of newly discovered proteins that have novel functions in heme assimilation in the model organism Schizosaccharomyces pombe. This tractable model allows the use of the power of genetics to selectively block heme biosynthesis, setting conditions to investigate the mechanisms by which external heme is taken up by the cells. Studies have revealed that S. pombe possesses two independent heme uptake systems that require Shu1 and Str3, respectively. Heme-bound iron is captured by Shu1 at the cell surface, triggering its internalization to the vacuole with the aid of ubiquitinated proteins and the ESCRT machinery. In the case of the plasma membrane transporter Str3, it promotes cellular heme import in cells lacking Shu1. The discovery of these two pathways may contribute to gain novel insights into the mechanisms whereby fungi assimilate heme, which is an essentially biological process for their ability to invade and colonize new niches.

The major facilitator transporter Str3 is required for low-affinity heme acquisition in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, acquisition of exogenous heme is largely mediated by the cell membrane-associated Shu1. Here, we report that Str3, a member of the major facilitator superfamily of transporters, promotes cellular heme import. Using a strain that cannot synthesize heme de novo (hem1Δ) and lacks Shu1, we found that the heme-dependent growth deficit of this strain is rescued by hemin supplementation in the presence of Str3. Microscopic analyses of a hem1Δ shu1Δ str3Δ mutant strain in the presence of the heme analog zinc mesoporphyrin IX (ZnMP) revealed that ZnMP fails to accumulate within the mutant cells. In contrast, Str3-expressing hem1Δ shu1Δ cells could take up ZnMP at a 10-µm concentration. The yeast Saccharomyces cerevisiae cannot efficiently transport exogenously supplied hemin. However, heterologous expression of Str3 from S. pombe in S. cerevisiae resulted in ZnMP accumulation within S. cerevisiae cells. Moreover, hemin-agarose pulldown assays revealed that Str3 binds hemin. In contrast, an Str3 mutant in which Tyr and Ser residues of two putative heme-binding motifs (530YX3Y534 and 552SX4Y557) had been replaced with alanines exhibited a loss of affinity for hemin. Furthermore, this Str3 mutant failed to rescue the heme-dependent growth deficit of a hem1Δ shu1Δ str3Δ strain. Further analysis by absorbance spectroscopy disclosed that a predicted extracellular loop region in Str3 containing the two putative heme-binding motifs interacts with hemin, with a KD of 6.6 µm Taken together, these results indicate that Str3 is a second cell-surface membrane protein for acquisition of exogenous heme in S. pombe.

Heme Assimilation in Schizosaccharomyces pombe Requires Cell-surface-anchored Protein Shu1 and Vacuolar Transporter Abc3.

The Schizosaccharomyces pombe shu1+ gene encodes a cell-surface protein required for assimilation of exogenous heme. In this study, shaving experiments showed that Shu1 is released from membrane preparations when spheroplast lysates are incubated with phosphoinositide-specific phospholipase C (PI-PLC). Shu1 cleavability by PI-PLC and its predicted hydropathy profile strongly suggested that Shu1 is a glycosylphosphatidylinositol-anchored protein. When heme biosynthesis is selectively blocked in hem1Δ mutant cells, the heme analog zinc mesoporphyrin IX (ZnMP) first accumulates into vacuoles and then subsequently, within the cytoplasm in a rapid and Shu1-dependent manner. An HA4-tagged shu1+ allele that retained wild-type function localizes to the cell surface in response to low hemin concentrations, but under high hemin concentrations, Shu1-HA4 re-localizes to the vacuolar membrane. Inactivation of abc3+, encoding a vacuolar membrane transporter, results in hem1Δ abc3Δ mutant cells being unable to grow in the presence of hemin as the sole iron source. In hem1Δ abc3Δ cells, ZnMP accumulates primarily in vacuoles and does not sequentially accumulate in the cytosol. Consistent with a role for Abc3 as vacuolar hemin exporter, results with hemin-agarose pulldown assays showed that Abc3 binds to hemin. In contrast, an Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. Taken together, these results show that Shu1 undergoes rapid hemin-induced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol.

Cell-surface copper transporters and superoxide dismutase 1 are essential for outgrowth during fungal spore germination.

During fungal spore germination, a resting spore returns to a conventional mode of cell division and resumes vegetative growth, but the requirements for spore germination are incompletely understood. Here, we show that copper is essential for spore germination in Schizosaccharomyces pombe Germinating spores develop a single germ tube that emerges from the outer spore wall in a process called outgrowth. Under low-copper conditions, the copper transporters Ctr4 and Ctr5 are maximally expressed at the onset of outgrowth. In the case of Ctr6, its expression is broader, taking place before and during outgrowth. Spores lacking Ctr4, Ctr5, and the copper sensor Cuf1 exhibit complete germination arrest at outgrowth. In contrast, ctr6 deletion only partially interferes with formation of outgrowing spores. At outgrowth, Ctr4-GFP and Ctr5-Cherry first co-localize at the spore contour, followed by re-location to a middle peripheral spore region. Subsequently, they move away from the spore body to occupy the periphery of the nascent cell. After breaking of spore dormancy, Ctr6 localizes to the vacuole membranes that are enriched in the spore body relative to the germ tube. Using a copper-binding tracker, results showed that labile copper is preferentially localized to the spore body. Further analysis showed that Ctr4 and Ctr6 are required for copper-dependent activation of the superoxide dismutase 1 (SOD1) during spore germination. This activation is critical because the loss of SOD1 activity blocked spore germination at outgrowth. Taken together, these results indicate that cell-surface copper transporters and SOD1 are required for completion of the spore germination program.

The Zap1 transcriptional activator negatively regulates translation of the RTC4 mRNA through the use of alternative 5' transcript leaders.

The zinc-responsive transcription activator Zap1 plays a central role in zinc homeostasis in the budding yeast Saccharomyces cerevisiae. In zinc-deficient cells, Zap1 binds to zinc responsive elements in target gene promoters and activates gene expression. In most cases, Zap1-dependent gene activation results in increased levels of mRNAs and proteins. However, Zap1-dependent activation of RTC4 results in increased levels of the RTC4 mRNA and decreased levels of the Rtc4 protein. This atypical regulation results from Zap1-mediated changes in the transcriptional start site for RTC4 and the production of a RTC4 transcript with a longer 5' leader. This long RTC4 transcript contains small upstream open reading frames that prevent translation of the downstream RTC4 ORF. The new studies with Zap1 highlight how a transcriptional activator can facilitate decreased protein expression.

An antisense RNA-mediated mechanism eliminates a meiosis-specific copper-regulated transcript in mitotic cells.

Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1(+) gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1(+) sense transcript, inhibiting cum1(+) expression in vegetative cells. Inhibition of cum1(+) transcription was not at the level of its initiation because fusion of the cum1(+) promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1(+)-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1(+) led to the exclusive production of a shorter iss1(+) mRNA isoform, thereby allowing accumulation of cum1(+) sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1(+) antisense mRNA could pair with cum1(+) sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1(+) sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells.

Shu1 is a cell-surface protein involved in iron acquisition from heme in Schizosaccharomyces pombe.

Iron is an essential metal cofactor that is required for many biological processes. Eukaryotic cells have consequently developed different strategies for its acquisition. Until now, Schizosaccharomyces pombe was known to use reductive iron uptake and siderophore-bound iron transport to scavenge iron from the environment. Here, we report the identification of a gene designated shu1(+) that encodes a protein that enables S. pombe to take up extracellular heme for cell growth. When iron levels are low, the transcription of shu1(+) is induced, although its expression is repressed when iron levels rise. The iron-dependent down-regulation of shu1(+) requires the GATA-type transcriptional repressor Fep1, which strongly associates with a proximal promoter region of shu1(+) in vivo in response to iron repletion. HA4-tagged Shu1 localizes to the plasma membrane in cells expressing a functional shu1(+)-HA4 allele. When heme biosynthesis is selectively blocked in mutated S. pombe cells, their ability to acquire exogenous hemin or the fluorescent heme analog zinc mesoporphyrin IX is dependent on the expression of Shu1. Further analysis by absorbance spectroscopy and hemin-agarose pulldown assays showed that Shu1 interacts with hemin, with a KD of ∼2.2 µm. Taken together, results reported here revealed that S. pombe possesses an unexpected pathway for heme assimilation, which may also serve as a source of iron for cell growth.

Characterization of Schizosaccharomyces pombe copper transporter proteins in meiotic and sporulating cells.

Meiosis requires copper to undertake its program in which haploid gametes are produced from diploid precursor cells. In Schizosaccharomyces pombe, copper is transported by three members of the copper transporter (Ctr) family, namely Ctr4, Ctr5, and Ctr6. Although central for sexual differentiation, very little is known about the expression profile, cellular localization, and physiological contribution of the Ctr proteins during meiosis. Analysis of gene expression of ctr4(+) and ctr5(+) revealed that they are primarily expressed in early meiosis under low copper conditions. In the case of ctr6(+), its expression is broader, being detected throughout the entire meiotic process with an increase during middle- and late-phase meiosis. Whereas the expression of ctr4(+) and ctr5(+) is exclusively dependent on the presence of Cuf1, ctr6(+) gene expression relies on two distinct regulators, Cuf1 and Mei4. Ctr4 and Ctr5 proteins co-localize at the plasma membrane shortly after meiotic induction, whereas Ctr6 is located on the membrane of vacuoles. After meiotic divisions, Ctr4 and Ctr5 disappear from the cell surface, whereas Ctr6 undergoes an intracellular re-location to co-localize with the forespore membrane. Under copper-limiting conditions, disruption of ctr4(+) and ctr6(+) results in altered SOD1 activity, whereas these mutant cells exhibit substantially decreased levels of CAO activity mostly in early- and middle-phase meiosis. Collectively, these results emphasize the notion that Ctr proteins exhibit differential expression, localization, and contribution in delivering copper to SOD1 and Cao1 proteins during meiosis.

Molecular basis of the regulation of iron homeostasis in fission and filamentous yeasts.

When iron load exceeds that needed by fission and filamentous yeasts, iron-regulatory GATA-type transcription factors repress genes encoding iron acquisition systems. In contrast, under iron starvation, optimization of cellular iron utilization is coordinated by a specialized regulatory subunit of the CCAAT-binding factor that fosters repression of genes encoding iron-using proteins. Despite these findings, there is still limited knowledge concerning the mechanisms by which these iron-responsive regulators respond to high- or low-iron availability. To provide a framework for understanding common and distinct properties of iron-dependent transcriptional regulators, a repertoire of their functional domains in different fungal species is presented here. In addition, discovery of interacting partners of these iron-responsive factors contributes to provide additional insight into their properties.

Transcriptional regulation of the copper transporter mfc1 in meiotic cells.

Mfc1 is a meiosis-specific protein that mediates copper transport during the meiotic program in Schizosaccharomyces pombe. Although the mfc1(+) gene is induced at the transcriptional level in response to copper deprivation, the molecular determinants that are required for its copper starvation-dependent induction are unknown. Promoter deletion and site-directed mutagenesis have allowed identification of a new cis-regulatory element in the promoter region of the mfc1(+) gene. This cis-acting regulatory sequence containing the sequence TCGGCG is responsible for transcriptional activation of mfc1(+) under low-copper conditions. The TCGGCG sequence contains a CGG triplet known to serve as a binding site for members of the Zn(2)Cys(6) binuclear cluster transcriptional regulator family. In agreement with this fact, one member of this group of regulators, denoted Mca1, was found to be required for maximum induction of mfc1(+) gene expression. Analysis of Mca1 cellular distribution during meiosis revealed that it colocalizes with both chromosomes and sister chromatids during early, middle, and late phases of the meiotic program. Cells lacking Mca1 exhibited a meiotic arrest at metaphase I under low-copper conditions. Binding studies revealed that the N-terminal 150-residue segment of Mca1 expressed as a fusion protein in Escherichia coli specifically interacts with the TCGGCG sequence of the mfc1(+) promoter. Taken together, these results identify the cis-regulatory TCGGCG sequence and the transcription factor Mca1 as critical components for activation of the meiotic copper transport mfc1(+) gene in response to copper starvation.

Copper transport and regulation in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has been successfully used as a model to gain fundamental knowledge in understanding how eukaryotic cells acquire copper during vegetative growth. These studies have revealed the existence of a heteromeric Ctr4-Ctr5 plasma membrane complex that mediates uptake of copper within the cells. Furthermore, additional studies have led to the identification of one of the first vacuolar copper transporters, Ctr6, as well as the copper-responsive Cuf1 transcription factor. Recent investigations have extended the use of S. pombe to elucidate new roles for copper metabolism in meiotic differentiation. For example, these studies have led to the discovery of Mfc1, which turned out to be the first example of a meiosis-specific copper transporter. Whereas copper-dependent transcriptional regulation of the Ctr family members is under the control of Cuf1 during mitosis or meiosis, meiosis-specific copper transporter Mfc1 is regulated by the recently discovered transactivator Mca1. It is foreseeable that identification of novel meiotic copper-related proteins will serve as stepping stones to unravel fundamental aspects of copper homoeostasis.

The monothiol glutaredoxin Grx4 exerts an iron-dependent inhibitory effect on Php4 function.

When iron is scarce, Schizosaccharomyces pombe cells repress transcription of several genes that encode iron-using proteins. Php4 mediates this transcriptional control by specifically interacting with the CCAAT-binding core complex that is composed of Php2, Php3, and Php5. In contrast, when there is sufficient iron, Php4 is inactivated, thus allowing the transcription of many genes that encode iron-requiring proteins. Analysis by bimolecular fluorescence complementation and two-hybrid assays showed that Php4 and the monothiol glutaredoxin Grx4 physically interact with each other. Deletion mapping analysis revealed that the glutaredoxin (GRX) domain of Grx4 associates with Php4 in an iron-dependent manner. Site-directed mutagenesis identified the Cys172 of Grx4 as being required for this iron-dependent association. Subsequent analysis showed that, although the thioredoxin (TRX) domain of Grx4 interacts strongly with Php4, this interaction is insensitive to iron. Fine mapping analysis revealed that the Cys35 of Grx4 is necessary for the association between the TRX domain and Php4. Taken together, the results revealed that whereas the TRX domain interacts constitutively with Php4, the GRX domain-Php4 association is both modulated by iron and required for the inhibition of Php4 activity in response to iron repletion.