Schizosaccharomyces Pombe As An Alternative Model System

3.1. Copper Uptake

3.1.1. "The Other Yeast"

Although use of S. cerevisiae has rapidly advanced the field of metal-ion metabolism, this organism has some limitations as a model system. One of them is the difficulty in expressing higher eukaryote cDNAs. Furthermore, once the protein is expressed, to ensure its physiological integrity and function within the cell (128). For instance, recently, a human gene for Cu uptake, hCTR1, has been isolated and expressed in bakers' yeast cells (95,97). Although this cDNA shows sequence similarity to yeast CTR1 and CTR3, the complementation of respiratory incompetence in bakers' yeast cells harboring ctr1A ctr3A mutations by hCTR1 was only partial. Therefore, based on this and other observations, we decided to develop an alternative model system, the fission yeast Schizosaccharomyces pombe, that will complement and add to ongoing work on Cu transport, trafficking, and sequestration in S. cerevisiae (8,37). Like S. cerevisiae, S. pombe has proved to be a tractable eukaryotic organism amenable to genetic analysis (129,130). There are a number of features of S. pombe that make the organism an especially attractive one. For example, because S. pombe cells normally exist in the haploid state, recessive mutations can be isolated (131). The fission yeast is a rod-shaped cell that divides by septation and medial cleavage in a manner more similar to higher eukaryotes than S. cerevisiae (132). Several other reasons explain the growing popularity to exploit the fission yeast as a model organism. S. pombe has been favored because cells have well-organized and morphologically defined organelles (e.g., distinct Golgi complexes, vacuoles which are similar to the lysosome of mammalian cells, etc.) (128). Moreover, posttranslational modifications such as isoprenylation and the addition of terminal galactose in glycoproteins that occur in S. pombe are more similar to mammalian cells than those of S. cerevisiae (128). This latter observation may become important with regard to the intracellular targeting of proteins, maturation processes, and functional activation. Although there is a large evolutionary gap between S. pombe and S. cerevisiae, almost as large as that separating them both from animals, in general, S. pombe genes and proteins are more similar to mammalian cells than is S. cerevisiae (128). For the study of the roles and mechanisms of action of genes critical for trace-metal-ion homeostasis, S. pombe represents an attractive model organism because the fission yeast exhibits a low level of Cu resistance, presumably as a consequence of lack of "typical" MT (133,134). In S. pombe, the only known molecules for sequestering excess metal ions are the phytochelatins (135,136). Furthermore, these short glutathione-related peptides appear to bind Cd predominantly (137,138). Therefore, much like the Chinese hamster cell line (139) and the Baby hamster kidney cell line (140) in which MT genes are not expressed, S pombe represents an attractive system for studying metal-ion transport because there is no interference from MTs (141,142), which considerably simplifies analysis. In S. pombe, three components involved in the high-affinity Fe-uptake system have been identified (143,144). Frpl, which shares amino-acid sequence similarity with the Frel reductase from S. cerevisiae, is required at the cell surface of the fission yeast to reduce Fe3+ (144,145). Once reduced, Fe2+ is taken up by a permease/oxidase complex, called Fipl/Fiol (143), and transported into the cell, as occurs with the Fet3/Ftrl complex in S. cerevisiae (53). Although Fiol is similar to Fet3, by itself Fiol cannot replace Fet3 in a S. cerevisiae fet3A mutant strain, thereby indicating that some molecular differences exist for high-affinity Fe uptake between the two species of yeast (9,143).

3.1.2. Cu-Transporter Machinery

Several studies suggest that the high-affinity Cu uptake into eukaryotic cells requires reduction of Cu2+ to Cu*+ by cell-surface metalloreductases (26,146-148). So far, analysis of genomic DNA sequences from the S. pombe Genome project has revealed two open reading frames (SPBC1683.09C, denoted frp1+, and SPBC947.05C) related to cell-surface Fre reductases found in S. cerevisiae. Although the frp1 +-encoded reductase can reduce Fe3+ to Fe2+, its role in the metabolism of other metal ions (e.g., Cu) is unknown. Regarding the second ORF, SPBC947.05C, its potential role in Fe3+/Cu2+ reductase activity is still uncharacterized. As mentioned previously, Cu uptake in S. pombe is tightly regulated in response to the availability of Cu in the growth medium (Fig. 2). When cells are grown during Cu scarcity, the nutritional Cu-sensing transcription factor Cufl (see Section 3.l.3.) fosters the activation of Cu uptake genes, including the ctr4+-encoded permease and the ctr5+-encoded cotransporter that acts as a partner with Ctr4 for Cu transport at the cell surface (37,93). Conversely, when Cu ions reach the physiological requirements for normal growth, there is an inac-tivation of Cu-uptake gene transcription. Ctr4 is a 289-amino-acid protein with 5 repeats of the putative Cu-binding Met-X2-Met-X-Met motif at the amino-terminus. This latter motif is predicted by

Fig. 2. A model for Cu homeostasis in S. pombe. Prior to uptake, Cu2+ is reduced to Cu1+ by a putative extracellular reductase. The Ctr4-Ctr5 high-affinity Cu-transport complex mediates the passage of Cu ions across the plasma membrane. Furthermore, this physical association between both Ctr4 and Ctr5 must occur for maturation and colocalization to the cell surface. As in Fig. 1, the S. pombe SPBC1709.10C, SPAC22E12.04, and SPBC26H8.14C genes encode putative chaperones orthologous to S. cerevisiae Atxl, CCS, and Cox17, respectively. At the transcriptional level, the nuclear protein Cuf1 directly binds the copper-signaling element (CuSE) under conditions of low Cu to activate expression of ctr4+ and ctr5+ genes, whereas elevated Cu concentrations negatively regulate Cufl.

Fig. 2. A model for Cu homeostasis in S. pombe. Prior to uptake, Cu2+ is reduced to Cu1+ by a putative extracellular reductase. The Ctr4-Ctr5 high-affinity Cu-transport complex mediates the passage of Cu ions across the plasma membrane. Furthermore, this physical association between both Ctr4 and Ctr5 must occur for maturation and colocalization to the cell surface. As in Fig. 1, the S. pombe SPBC1709.10C, SPAC22E12.04, and SPBC26H8.14C genes encode putative chaperones orthologous to S. cerevisiae Atxl, CCS, and Cox17, respectively. At the transcriptional level, the nuclear protein Cuf1 directly binds the copper-signaling element (CuSE) under conditions of low Cu to activate expression of ctr4+ and ctr5+ genes, whereas elevated Cu concentrations negatively regulate Cufl.

topological analysis to reside extracellularly (37). Interestingly, this Met-rich motif is also found two times in the human Ctrl protein and eight times in the S. cerevisiae Ctrl protein (8). Likewise in these proteins, the Met motif is present at the amino-terminus and it is thought to be involved in the capture of extracellular Cu as part of the uptake mechanism. Interestingly, the carboxyl-terminal residues 111-248 of the S. pombe Ctr4 exhibit strong homology to the S. cerevisiae Ctr3 Cu transporter, especially with respect to several residues within the predicted transmembrane domains (37). Therefore, based on sequence homology, it appears that the S. pombe Ctr4 protein displays properties of both Ctr1 and Ctr3, which are the two high-affinity Cu transporters in bakers' yeast that are functionally redundant, but structurally distinct. Perhaps the S. pombe Ctr4 is the product of a gene, which was issued from a biological event in which CTR1 and CTR3 were fused into a single open reading frame. Using a fully functional Ctr4-GFP fusion protein, the transporter was localized to the plasma membrane (37). As would be expected, chromosomal disruption of the ctr4+ gene renders S. pombe cells respiratory deficient because of the lack of Cu as a cofactor for the cytochromes oxidase enzyme (37). Surprisingly, the Ctr4 protein from S. pombe was unable to complement the respiratory defect in S. cerevisiae ctrlA ctr3A cells, unless a specific component of the Cu-transport machinery from S. pombe, denoted Ctr5, was provided (93). In fact, the Ctr5 protein, which is an integral membrane protein, was isolated as an indispensable partner for proper localization and function of S. pombe Ctr4 when expressed either in S. cerevisiae or S. pombe cells (93). Ctr5 is a 173-amino-acid protein harboring two putative Cu-binding Met-X2-Met-X-Met motifs in its amino-terminal region similar to those found in the Ctr4 (five times), S. cerevisiae Ctrl (eight times), and human Ctrl (two times) (8,93).

Furthermore, like Ctr4 and human Ctrl, the Ctr5 predicted multimembrane-spanning region is homologous to S. cerevisiae Ctr3 (93). Elegant biochemical and genetic experiments revealed that Ctr4 and Ctr5 form an heteromeric complex at the cell surface (93). Furthermore, this association between Ctr4 and Ctr5 appears to be critical for protein maturation and secretion of the heteroprotein complex to the plasma membrane (93). Within this complex, the exact function of each protein is unclear. This aspect certainly consists of an exciting area for future investigation. Once inside fission yeast cells, Cu ions are presumably taken by putative Cu chaperones, yet uncharacterized at the molecular level. The open reading frame SPBC1709.10C encodes a putative Atxl ortholog in S. pombe. The protein from fission yeast exhibits 57% identity to the S. cerevisiae Atxl. Furthermore, the Cu-binding MXCXXC motif is conserved between the two proteins, consistent with a role in Cu delivery within the cell for the fission yeast protein. Based on the S. pombe Genome database, SPAC22E12.04 encodes a putative ortholog of S. cerevisiae CCS. Although this putative ortholog bears 30% identity and 47% similarity to its bakers' yeast counterpart, notable differences exist between the two molecules. For instance, the amino-terminal Domain I of the S. pombe CCS ortholog lacks the Cu-binding MXCXXC motif. Instead, the protein harbors an extra domain at the carboxyl terminus that contains a series of cysteine residues, which are arranged in Cys-Cys configurations in a manner similar to MTs. Although the exact role of this extra domain in the S. pombe CCS ortholog is unknown, one would expect that it may serve as a Cu-binding site for cytoplasmic Cu prior to transfer into Sodl. This example of molecular difference between the S. pombe and S. cerevisiae CCS chaperones suggests that, although similar in function, these proteins may have mechanistic distinctions. The Coxl7 Cu chaperone from S. cerevisiae, which delivers Cu to mitochondria for cytochrome-c oxidase assembly and function, has a related protein in S. pombe encoded by SPBC26H8.14C. The Coxl7 ortholog in S. pombe exhibits a slightly more overall homology to human Coxl7 (56% identity) than to bakers' yeast Coxl7 (47% identity). Interestingly, the S. pombe SPBC119.06 and SPAC1420.04 genes encode proteins that exhibit a strong sequence similarity to S. cerevisiae Scol and Coxll, respectively. It is likely that these S. pombe components function in the mitochondrial Cu-delivery pathway. Based on these observations, it is important to note that the high degree of structural similarity among the budding yeast, fission yeast, and human components involved in Cu transport suggests that different carriers with specific Cu-binding motifs for metal-ion delivery are well conserved through evolution.

3.1.3. Nutritional Cu-Sensing Transcription Factor Cufl

The cuf1+ gene encodes a nuclear protein that occupies a central role in the S. pombe high-affinity Cu transport system (37). Indeed, we have shown previously that the deletion of the cuf1+ gene (cuf1A) gives at least three phenotypes associated with Cu starvation in yeast cells: inability to use respiratory carbon sources (owing to lack of Cu incorporation into cytochrome-c oxidase), impaired superoxide dismutase activity (owing to lack of Cu incorporation into Cu,Zn-Sodl), and failure in Fe accumulation (owing to lack of Cu incorporation into multi-Cu oxidase Fiol) (37). Because these phenotypes are specifically corrected by the addition of exogenous Cu to the medium, this is consistent with cuf1A strains being defective in the expression of genes required for high-affinity Cu trans port. Furthermore, the observation that, in S. pombe, the high-affinity Cu transporters Ctr4 and Ctr5 mRNA levels are dependent on the Cuf1 protein implies a role for trans-activation of Cu transport genes by Cuf1 (37,93). Although the Cuf1 protein is required for S. pombe high-affinity Cu transport, Cuf1 shows at its amino-terminus a strong homology (amino acid residues 1-61) to the amino-terminal 63 and 62 amino acids of the S. cerevisiae Ace1 and C. glabrata Amt1 class of Cu-detoxifying transcription factors and much less similarity to Mac1, its functional ortholog (37). Consistently, we have demonstrated that Cuf1 acts through a closely related MRE-like element to regulate expression of fission yeast genes-encoding components of the Cu-transport machinery (l49). The carboxyl-terminal region of Cuf1 contains one cysteine-rich motif Cys328-X-Cys330-X3-Cys334-X-Cys336-X2-Cys339-X2-His342, which is absent in Ace1/Amt1 but found duplicated in both Mac1 and Grisea of Podospora anserina (37,l02,l50,l5l). Whether this motif at the carboxyl-terminus of Cuf1 constitutes by itself the Cu-sensing domain remains to be elucidated. The transcriptional control of Cutransport gene expression in S. pombe by the regulator Cuf1 occurs through a specific cis-acting element termed copper-signaling element (CuSE) (l49). The sequence 5'-D(T/A)DDHGCTGD-3' (D = A, G or T; H = A, C or T) referred to as CuSE is found in multiple copies on the ctr4+ and ctr5+ promoters and is necessary for appropriate repression and induction of gene transcription in response to Cu and Cu starvation, respectively (l49). Although the overall magnitude of the response to Cu is optimal with the presence of multiple elements, the presence of only one element is sufficient to confer regulation in response to Cu changes. The CuSE is strikingly similar in sequence to the MRE, which is defined by the consensus sequence 5'-HTHNNGCTGD-3' (D = A, G or T; H = A, C or T; N = any residue) (ll8). When a consensus MRE from S. cerevisiae is introduced into S. pombe, transcription is induced by copper deprivation in a Cuf1-dependent manner, similar to regulation by Mac1, the nuclear sensor for regulating the expression of genes-encoding components involved in copper transport in S. cerevisiae. Consistently, when the cufl+-encoded transcription factor of S. pombe is expressed into S. cerevisiae in which the ACEl gene was deleted (acelA), the CUPl-encoded MT gene is regulated in opposite direction in response to copper, being induced under copper-starvation conditions (l49). This upregulation is just opposite to what is normally seen in the wild-type S. cerevisiae strain, whereas Cu deprivation inactivates CUPl, whereas elevated Cu concentrations strongly activates expression of CUPl through Ace1. Using a cufl+-GFP fusion allele (37) and a cufl+-FLAG2 epitope-tagged allele (l49), which both retain wild-type function, we observed that the presence of Cu ions has little if any effect on the stability of the steady-state levels of the Cuf1 protein. This observation suggests that Cu ions modulate the activity of Cuf1 at a posttranslational level, perhaps by promoting conformational changes that would negatively regulate its DNA-binding function, therefore resulting in the inactivation of the expression of the Cu-uptake genes. Using a S. cerevisiae yeast system for expression of heterologous proteins in which the endogenous ACEl gene was knocked out, we determined that the Cuf1 protein interacts directly with CuSE (l49). Furthermore, no CuSE-Cuf1-dependent complex was observed when extract preparations were derived from cells grown in the presence of Cu prior to extract preparation. Conversely, a specific interaction between CuSE and Cuf1 was seen when extract preparations were obtained from cells grown under conditions of low Cu availability, suggesting an occupancy of the CuSEs in cells deprived of Cu by Cuf1 (l49).

3.2. Relationship Between Copper and Iron

3.2.1. Cufl Oppositely Regulates Cu and Fe Metabolic Genes

In S. pombe, the Fio1 protein is a Cu-dependent Fe oxidase required for high-affinity Fe transport that acts in concert with the Fe permease Fip1 at the cell surface (l43). Because of this requirement for Cu as a cofactor for the function of Fio1, Fe transport and mobilization rely on the high-affinity Cu-uptake system. In the S. pombe genome, fiol+ and fipl + genes share a common promoter, but are divergently transcribed (37,l43). Previous results suggest that fiol+ and fipl+ gene expression is regulated by Fe availability through a putative Fe-sensing transcription factor (l43). Recently, we have also demonstrated that an additional level of transcriptional control takes place to regulate Fe-transport gene expression (37). Although Cufl activates the high-affinity Cu uptake gene expression under Cu starvation conditions, under these same conditions Cufl directly represses expression of genes encoding components of the Fe transport machinery. This Cu-dependent homeostatic control offrp1+, fio1+, andfip1+ gene expression by Cufl occurs by direct binding of this latter transcription factor to repeated cis-acting elements found in multiple copies in both frp1+ promoter and the fio1+-fip1+ intergenic promoter regions (37) (Beaudoin and Labbé, unpublished data). Importantly, this regulated Cu-starvation-mediated repression of the Fe-responsive genes is also observed in bakers' yeast S. cerevisiae, where the FET3 gene is repressed when cells are starved for Cu and activated during Cu repletion (37). Moreover, this downregulation of Fet3 gene expression under conditions of Cu starvation requires a functional MAC1 gene. Indeed, mac1A cells exhibit elevated expression of Fe-uptake genes (37). On the other hand, isogenic cells with the MAC1up1 allele, which encodes a Cu-insensitive Macl protein that is constitutively bound to the promoter elements (99), exhibit a very low level of expression of FET3, consistent with the notion that Mac1up1 represses expression of Fe-uptake genes (37). Clearly, the regulation of Fe-transport genes by nutritional Cu-sensing transcription factors is a useful means for yeast cells to prevent futile expression of the Fe-transport systems under conditions of Cu starvation.

0 0

Post a comment