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Mol Microbiol. Author manuscript; available in PMC Jul 1, 2012.
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PMCID: PMC3133879
NIHMSID: NIHMS298857

Functional Characterization of the Ferroxidase, Permease High Affinity Iron Transport Complex from Candida albicans

Summary

Saccharomyces cerevisiae expresses two proteins that together support high-affinity Fe-uptake. These are a multicopper oxidase, Fet3p, with specificity towards Fe2+ and a ferric iron permease, Ftr1p, which supports Fe-accumulation. Homologs of the genes encoding these two proteins are found in all fungal genomes including those for the pathogens, Candida albicans and Cryptococcus neoformans. At least one of these loci represents a virulence factor for each pathogen suggesting that this complex would be an appropriate pharmacologic target. However, the mechanism by which this protein pair supports Fe-uptake in any fungal pathogen has not been elucidated. Taking advantage of the robust molecular genetics available in S. cerevisiae, we identify the two of five candidate ferroxidases likely involved in high-affinity Fe-uptake in C. albicans, Fet31 and Fet34. Both localize to the yeast plasma membrane and both support Fe-uptake along with an Ftr1 protein, either from C. albicans or S. cerevisiae. We express and characterize Fet34, demonstrating that it is functionally homologous to ScFet3p. Using S. cerevisiae as host for the functional expression of the C. albicans Fe-uptake proteins, we demonstrate that they support a mechanism of Fe-trafficking that involves channeling of the CaFet34-generated Fe3+ directly to CaFtr1 for transport into the cytoplasm.

Keywords: iron transport, ferroxidase, copper oxidase, iron channeling, Candida albicans

Introduction

Saccharomyces cerevisiae expresses two proteins that associate in the plasma membrane to form a high-affinity iron uptake complex (Kwok & Kosman, 2006). The iron permease component in this complex is Ftr1p which has seven transmembrane domains with an amino-terminal out, carboxy-terminal in orientation (Singh et al., 2006, Severance et al., 2004, Stearman et al., 1996). The other component is a multicopper oxidase (MCO), Fet3p (Kosman, 2010); Fet3p is a Type Ia membrane protein whose catalytic, MCO domain is exocytoplasmic (Singh et al., 2006, Kwok et al., 2006, De Silva et al., 1995). Fet3p has a specific reactivity towards ferrous iron, Fe2+, as reducing substrate catalyzing what is known as the ferroxidase reaction (reaction 1).

4Fe2++4H++O24Fe3++2H2O
(1)

The ferric iron product of this Fet3p-catalyzed reaction is ligand for the permease for Fe-uptake (Kwok et al., 2006).

Detailed structure-function studies have identified the residues in both proteins that are essential to this ferroxidation-permeation pathway. These studies also have examined the mechanism by which the Fet3p-produced Fe3+ is trafficked to Ftr1p for uptake (Kwok et al., 2006). An early and key observation was that exogenous ferric iron was not ligand for Ftr1p, that is, only Fe3+ produced by Fet3p was substrate for Ftr1p-mediated uptake (Stearman et al., 1996). Putative iron-binding ligands on both proteins were identified that were required for this trafficking; kinetic studies indicated that iron was channeled from Fet3p to Ftr1p, a mechanism in which the Fet3p-produced Fe3+ did not equilibrate with bulk solvent before becoming associated with Ftr1p for membrane permeation (Stoj et al., 2006, Kwok et al., 2006, Severance et al., 2004).

Fet and Ftr homologues populate all archived fungal genomes and some algal ones (Terzulli & Kosman, 2010). In addition, MCOs with ferroxidase activity are essential to iron trafficking in mammals, i.e., ceruloplasmin (Cp) and hephaestin (Hp) (Anderson & Vulpe, 2009). Although Cp is structurally dissimilar to Fet3p overall, it shares with the fungal ferroxidase acidic residue types which likely are linked to the iron-binding and trafficking processes for which these mammalian proteins are responsible (Bento et al., 2007). However, these structural similarities among fungal, algal and mammalian homologs have not been experimentally linked to a similar mechanism of action. The research here addresses this lack in regards to high-affinity Fe-uptake in pathogenic fungi.

The Fet, Ftr1 system has been identified in several fungi including Candida albicans (Fang & Wang, 2002, Knight et al., 2002, Ramanan & Wang, 2000, Eck et al., 1999), Cryptococcus neoformans (Jung et al., 2008, Weissman et al., 2002, Jacobson et al., 1998), and Aspergillus fumigates (Schrettl et al., 2004), all of which are human pathogens; Fusarium graminearum (Park et al., 2007) and Ustilago maydis (Eichhorn et al., 2006) which are important plant pathogens; and Schizosaccharomyces pombe (Askwith & Kaplan, 1997), Laccaria bicolor (Courty et al., 2009), and Phanerochaete chrysosporium (Larrondo et al., 2007). Structure-function studies have been reported only for the Ftr1 component of this complex in C. albicans (Fang & Wang, 2002). In fact, the genomes of both C. albicans and C. neoformans encode two Ftr proteins each but in each organism the Ftr1 ortholog and not the Ftr2 one supports high affinity iron uptake while contributing to virulence (Jung et al., 2008, Ramanan & Wang, 2000). Both genomes also encode two or more Fet proteins, five in C. albicans, two in C. neoformans; in the latter fungus, only Cfo1 is associated with pathogenic fitness (Jung et al., 2009). None of the Fet species encoded in other fungal genomes has been examined at the molecular level, and there has been no validation of the mechanism by which iron is trafficked from ferroxidase to permease in any of these other systems. The ability of these fungi to compete successfully with host iron binding agents (transferrin, ferritin, heme) likely stems in part from the tight coupling of ferrous iron oxidation by the Fet protein and ferric iron permeation by the Ftr protein. This assumption holds, of course, only if the channeling mechanism determined for the ScFet3p, Ftr1p system is true for all homologous systems, fungal or otherwise.

Thus, we have used S. cerevisiae as host to establish the mechanism by which the high-affinity iron uptake proteins from C. albicans support iron accumulation and thus to indicate the likelihood that all Fet, Ftr complexes share this iron channeling characteristic. We have examined the structure-activity relationships in CaFet34, the MCO homolog in this fungus most similar to ScFet3p and, as we show here, the one that best complements the absence of Fet3p in budding yeast. For these studies we have produced in S. cerevisiae the soluble MCO domain of wild type and site-specific mutants of CaFet34 and quantified the kinetic behavior of these several species. We have examined the kinetics of Fe-uptake supported by these mutant forms when expressed as full-length proteins in the yeast plasma membrane. Complementary mutations in predicted CaFtr1 iron-trafficking (iron channeling) residues were made and examined for function also. The data confirm that the ferroxidase-permease iron uptake pathway expressed by this pathogenic fungus mirrors the one well-characterized in S. cerevisiae. These data support the inference that this iron channeling mechanism is a characteristic of all such systems whether expressed in fungi, algae or humans.

Results

Expression, localization and function of C. albicans Fet proteins in S. cerevisiae

CaFtr1 has been expressed in S. cerevisiae where it complements the iron-uptake minus phenotype of an ftr1Δ strain (Ramanan & Wang, 2000). Like all Ftr1 homologs (Kwok & Kosman, 2006), CaFtr1 possesses the two essential REXLE motifs located in transmembrane domains 1 and 4 of these permeases (Fang & Wang, 2002). Since CaFtr1 and not CaFtr2 is required for the virulence of this pathogen (Ramanan & Wang, 2000) it was selected for structure-function studies in regards to the mechanism of Fe-uptake supported by this ferric iron permease.

The ferroxidase (Fet) partner to CaFtr1 is not clear. The C. albicans genome encodes five ScFet3p homologs; these are Fet3 (orf19.4211), Fet31 (19.4213), Fet33 (19.943), Fet34 (19.4215) and Fet99 (19.4212) (systematic names from CGD Assembly 21). The possible function of two of these proteins has been examined, Fet31 and Fet99. A homozygous fet31Δ strain was shown to have a limited growth sensitivity to iron limitation and no loss of pathogenic fitness indicating the Fet protein expressed from this locus plays some but not an essential role in C. albicans Fe-uptake. In addition, complementation by FET31 of a Fe-deficient phenotype in an S. cerevisiae fet3Δ strain was limited to conditions of minimal Fe-deprivation, again indicating some but a limited role in Fe-accumulation (Eck et al., 1999). [Note that the authors of this work mistakenly referred to the locus as FET3; inspection of the sequence they report for the open reading frame (ORF) used in their studies shows it to be that of FET31.] Knight et al unsuccessfully screened a C. albicans cDNA library for clones complementing the Fe-deficient phenotype of a S. cerevisiae fet3Δ strain (Knight et al., 2002). Subsequently choosing FET99 as encoding a Fet homolog similar to Fet3p, the authors were unsuccessful in demonstrating any rescue by this species. In neither of these attempts at genetic complementation was protein expression or localization examined.

On the other hand, all five of these ORFs encode MCO proteins that contain also the ferrox-idase-specific residues shown to provide ScFet3p its reactivity towards ferrous iron (Stoj et al., 2006, Quintanar et al., 2004) (see Fig. S1). The failure of Fet31 and Fet99 to complement in S. cerevisiae was likely due to a failure to assemble with an Ftr protein in the endoplasmic reticulum (ER) and to traffic to the plasma membrane (PM). With this hypothesis in mind, we first constructed low-copy expression plasmids for each of the five CaFet proteins; in these episomes, the CaFet ORF was fused to YFP and was placed under control of the ScFET3 promoter. These fusions were then expressed in the fet3Δftr1Δ strain, AJS-05, along with ScFTR1 expressed from a homologous plasmid. By this simple experiment, both the expression and localization of the five C. albicans Fet proteins could be examined when produced along with the ScFtr1 protein. The results of this screen are shown in Fig. 1; localization of ScFet3:GFP when expressed with its partner permease was used as positive control (last panel).

Fig. 1
Localization of CaFet species in S. cerevisiae. Plasmids encoding carboxyl-terminal YFP or CFP fusions of the five CaFet species under control of the ScFET3 promoter were expressed along with ScFTR1 in AJS-05, the double fet3ftr1 deletion strain carrying ...

The images demonstrate that only CaFet34 compared favorably to the S. cerevisiae proteins in its localization to the plasma membrane with little evidence of protein retention in intracellular compartments. CaFet31 exhibited a distinct PM localization, also, but in addition was retained in what is likely the ER. In contrast, the other three CaFet proteins were restricted exclusively to intracellular compartments; previous studies have indicated this type of distribution correlates to Fet protein that fails to exit the ER (Singh et al., 2006, Severance et al., 2004, Sato et al., 2004). The result with Fet99 is consistent with the inference noted above that the failure of this C. albicans Fet species to complement the absence of Fet3p in S. cerevisiae was due to its failure to assemble and traffic with the endogenous iron permease, ScFtr1p. The result here for CaFet31 correlates also with previous observations in that the weak complementation reported mirrors the relatively limited PM localization demonstrated here. In contrast, the apparently efficient PM localization of Fet34 reflected sequence comparisons of this C. albicans gene product and ScFet3p which included conservation of N-linked glycosylation sites, homology of transmembrane domains, and sequences at the likely cytoplasmic face of this domain shown to be critical to assembly of the Fet3p, Ftr1p complex in budding yeast (Singh et al., 2006). Also noteworthy is that Fet34 mRNA levels are increased 3.3-fold in C. albicans when grown on limiting iron; the transcript for no other Fet species was altered by this growth condition indicating that Fet34 likely played a specific role in accumulation of extracellular iron (Lan et al., 2004). Thus, the trafficking of CaFet34 was subsequently examined in more detail (see below).

The localization of these various C. albicans Fet proteins was correlated with their ability to partner together with the native yeast permease, Ftr1p, in support of 55Fe-uptake. For this screen we used [Fe] = 0.2 µM, the KM for iron in this process. Uptake was performed in the presence of 20 mM dihydroascorbic acid, making Fe-accumulation independent of plasma membrane reductase activity (Singh et al., 2006, Kwok et al., 2006). Figure 2 shows the relative 55Fe-accumulation supported by these putative ferroxidase, permease complexes; these data are given as the percent of accumulation supported by the native ScFet3p, Ftr1p complex. The results correlate with the data on localization summarized in Fig. 1, namely that together with ScFtr1p, CaFet31 and Fet34 support Fe-uptake (20 and 40% of control, respectively) whereas the other three C. albicans proteins fail to support any quantifiable uptake above background cell association of 55Fe. As noted, our results for Fet31 and Fet99 are consistent with published data in that Fet31 complemented in a S. cerevisiae fet3Δ strain (albeit weakly) (Eck et al., 1999) while Fet99 did not (Knight et al., 2002).

Fig. 2
Support of Fe-uptake by CaFet species in S. cerevisiae. Transformants as in Fig. 1 were examined for reductase-independent 55Fe-uptake. The conditions were: pH 6.0 MES (100 mM), [55Fe] = 0.2 µM, [dihydroascorbic acid] = 20 mM. Uptake values are ...

Localization of Fet34 and Fet33 in C. albicans

In our studies we took advantage of the haploid nature of S. cerevisiae to assess the ability of the five C. albicans Fet proteins to function in S. cerevisiae at the plasma membrane in Fe-uptake. We complemented this approach by examining the cell localization of two of the CaFet proteins in C. albicans to confirm that the cellular trafficking of these two paralogs at the least was not different in the two fungi. We choose Fet34 as a CaFet which was PM-localized in S. cerevisiae (and supported Fe-uptake) and Fet33 as a paralog which failed to complement in this fashion. Fet33 was chosen also because sequence analysis indicated that it might be a homolog of Sc Fet5, the ferroxidase protein in budding yeast that localizes to the vacuolar membrane (see Fig. S1) (Urbanowski & Piper, 1999). To establish the cell locale of these two ferroxidases in C. albicans, the GFP ORF was inserted in-frame at the 3’-end of the coding sequences for the two Fet proteins using a plasmid that allowed for Ura+ selection of integrants (Gerami-Nejad et al., 2001). The fidelity of this integration was confirmed by sequencing genomic DNA recovered from these recombinants. These strains were then examined by fluorescence microscopy; note that in these constructions, both fusions remained under control of the endogenous promoters associated with the two loci.

The images in Fig. 3 demonstrate first that both integrants support expression of a GFP fusion protein and second that Fet34:GFP specifically localizes to the PM in C. albicans. In contrast, Fet33:GFP is retained in the cell in a pattern that suggests vacuolar membrane localization. This conclusion was tested successfully by co-staining the cells with FM4-64 (Fig. 3); FM4-64 specifically localizes to the fungal vacuolar membrane following its uptake by endocytosis (Vida & Emr, 1995). The efficacy of PM localization of Fet34 in C. albicans indicates that the GFP tag does not compromise trafficking; therefore, the somewhat less efficacious PM localization of the same construct in S. cerevisiae likely is due to species differences. Also, the fluorescent images qualitatively reflect a greater expression of Fet34:GFP in comparison to Fet33:GFP. This difference is ascribed to the fact that in the former cells, expression of FET34 was induced by growth in the presence of the iron chelator, BPS. In a comprehensive analysis of genes induced in C. albicans under iron restriction conducted by Lan et al, a 3-fold increase in FET34 transcript abundance was detected; none of the other four FET loci were up-regulated in this fashion, including FET33 (Lan et al., 2004). The images in Fig. 3 are consistent with this expression data.

Fig. 3
Localization of Ca Fet33 and Fet34 in C. albicans. A URA3 cassette containing the GFP ORF followed by the ADH1 terminator was integrated at the 3’-end of either the FET33 or FET34 locus in C. albicans strain BWP17 (Gerami-Nejad et al., 2001). ...

Expression, purification and characterization of sFet34

Based on the data above, we chose to further characterize CaFet34 with the objective of delineating the likely mechanism by which this C. albicans protein supports Fe-accumulation. Note that the fluorescence images collected for the five CaFet fusion proteins in S. cerevisiae indicated that their expression from the ScFET3 promoter was comparable; western blot analysis directed towards the fluorescent protein tag on these proteins confirmed this observation (data not shown).

ScFet3p has been expressed, purified and structurally analyzed as a soluble form secreted from the yeast cell, sFet3p. This construct had a truncation at G555 that removed the carboxy-terminal transmembrane domain tethering native Fet3p to the yeast PM (Hassett et al., 1998). A similar strategy was used to determine the structure-function properties of Fet34; the soluble, Ca sFet34 species was generated by truncation of the FET34 ORF to produce a protein with a carboxy-terminal G550; as with the S. cerevisiae sFet3 species, a FLAG tag was included up-stream of the stop codon. This sFet34 was expressed from the FET3 promoter and recovered from conditioned media using a purification scheme identical to that used for sFet3p. The yield of pure protein compared favorably as well, ca 4 mg/L.

The SDS-PAGE protein gels shown in Fig. 4 illustrate the purity of sFet34 and its electrophoretic behavior in comparison to sFet3. The left panel illustrates the purification of sFet34 including the result of EndoH treatment of the purified protein. Lane 2 contains the conditioned growth medium harvested for sFet34 purification (sample concentrated 20-fold). Lanes 3–5 contain sFet34 after MonoQ chromatography, EndoH treatment, and MonoQ chromatography of the EndoH-treated protein, respectively. The right panel compares the effect of EndoH treatment on sFet3p in comparison to sFet34. Lanes 1 and 2 contain sFet3p before and after EndoH-treatment; lanes 3 and 4 contain the corresponding sFet34 species. The gels indicate two facts. First, the sFet34 protein used in the following experiments was >95% pure. Second, the EndoH-treated proteins exhibited identical electrophoretic behavior while the native ones did not. Specifically, sFet3p migrated as a larger molecular mass and more diffuse species; this behavior is likely due to the ~30% by weight carbohydrate that native sFet3p possesses (Hassett et al., 1998). sFet34 appears to contain somewhat less glycan than sFet3 which is consistent with the fact that it is predicted to have 10 N-linked sites in comparison to the 13 found in sFet3p (Taylor et al., 2005, Hassett et al., 1998, Ziegler et al., 2010) (see also Fig. S1).

Fig. 4
Purification and electrophoretic behavior of sFet3 species. Ca sFet34 expressed in S. cerevisiae is evaluated by SDS-PAGE as a function of purification stage (left panel). The samples are (lane): 1) MW markers; 2) conditioned medium; 3) MonoQ eluate; ...

The visible absorbance spectrum of sFet34 was equivalent to that of sFet3p (Fig. 5A). The near UV absorbance shoulder at 330 nm is due to these proteins’ type 3 Cu2+ pair known as the binuclear Cu-cluster, while the absorbance at ~600 nm is due to the T1 Cu2+, an absorbance that imparts to all MCO proteins their blue hue (Stoj & Kosman, 2005, Hassett et al., 1998, Kosman, 2010). The molar extinction coefficients for these two transitions in Ca sFet34 were 4250 and 4920 M−1 cm−1, equivalent to the values for Sc sFet3 (Hassett et al., 1998). These values indicated that sFet34 as isolated contained its full complement of 4 Cu atoms/per molecule; this was confirmed by Cu analysis using flameless atomic absorption spectrophotometry (data not shown). In addition to the T1 and T3 Cu2+ atoms, MCO proteins possess a fourth Cu2+ known as a T2 Cu (Kosman, 2010, Stoj & Kosman, 2005). This T2 Cu2+ has a very low visible absorbance (<100 M−1 cm−1) but does exhibit distinct electron spin transitions in an X-band, cwEPR spectrum (Blackburn et al., 2000). This spectrum for Ca sFet34 is shown in Fig. 5B. These spectra illustrate the spectral features of both the T1 or “blue” Cu2+ and the T2 Cu2+ atoms which both possess. The spectral data for Ca sFet34 are compiled in Table 1; the data for Sc sFet3p are provided also for comparison. These near-UV, visible and EPR spectral features confirm that Ca Fet34 is an MCO.

Fig. 5
Spectral properties of Ca sFet34. A) Near-UV, visible absorbance spectrum illustrates the shoulder at 330 nm (T3 Cu) and transition centered at 608 nm (T1 Cu) characteristic of all MCO proteins. B) cwEPR spectrum; the insert C) illustrates the anisotropy ...
Table 1
Near-UV, visible and EPR spectral properties of Ca sFet34 and Sc sFet3p All spectra were obtained on samples in 100 mM MES buffer, pH 6.0.

The enzymatic activity of sFet34 towards ferrous iron was determined by quantifying the velocity of O2 uptake using standard O2-electrode protocols. The rate data as a function of [Fe2+] are shown in Fig. 6 with the non-linear fit of these data to the Michaelis-Menten equation indicated by the smooth curve. The values of KM for Fe2+ and kcat for Fe2+ turnover by wild type Ca sFet34 are given in Table 2; the corresponding values for turnover of a standard oxidase substrate, hydroquinone (HQ) are given for comparison as are the values for the oxidation of these two substrates by Sc sFet3p (Stoj et al., 2006). These comparisons demonstrate quantitatively that sFet34 is a ferroxidase, that is, has a specific reactivity with Fe2+ as electron donor. sFet34 is only the second fungal ferroxidase purified and characterized in this way and the first from a human pathogen.

Fig. 6
Steady-state turnover of Fe2+ by Ca sFet34. The oxidation of Fe2+ was quantified by the consumption of dioxygen using an Oxygraph O2-electrode. The line represents the non-linear fit of the velocity data to the Michaelis-Menten equation. The fitted kinetic ...
Table 2
Steady-state kinetic constants for Ca sFet34 and Sc sFet3p Kinetic constants were obtained by non-linear fit of v versus [S] data provided by O2-uptake rates using an Oxygraph oxygen electrode and OXYGRAPH software. The fits were provided by PRISM5 (GraphPad). ...

Origin of ferrous iron specificity of Ca sFet34

The ferrous iron specificity of ScFet3p is due to two acidic residues which in the structure are spatially adjacent to the T1 Cu2+ site at which electron-transfer occurs from the reducing substrate in the reaction, e.g. Fe2+ or hydroquinone (HQ). By binding to the carboxylate side chains of E185 and D409, Fe2+ becomes a better reductant by at least 250 mV or ~−5.6 Kcal/mol in driving energy for the reaction (Stoj et al., 2006, Quintanar et al., 2007). This insight was obtained by the quantification of the reactivity of E185A and D409A sFet3p mutants. A similar strategy was employed to demonstrate that E184 and D408 make a similar contribution to the specific reactivity of CaFet34. These residues are clearly homologous to ferroxidase-specific ones in ScFet3 (Fig. S1). The relevant kinetic data for these mutants are given in Table 2 again with comparison to the values obtained for sFet3p. In this steady-state analysis, the effect on reactivity is given by the increased values for the Fe2+ Michaelis constant; as this value increases the rate of electron-transfer from Fe2+ to the T1 Cu2+ decreases proportionately and the ferrous iron specificity of the protein is lost (Quintanar et al., 2007, Stoj et al., 2006). Note that the kinetic values for HQ turnover are unaffected by these mutations demonstrating the specific role they play in electron transfer from Fe2+. In summary, CaFet34 and ScFet3p present structurally homologous active sites to the reducing substrate, an active site designed to be most reactive with Fe2+.

Wild type and mutant forms of CaFet34 and CaFtr1 in S. cerevisiae

As noted, CaFtr1 was demonstrated to complement in an ftr1Δ strain; in this case, it functioned with the endogenous ScFet3p (Ramanan & Wang, 2000). Our objective was to use S. cerevisiae as the host in which to characterize the mechanism by which the C. albicans Fet and Ftr proteins supported high-affinity Fe-uptake. Thus, CaFTR1 was expressed from the ScFET3 promoter as was CaFet34 (as in the experiments above) in the ftr1Δfet3Δ strain of S. cerevisiae, AJS-05 (Kwok et al., 2006). The hypothesis tested in these uptake experiments was that residues in both proteins, as in the S. cerevisiae homologs, provided a pathway for the Fe3+ produced by the Fet component to traffic to the Ftr one. We proposed that in CaFet34 these residues were E184 and D409 and in CaFtr1, D243 and E246. These latter two residues are predicted to be located in a large exocytoplasmic loop and are homologous to D246 and E249 in ScFtr1p (Severance et al., 2004). The role these residues played in this Fe-trafficking pathway was indicated by mutant sensitivity to added Fe3+ chelators, e.g citrate, NTA and HEDTA (Kwok et al., 2006). For example, whereas 55Fe-uptake through a wild type ScFet3p, Ftr1p complex is insensitive to citrate, a complex assembled from Fet3p(E185A) and Ftr1p(D246N/E249Q) is inhibited 50% by 300 µM citrate (Kwok et al., 2006). We used citrate as the Fe3+-chelator to test Fe-trafficking in the CaFet34, Ftr1 Fe-uptake system functioning in S. cerevisiae.

We first demonstrated that the C. albicans Fe-uptake proteins were expressed in the heterologous fungal host and that both proteins trafficked to the plasma membrane. As described above for the initial analysis of the C. albicans Fet proteins, these two objectives were accomplished by constructing a carboxy-terminal CFP fusion of Fet34 and a comparable YFP fusion of CaFtr1. These constructs were then expressed in the fet3Δftr1Δ S. cerevisiae strain AJS-05; the corresponding S. cerevisiae fusions were used also as they had been in Fig. 1 (Singh et al., 2006, Kwok et al., 2006). As above, all fusions were expressed under control of the ScFET3 promoter which is maximally, but physiologically, activated by the Aft1UP transcription factor encoded in this host. The fluorescent images of the cells expressing these various combinations are shown in Fig. 7.

Fig. 7
Localization of Ca and Sc Fe-uptake proteins in S cerevisiae. Fet and Ftr fusions with CFP, YFP and GFP were expressed from the FET3 promoter in AJS-05, the double fet3ftr1 deletion strain carrying the AFT1UP allele (Singh et al., 2006). Cells were examined ...

The images in panels A–C show that the C. albicans proteins do traffic to the PM in S. cerevisiae, albeit with a variable efficiency. Thus, both ScFet3:CFP and CaFtr1:YFP cleanly localize to the PM with little if any residual vesicular compartmentalization (Fig. 7A). CaFet34:CFP serves as an effective trafficking partner for ScFtr1:YFP but a fraction of both fusion proteins is retained within an intracellular compartment (Fig. 7B). This intracellular localization is exaggerated in the partnership of CaFet34:CFP and CaFtr1:YFP (Fig. 7C).

Two sets of controls were examined to demonstrate two aspects of these localization experiments. First, localization of ScFet3:GFP was characterized; this fusion was expressed with or without ScFtr1p (untagged) and thus was used as a positive and negative control for trafficking. These controls are shown in Figs. 7D and 7E which demonstrate the fact that trafficking of Fet3:GFP to the PM (panel D) requires the co-expression of Ftr1p; without this trafficking partner, Fet3:GFP is retained in the perinuclear space (Singh et al., 2006, Kwok et al., 2006) where it is subsequently substrate for degradation by the proteosome (Sato et al., 2004). The second set of controls demonstrated that like ScFtr1p, CaFtr1p, when expressed in S. cerevisae, required the co-expression of a Fet protein for efficient localization to the PM. This dependence is illustrated in Figs. 7F and G. In both cases, the permease protein is retained entirely in intracellular compartments, primarily perinuclear in locale.

Note that expression of the FET and FTR loci in all cases is from the FET3 promoter and thus at least at theif transcript abundance should be comparable; the fluorescence data indicate a similar level of protein production as well. Thus, despite some variability in trafficking efficiency, these images indicate two facts: 1) comparable expression of C. albicans and S. cerevisiae proteins in this host, and 2) (some) localization of the high-affinity Fe-uptake complex at the PM irrespective of the protein pairs expressed.

These various combinations of co-transformants were then used to quantify 55Fe-uptake rates in normal Fe-uptake medium that includes citrate (20 mM) (as in Fig. 2) as well as in the same medium lacking this Fe3+-chelator. This comparison, as noted, provides a simple screen for the contribution a specific residue on the ferroxidase or permease makes to the trafficking of Fe3+ in the iron uptake complex. The 55Fe-uptake data for these wild type (non-mutant) Fe-uptake complexes are compiled in Table 3; the ScFet3, Ftr1 complex was used positive control (first entry).

Table 3
55Fe-uptake velocities for Fet, Ftr species. Uptake velocities were obtained at 0.2 µM 55Fe2+ in 100 mM MES buffer, pH 6.0 in the presence of 100 mM dihydroascorbic acid; when present, [citrate] = 20 mM. The strain used as host for the Fet, Ftr ...

The first result evident from these data is that the Fe-uptake activity of C. albicans protein(s) in S. cerevisiae is not strongly different from the activity of the host proteins; that is, the difference in 55Fe accumulated via the four complexes qualitatively correlates with their relative abundance in the PM (Fig. 7). This clearly is true of CaFtr1 in comparison to ScFtr1; the uptake data for the ScFtr1 and CaFtr1-containing complexes (Table 3, first two entries) are the quantitatively identical and comparable to those reported by Ramanan and Wang (Ramanan & Wang, 2000). The relatively wild type uptake supported by CaFtr1 in combination with the host Fet3p correlates with the essentially wild type localization of this complex (Fig. 7A). Also reasonably consistent with the protein localization patterns (Fig. 7B, C), the CaFet34, ScFtr1p combination exhibits ~50% of wild type Fe-uptake activity (Table 3, third entry), while the fully C. albicans protein complex supports ~30% (last entry). Taken together, the protein localization and Fe-uptake data indicate that CaFtr1 is fully functional in S. cerevisiae; in contrast, CaFet34 does not fully support localization of the Fe-uptake complex at the PM and thereby fails to fully complement the absence of ScFet3p in Fe-uptake. The second result is that irrespective of the protein composition of the Fe-uptake complex, 55Fe-uptake is insensitive to the presence of citrate. This result strongly indicates that in the either mixed S. cerevisiae/C. albicans or fully C. albicans protein complexes, the Fe-channeling mechanism in uptake characterized in the wild type S. cerevisiae complex is preserved.

This inference was tested by quantifying the citrate sensitivity of Fe-uptake complexes containing mutant forms of the CaFet34 and Ftr1 proteins. The residues targeted were those considered likely to be components of the Fe-trafficking pathway that linked ferroxidation by CaFet34 to Fe-permeation by CaFtr1. These included the CaFet34 residues examined above for their role in the ferroxidase reaction as well as the acidic residues in the CaFtr1 motif projected to be in an exocytoplasmic loop, 243DASE246; a homologous 246DASE249 motif is found in S. cerevisiae (Severance et al., 2004). The 55Fe-uptake data for the various combinations examined are given in Fig. 8 as a percent of the values in Table 3 for the wild type CaFet34, CaFtr1 complex.

Fig. 8
Citrate sensitivity of CaFet34 and Ftr1 mutants demonstrates channeling mechanism of Fe-uptake. Strain AJS-05 was used as host for the CaFet34, Ftr1 Fe-permease complexes as indicated. Uptake velocities were obtained as described in Table 3; the values ...

The pattern of citrate-sensitivity of mutant C. albicans Fe-uptake complexes was identical to the pattern for the corresponding S. cerevisiae ones (Severance et al., 2004). Mutation at either of the CaFet34 carboxylate residues that contributed to the ferroxidase specificity of sFet34 (Table 2) rendered the Fe-uptake complex strongly sensitive to citrate inhibition (>95% inhibition at 20 mM citrate) (first two sets of bars). In contrast, single mutations in the DASE motif in CaFtr1 had little effect on the sensitivity (second two sets of bars). However, substitution of both acidic residues led to inhibition of Fe-uptake >95% at this concentration of citrate (last set of bars); a quantitatively similar result is obtained for the 246NASQ249 ScFtr1p mutant (Severance et al., 2004). In summary, we propose that tight coupling of ferrous iron oxidation and ferric iron permeation is a fundamental component of the acquisition of iron by all fungi expressing these proteins, including the pathogens C. albicans and C. neoformans.

Discussion

The data presented here support the model that the C. albicans Fet34, Ftr1 proteins assemble in the plasma membrane in S. cerevisiae as components of a high affinity Fe-uptake complex homologous to the one formed by the native Fet3, Ftr1 proteins. A cartoon depicting this proposed complex is shown in Fig. 9. We suggest that this model describes the high affinity Fe-uptake complex involving these two proteins in the C. albicans plasma membrane as well.

Fig. 9
Model of CaFet34 and Ftr1 in the fungal plasma membrane. The topology and orientation are based on homology with the protein pair from S. cerevisiae. The RExxLE motifs in Ftr1 TM1 and TM4 are essential for Fe-permeation (Severance et al., 2004, Fang & ...

This model highlights two features tested here by mutagenesis, namely the acidic residues in CaFet34 required for supporting the ferroxidase and Fe-trafficking activity of this MCO, and the exocytoplasmic acidic residues in CaFtr1 required for wild type Fe-trafficking in Fe-uptake. These latter two essential residues are in addition to the 15RESLE19 and 157REGLE161 motifs in CaFtr1 transmembrane domains 1 and 4 also required for wild type function (Fang & Wang, 2002). At this level, the structure-function homology between the S. cerevisiae and C. albicans high-affinity Fe-uptake components is exact.

CaFtr1:GFP previously was shown to traffic to the PM of C. albicans (Ramanan & Wang, 2000); there have been no localization studies reported for any of the five Ca Fet proteins. Now we have shown that Fet34 localizes to the PM in C. albicans whereas Fet33 appears to be a vacuolar membrane protein. The data here show also that CaFet34 and Ftr1 traffic to the plasma membrane only when produced together or with a partner protein from S. cerevisiae. This pattern corresponds to the results for the S. cerevisiae protein pair (Singh et al., 2006) and shows that CaFet34 and CaFtr1 likely associate early in the secretory pathway, also. Clearly, however, the C. albicans proteins vary in their trafficking efficiency when expressed in S. cerevisiae with CaFtr1 nearly “wild type” in this respect and CaFet34 functioning at ≤50%.

We propose that the wild type C. albicans proteins are in close proximity if not in a protein complex in the S. cerevisiae PM whether expressed together or with an endogenous partner protein. This would allow for the tight coupling of ferroxidation and permeation indicated by the insensitivity of 55Fe-uptake supported by these proteins to the presence of a ferric iron chelator like citrate. This insensitivity to citrate suggests that the Fe3+ product of the ferroxidase reaction does not equilibrate with bulk solvent prior to being substrate for Fe3+-permeation through the Ftr1 partner (Kwok et al., 2006). The fact that this Fe3+-trafficking pathway can be made “leaky” by substitution at specific acidic residues on the ferroxidase and permease partners reinforces the premise that the C. albicans proteins support an iron channeling mechanism like the one well-characterized in the S. cerevisiae system (Kwok et al., 2006). The structural basis for this mechanism lies in the homologous residues highlighted in Fig. 9 and in a physical contiguity of ferroxidase and permease that is consistent with the fluorescence data.

Our biochemical analyses of Ca sFet34 show this protein to be an MCO with specificity towards Fe2+ that closely mirrors the specificity demonstrated by ScFet3p. The mutagenesis results demonstrate also the close functional homology between the two proteins, which is not surprising given their close phylogenetic relationship. Both are of the Archiascomycetes lineage Saccharomycetales, and within that lineage are the most closely related (Liu et al., 1999). In contrast, we have been unable to engineer plasmids for functional expression in S. cerevisiae of the Fet, Ftr homologs from Cryptococcus neoformans, Cfo1 and Cft1 (Singh and Kosman, unpublished). This negative result was perhaps not surprising since while ScFet3p and CaFet34 are 59% identical and 75% similar in sequence, these values for Fet3p and Cfo1 are only 36 and 52%, respectively. Furthermore, Cfo1 lacks all four of the asparagines that in Fet3p are sites of the essential N-linked core glycosylation required for protein trafficking from the endoplasmic reticulum (Ziegler et al., 2010). Fet34 retains all four of these N-linked sites (see Fig. S1). On the other hand, Cfo1 and Cft1 contain all of the acidic residues required for the Fe trafficking and uptake supported by the ferroxidase and permease homologs from S. cerevisiae and C. albicans, e.g. Fig. 9 (Stoj et al., 2006, Singh et al., 2006, Kwok et al., 2006, Severance et al., 2004); we conclude these residues likely play equivalent roles in high affinity Fe-uptake in C. neoformans (Jung et al., 2009, Jung et al., 2008).

Of the remaining four CaFet proteins, only CaFet31 localized to the PM and supported Fe-uptake when expressed with ScFtr1p. However, all four proteins possess residues homologous to E184 and D408 as found in Fet34; all exhibit the four sequence motifs specific to the MCO copper-ligand arrays; and each has a likely transmembrane motif in the carboxyl-terminal domain (see Fig. S1).1 With the exception of Fet33 (which is likely a ferroxidase targeted to the vacuole), we predict that these proteins, too, are MCOs with ferroxidase activity targeted to the PM; CaFet33 lacks a cleavable signal sequence and most of the predicted N-linked glycosylation sites found in Fet proteins targeted to the PM (Ziegler et al., 2010). CaFet33 also lacks a serine residue within its transmembrane domain that is shared by the other four CaFet proteins; this serine is homologous to S567 that is required for the quality control of ScFet3p in the ER likely via an interaction with a partner Ftr permease (Sato et al., 2004). Our data on the likely vacuolar membrane localization of Fet33:GFP is fully consistent with those conclusions drawn solely from the motif differences as noted.

Possibly, the four Fet “isoforms” likely targeted to the PM could complement the absence of one another as partners for CaFtr1 resulting in the observation that deletion of the gene encoding any one of them (e.g. FET3 or FET99) had no effect on fungal virulence in vivo (Knight et al., 2002, Eck et al., 1999). In other words, ferroxidase activity, in addition to the permease, is likely to be a virulence factor as would be expected of an up-stream function in an essential metabolic pathway. This is true of Cfo1 in C. neoformans, which alone is the ferroxidase required for reductive iron uptake; a cfo1 strain exhibits attenuated virulence in a mouse model of pathogenic fitness (Jung et al., 2009). Note, however, by using S. cerevisiae primarily as our model we were unable to test the possibility that Fet34, specifically, is a virulence factor in C. albicans infection. Irrespective of whether any one of the likely PM Fet proteins does or does not assemble with a Ftr one in the Candida PM, that permease would be Ftr1 in as much as Ftr2 plays no quantifiable role in this process (Ramanan & Wang, 2000). Possibly Ftr2 assembles with Fet33 in the vacuolar membrane, much as Fth1 and Fet5 do in S. cerevisiae (Urbanowski & Piper, 1999).

Of course, the primary questions is, Does this model of structure-function in a metabolic pathway that confers fitness to a human pathogen give us any clues as to effective pharmacologic intervention? Certainly, making iron less available to disseminated pathogens has been considered as a therapeutic strategy (Ibrahim et al., 2008). One iron chelator, deferiprone, has been approved for clinical use (Neufeld, 2006); in contrast, deferiprone has been shown to inhibit growth of non-pathogenic but not pathogenic C. albicans strains (Holbein & Mira de Orduna, 2010).

As a pharmacologic target, the ferroxidase-permease system of high-affinity Fe-uptake is unique to fungi (and algae); drugs specific to this system would be less likely compromised by adverse iatrogenic complications. That is, the key step in this pathway is the non-dissociative channeling of Fe3+ from ferroxidase to permease (Terzulli & Kosman, 2010, Kwok et al., 2006). In the wild type complex, this Fe3+ is inaccessible to common Fe3+ chelating agents that form thermodynamically stable ferric iron complexes. Channeling is a kinetic phenomenon and, consequently, a pharmacologically efficacious inhibitor will be one that diverts the trafficking trajectory of the ferroxidase-generated Fe3+ and not one that is simply a strong chelating agent that sequesters iron from the host. Such a compound would, in effect, be competing with the fungal permease for Fe3+, and not with, for example, a ferric iron-binding host protein for iron, e.g. apotransferrin. Investigations evaluating the structure-activity of such compounds are in progress.

Experimental procedures

Strains, media and culture conditions

Two S. cerevisiae strains were used in these studies, DY3260 (MATα trp1-63 leu2-3,112 gcn4-101 his3-609 ura3-52 AFT1-1up) and AJS-05 (MATα can1 his3 leu2 trp1 ura3 ade6 fet3::HIS ftr1::TRP1 aft1::AFT1-1upKAN) which was derived from DEY1457 (Singh et al., 2006). The AFT1-1up allele codes for a constitutively active form of the Aft1p transcription factor that we have used to drive expression of recombinant FET and FTR loci cloned downstream from the FET3 promoter (Yamaguchi-Iwai et al., 1996). In this background, production in S. cerevisiae of episomally-expressed wild type and mutant alleles of CaFet and Ftr1 proteins was maintained at a high but physiologic level equivalent to strong iron deprivation. Early log phase cells (OD660 nm = 0.8 – 2.0) grown in selective media (6.67 g/L yeast nitrogen base w/o amino acids, 2 % glucose plus the appropriate drop-out mixture of amino acids) were used for all experiments. The C. albicans strain used to demonstrate the localization of Fet33 and Fet34 was BWP17 (ura::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG) (Wilson et al., 1999).

CaFET and FTR1 plasmids

All CaFet and Ftr1 proteins were expressed from the FET3 promoter in low-copy pRS vectors equivalent to the cassettes used for the episomal expression of ScFET3 and FTR1 (Singh et al., 2006, Kwok et al., 2006). These cassettes included (5’ to 3’): the FET3 promoter, cloning sites, in-frame GFP, YFP, or CFP ORF, followed by a stop codon. Fet species were cloned into the pRS416 cassette for Ura+ selection; Ftr species were cloned into the pRS415 cassette for Leu+ selection. The CaFET34 and FET99 vectors were constructed using cDNAs provided by Drs. A. Dancis and S. Knight (Knight et al., 2002). The FET3, FET31, and FET33 vectors were constructed using cDNA PCR amplified from genomic C. albicans DNA provided by Dr. M. Edgerton. The primers used for this cloning are listed in Table 4 and contained the indicated restriction sites for subsequent vector construction. PCR reactions were performed with approximately 200–300 ng genomic DNA using the Invitrogen Pfx PCR kit containing 1X Enhancer solution and the following thermal-cycling conditions: 94°C for 2 min (x1); 94°C for 15 s, 50°C for 45 s, 68°C for 2.5 min (x30); and 68°C for 8 min (x1). Each PCR product was gel-purified, digested with the appropriate pair of restriction enzymes and cloned into the BglII and XbaI restriction sites upstream of the YFP ORF contained in pAJS21-01, one of the pRS cassettes noted above (Singh et al., 2006). For examination of localization and Fe-uptake activity of the five CaFET species, these were each co-transformed with p703FTR1-myc (Severance et al., 2004) into AJS-05. For experiments involving CaFTR1, a CaFET plasmid was co-transformed into AJS-05 along with the pRS415 cassette containing the CaFTR1 ORF.

Table 4
Primers for construction of FET expression plasmids in S. cerevisiae

Constructing Fet33:GFP and Fet34:GFP expressing C. albicans strains

The cassettes for integration of the GFP-encoding sequence at the 3’ end of the coding regions for Fet33 and Fet34 were constructed by a modification of the method described by Gerami-Nejad et al; the template used for these constructions was plasmid pGFP-URA3 (Gerami-Nejad et al., 2001). For each construct, forward and reverse primers (~100 bp each) were designed such that the5’ and 3’ ends of the PCR product contained ~70 bp that were homologous to the target gene locus; the remaining sequence within the primer was used to anneal to cassette DNA. The resulting PCR product was subcloned into pJet (Thermo/Fermentas) and then amplified such that a linear fragment with locus-specific flanking regions could be excised by restriction digest and used to transform BWP17 (Wilson et al., 1999), selecting for a ura+ phenotype. The flanking regions directed homologous recombination at the 3’ end of the Fet ORF such that an in-frame fusion between the carboxyl-terminal residue of the ORF and the amino-terminal residue of GFP was achieved. These junctions were confirmed by sequencing genomic DNA recovered from the integrant strain. The primers used for these two constructions are given in Table 5. Note that the integrants were heterozygous with respect to the FET locus targeted by the cassette, and that FET::GFP expression was under control of the endogenous FET promoter.

Table 5
Primers for construction of GFP carboxyl-terminal Fet fusions in C. albicans

Expression and purification of soluble Fet34 proteins

Strain DY3260 carrying plasmid pFET34 was used as the expression system for the purification of soluble wild type and mutant Fet34 proteins; this vector was a derivative of the expression plasmid used for the production of wild type and mutants forms of sFet3p (Hassett et al., 1998). Thus, pFET34 is a high-copy vector that carries the FET34 ORF truncated at nucleotide +1650 (at amino acid residue 550) with an in-frame, carboxy-terminal FLAG epitope; this transcription unit is under control of the ScFET3 promoter. Truncation removes the likely membrane-spanning domain found in the carboxy-terminal region, residues 555–576 (see Fig. S1). The data shown here demonstrate that expression from pFET34 results in sFet34 that is secreted directly into the growth medium rather than being retained in the plasma membrane. sFet34 protein production and purification followed the protocols used for sFet3p which have been described in some detail (Hassett et al., 1998).

Construction of Fet34 and FTR1 mutants

Mutant FET34 alleles were constructed directly in pFET34 (for secreted, soluble Fet34) and in CaFET34:CFP expressing a Fet34:CFP fusion for the native, membrane-associated Fet34 protein. Site-directed mutagenesis employed the QuikChange kit from Stratagene (Santa Rosa, CA). A similar strategy was used to express wild type and mutant forms of CaFTR1:YFP as has been reported in detail for ScFtr1 (Severance et al., 2004). All sequences were confirmed by automated fluorescence sequencing on an ABI PRISM 377 instrument.

Electrophoretic and western blot analyses

Following electrophoretic separation, gels were soaked in 0.1% SDS followed by transfer to PVDF membranes for western analysis using rabbit anti-FLAG antibody directed towards the carboxy-terminal FLAG epitope carried by sFet protein species (as noted above) and, for membrane-bound Fet proteins, rabbit anti-GFP antibody directed towards the carboxy-terminal fluorescent protein tag.

Protein and kinetic characterization

Room-temperature UV-visible absorption spectra were recorded using a Varian Cary 50 spectrophotometer. cwEPR spectra were obtained at ~9.5 GHz (X-band) on a Bruker EMXplus spectrometer at 120° K. Protein concentration was determined using the standard dye-binding Bradford assay using BSA as the protein standard (Bradford, 1976). Steady-state kinetic analyses were based on oxygen consumption using an Oxygraph (Hansatech, www.hansatech-instruments.co.uk) (Stoj et al., 2006). Rates of O2 uptake were evaluated using the OXYG32 software provided by Hansatech. All initial velocity, v versus [S] data were subsequently analyzed by direct fitting to the Michaelis-Menten equation using Prism 5 software (GraphPad Software, La Jolla, CA). Ferrous ammonium sulfate (Sigma-Aldrich, St. Louis, MO) was used for kinetic analysis of the metallo-oxidase activity of Fet proteins. Substrate stock solutions were freshly prepared in nitrogen-purged 100 mM MES at pH 6.0. All transfers from these stock solutions were done using gas-tight syringes. The buffer used for O2-uptake measurements was air-saturated 100 mM MES at pH 6.0.

Trafficking and 55Fe-uptake activity Fet, Ftr species

These experiments were carried out in strain AJS-05 expressing carboxyl-terminal Fet:CFP and Ftr:YFP fusion proteins; these fusions have native iron uptake activity in vivo (Singh et al., 2006). Yeast cells were grown into early log phase, pelleted, washed once with phosphate-buffered saline, and resuspended in 100 µl of the same buffer for examination of fusion protein localization by epifluorescence using a Zeiss Axio Observer Z1 fluorescence microscope. 55Fe-uptake in these cells was quantified at [55Fe] = 0.2 µM at pH = 6.0 in the presence of 20 mM dihydroascorbic acid (reductase-independent uptake). This concentration of iron is comparable to the kinetically-determined KM value for reductase-independent Fe-uptake in this strain background, 0.36 µM (Kwok et al., 2006). Localization of Fet33:GFP and Fet34:GFP in C. albicans strain BWP17 modified by the appropriately targeted GFP-containing cassette was visualized using the Axio-Observer, also; co-staining the vacuolar membrane with FM4-64 (Molecular Probes/Invitrogen) followed a literature protocol (Vida & Emr, 1995).

Supplementary Material

Supp Fig S1

Acknowledgements

We thank Drs. Andrew Dancis and Simon Knight for the plasmids containing the FTR1, FET34, and FET99 ORFs and Dr. Mira Edgerton for the genomic DNA used as the starting material for the C. albicans reagents described in this research. We thank Dr. Wade Sigurdson for his continuing assistance in the use of fluorescence microscopy in the examination of protein localization in yeast. This work was supported by Grants DK053820 and DK077826 from the National Institutes of Health.

Footnotes

Supporting information can be found in the online version of this article.

1In a recent review, Almeida, Wilson and Hube erroneously report that Fet34 lacks a signal peptide and copper oxidase domains (Almeida et al., 2009). As noted here (see also Fig. S1) all five Candida Fet homologs contain all of the structural features that make them close structural homologs of ScFet3p.

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