![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 18, Issue 8, 2980-2990, August 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
*Department of Applied Molecular Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan; Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan; and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Saitama 322-0012, Japan
Submitted November 29, 2006;
Revised May 15, 2007;
Accepted May 18, 2007
Monitoring Editor: Karsten Weis
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; De Freitas et al., 2003). The yeast Saccharomyces cerevisiae has served as a model organism for the investigation of iron homeostasis (De Freitas et al., 2003). The high-affinity iron uptake system of S. cerevisiae consists of two pathways. One pathway involves the iron permease Ftr1p and the multicopper ferroxidase Fet3p, which transports ferrous ion that has been reduced by the ferric reductases Fre1p and Fre2p (Dancis, 1998). The other pathway includes four siderophore transporters, each of which mediates the uptake of iron chelated to siderophores (Philpott et al., 2002). One of these transporters, Arn3p/Sit1p, is a ferrioxamine B (FOB) transporter (Lesuisse et al., 1998; Yun et al., 2000). The transcriptional activator Aft1p plays a central role in the homeostatic regulation of iron metabolism in S. cerevisiae by regulating the expression of genes whose products are involved in iron uptake and iron use, including Ftr1p, Fet3p, and Arn3p/Sit1p, in an iron-responsive manner (Yamaguchi-Iwai et al., 1995; Yamaguchi-Iwai et al., 1996; Philpott et al., 2002; Puig et al., 2005). We have shown that the iron-dependent alteration of Aft1p localization is important for this regulation: Aft1p accumulates in the nucleus and activates the transcription of its target genes in the iron-starved condition, whereas it is localized in the cytoplasm when there is excess iron (Yamaguchi-Iwai et al., 2002). Importins and exportins mediate the nucleocytoplasmic localization of proteins by regulating nuclear import and export, respectively (Hood and Silver, 1999; Jans et al., 2000; Komeili and O'Shea, 2000). We have previously shown that Pse1p is the importin that translocates Aft1p and that Pse1p-mediated nuclear import of Aft1p is not affected by the iron status of cells (Ueta et al., 2003). This observation strongly suggests that the nuclear export of Aft1p is regulated by the availability of iron. The identification of the exportin for Aft1p will therefore be crucial for understanding the mechanism underlying the iron-regulated nucleocytoplasmic localization of Aft1p, and, therefore, iron homeostasis.
Four exportins have been identified in S. cerevisiae (Strom and Weis, 2001), each of which has a different mechanism of cargo recognition (Pemberton and Paschal, 2005). However, the precise mechanisms for target recognition have not been elucidated, with the exception that Xpo1p, also known as Crm1p, has been shown to recognize the well-defined short amino acid sequences referred to as the leucine-rich nuclear export signal (Fornerod et al., 1997; Fukuda et al., 1997; Ossareh-Nazari et al., 1997; Stade et al., 1997). In this study, we show that the nuclear export of Aft1p is crucial for its iron-regulated changes in localization, and we identify Msn5p as the exportin responsible for the iron-dependent nuclear export of Aft1p. Analysis of the iron-dependent recognition of Aft1p by Msn5p indicates that the phosphorylation and intermolecular interaction of Aft1p are important for the iron-dependent nucleocytoplasmic trafficking of Aft1p. Moreover, Cys291 of Aft1p, whose mutation to Phe in the Aft1-1up variant has been shown to render Aft1p constitutively active in iron-replete cells, is critical for the intermolecular interaction of the protein.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) in YPD or SD medium supplemented with amino acids. The media used for iron repletion or iron starvation have been described previously (Ueta et al., 2003). FeSO4 (200 µM) was added to the medium to create excess iron conditions for a two-hybrid analysis using PJ69-4A
fet3 cells (Y22). To use FOB as an iron source, 0–200 µM each of FeCl3 and deferoxamine were added to SD-Fe medium containing 100 µM bathophenanthroline disulfonate (BPS).
|
), were subcloned into pRS416 containing the Aft1 promoter. Plasmids for the yeast two-hybrid assay were constructed as follows. To construct pAD-AFT1 and its derivatives, NcoI–XhoI fragments encoding the entire AFT1 coding region, or fragments thereof, were inserted into the NcoI–XhoI site of pACT2 (Clontech, Mountain View, CA). pBD-MSN5 was created by inserting an EcoRI–XhoI fragment containing the open reading frame of MSN5 into the EcoRI–XhoI site of pGBKT7 (Clontech). To construct the expression plasmids for Gal4 DNA-binding domain (BD)-fused fragments of Aft1p, NcoI–XhoI fragments encoding Aft1p regions were inserted into the NcoI–SalI site of pGBKT7. The expression plasmid for simian virus 40 (SV40) nuclear localization signal (NLS)-protein kinase inhibitor (PKI) nuclear export signal (NES)-green fluorescent protein (GFP) (pPS1372) (Taura et al., 1998) was a gift from Dr. P. A. Silver (Harvard Medical School). Expression plasmids for GFP-fused Aft1p fragments from the MET25-driven promoter were constructed by inserting DNA fragments encoding various regions of Aft1p between the ClaI and EcoRI sites of pMET25-SV40 NLS-GFP (Ueta et al., 2003). The pET-GSP1G21V and pET-GSP1T26N vectors used to express mutant histidine-tagged Gsp1 proteins have been described previously (Ueta et al., 2003). The ORF of Msn5 was amplified from BY4741 genomic DNA by polymerase chain reaction (PCR). The Msn5 ORF was C-terminally myc-tagged and subcloned into pFastBac HT-A (Invitrogen, Carlsbad, CA) to generate the pFastBac-His6-Msn5-myc vector.
Yeast Two-Hybrid Assays
Yeast two-hybrid assays were performed by examining the growth of PJ69-4A or PJ69-4Afet3 (Y22) strains expressing both Gal4 transactivation domain (AD)- and BD-fused proteins in medium lacking adenine and histidine. In some experiments, 3-aminotriazole (3-AT; 10 mM final concentration) was added to the medium. Cells were spotted using 10-fold serial dilutions beginning at OD600 = 0.6. Cells showed similar growth on medium containing adenine and histidine. Adequate expression of fusion proteins in cells was confirmed by immunoblotting using anti-HA (12CA5) (Roche Diagnostics, Mannheim, Germany) and anti-Myc (9E10) (Covance, Princeton, NJ) antibodies.
Immunoblotting
Lysates of yeast cells, prepared by the bead lysing method, were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes. After blocking in phosphate-buffered saline (PBS) containing 0.1% Tween 20 and 5% skim milk, the membrane was incubated with the appropriate primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and visualization with the SuperSignal chemiluminescent detection system (Pierce Chemical, Rockford, IL). Signals obtained by immunoblotting were quantified by the LAS1000 luminescent image analyzer (Fuji Film, Tokyo, Japan).
Detection of Aft1p Phosphorylation
To detect Aft1p phosphorylation, a cell lysate was prepared using the bead lysing method from cells expressing Aft1p-HA or its derivatives in buffer (50 mM HEPES-KOH, pH 7.5, 50 mM NaCl, and 1 mM EDTA) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A) and 10 mM 
-glycerophosphate. The lysate was cleared by centrifugation, and the buffer was changed to calf intestine alkaline phosphatase (CIAP) buffer (10 mM Tris-Cl, pH 8.0, 50 mM NaCl, and 10 mM MgCl2) using protein desalting spin columns (Pierce Chemical). CIAP-treated samples were incubated with 1 U of CIAP (Takara, Kyoto, Japan) at 37°C for 1 h before the addition of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, and 1% -mercaptoethanol) and boiling. Proteins samples were separated by 4% SDS-PAGE and subjected to immunoblotting with an anti-HA antibody.
Indirect Immunofluorescence and GFP Microscopy
Subcellular localization of proteins was examined by indirect immunofluorescence microscopy or by GFP microscopy, essentially as described previously (Ueta et al., 2003). Briefly, for indirect immunofluorescence microscopy, cells expressing HA-tagged wild-type or mutant Aft1p were fixed in 4% formaldehyde. Cell walls were digested with 300 U of Zymolyase (Seikagaku Kogyo, Tokyo, Japan), followed by the addition of 2% SDS. Spheroplasts were fixed on poly-lysine–coated coverslips, permeabilized with 0.05% saponin, and then incubated with an anti-HA antibody. Signals were amplified and visualized using an Alexa Fluor 594 signal amplification kit (Invitrogen). Nuclei were stained by incubation with 500 ng/ml 4',6'-diamino-2-phenylindole (DAPI) for 5 min. For GFP microscopy, cells were cultured in medium lacking methionine to induce the expression of the GFP-fused proteins. Cells were fixed in 70% ethanol for 5 min and suspended in mounting medium containing 50 ng/ml DAPI. GFP-fused proteins and nuclei were visualized using an IX81 fluorescence microscope or an FV-1000 confocal microscope (Olympus, Tokyo, Japan). Expression of the tagged proteins was measured by immunoblotting and found to be similar in each of the strains under the different iron conditions used.
Purification of Recombinant Proteins
His-GSP1G21V and His-GSP1T26N were expressed in Escherichia coli, purified, and loaded with GTP or GDP as described previously (Ueta et al., 2003). Recombinant baculovirus expressing His6-Msn5-myc was generated using the Bac-to-Bac Baculovirus Expression system (Invitrogen). Hi Five cells were cultured in Grace's insect medium plus 10% fetal bovine serum at 27°C and infected with the recombinant virus. Sixty hours after infection, cells were lysed with 50 mM Tris-Cl, pH 7.5, and 2 mM PMSF and homogenized with a Dounce homogenizer. His6-Msn5-myc was purified from the soluble fraction using nickel-nitrilotriacetic acid agarose (QIAGEN, Valencia, CA). The His6-tag was cleaved using the AcTEV protease (Invitrogen).
Detection of Intermolecular Interaction of Aft1p
Cells expressing Aft1p-HA and Aft1p-TAP or Aft1pC291F-HA and Aft1pC291F-TAP were lysed in 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 2 mM PMSF using Multi-Beads Shocker (Yasui Kikai, Osaka, Japan). The soluble fraction was incubated with IgG-Sepharose (GE Healthcare) in 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 2% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS at 4°C for 2 h. The beads were washed five times with the same buffer, and precipitates were separated by 6% SDS-PAGE, and subjected to immunoblotting using an anti-HA antibody (16B12) (Covance).
Detection of Aft1p–Msn5p Interaction In Vitro
Lysates (2 mg) of cells expressing Aft1p-HA or Aft1pC291F-HA, prepared as above, were incubated with 1.5 µg of purified Msn5p-myc, in the presence of 1.5 µg of either GTP-loaded His6-Gsp1pG21V or GDP-loaded His6-Gsp1pT26N in 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, and 10% glycerol at 4°C for 2 h, followed by immunoprecipitation of Msn5p-myc by using an anti-myc antibody and protein A-Sepharose (GE Healthcare). The beads were washed four times with the same buffer, and precipitates were separated by 6% SDS-PAGE and subjected to immunoblotting using an anti-HA antibody.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), which raises the possibility that nuclear export of the protein occurs in an iron-dependent manner. To examine whether the nuclear export of Aft1p is regulated by iron, we used pse1-1 mutant cells carrying a temperature-sensitive PSE1 gene (Stade et al., 1997). We first grew pse1-1 cells expressing Aft1p-HA at the permissive temperature (25°C) in the absence of iron to accumulate Aft1p in the nucleus, followed by cultivation for 4 h at the restrictive temperature (37°C) in the continued absence of iron to inhibit additional nuclear import of Aft1p (Figure 1A). Cycloheximide was added to the culture at the restrictive temperature for the last 30 min to inhibit further synthesis of Aft1p. Aft1p accumulated in the nuclei of cells cultured in iron-deficient medium at the restrictive temperature (Figure 1B1). The culture was then divided into two aliquots, and cells were cultured in the presence or absence of 200 µM iron in the continuous presence of cycloheximide at the restrictive temperature, and the effect of iron on the subcellular localization of Aft1p was monitored by immunofluorescence microscopy. Aft1p was exported to the cytoplasm within 30 min of the addition of iron (Figure 1B2), whereas the protein remained in the nucleus in the absence of iron (Figure 1B3). Anti-HA immunoblotting revealed that the amount of Aft1p was not affected by iron treatment, although it decreased by 30 min in both iron-rich and -depleted cells (Figure 1C). These results suggest that Aft1p is exported from nucleus in an iron-dependent manner.
|
). Wild-type cells and xpo1-1 cells expressing Aft1p-HA were cultured to the logarithmic growth phase in iron-replete or iron-depleted conditions at the permissive temperature (25°C) and further cultured for 3 h at the restrictive temperature (37°C). As a control, we examined GFP-fused PKI NES, which is exported by Xpo1p (Wen et al., 1995). The cells were fixed, and the subcellular localization of Aft1p was examined by immunofluorescence microscopy. Aft1p was localized in the nucleus in iron-depleted wild-type cells as well as in xpo1-1 cells (Figure 2A, 1–4). In iron-replete xpo1-1 cells, Aft1p localized to the cytoplasm. In contrast, nuclear export of the control GFP-fused PKI NES was inhibited (Figure 2B, compare 1 and 2). These results indicate that Xpo1p is not the exportin for Aft1p. We next probed the subcellular localization of Aft1p in msn5 cells to assess the involvement of Msn5p in nuclear export of Aft1p. In msn5 cells, Aft1p accumulated in the nucleus regardless of iron availability (Figure 2A, 5 and 6), although nuclear export of GFP-fused PKI NES was not perturbed (Figure 2B3). These results indicate that Msn5p is required for the nuclear export of Aft1p.
|
; Ossareh-Nazari et al., 2001). Because Aft1p is exported to the cytoplasm only in the iron-replete condition, we examined whether the interaction between Aft1p and Msn5p is regulated by the iron status of cells using a yeast two-hybrid analysis. Aft1p was fused to the Gal4 transactivation domain (AD–Aft1p) and Msn5p was fused to the DNA-binding domain (BD–Msn5p). PJ69-4A cells, which possess GAL1–HIS3 and GAL2–ADE2 reporter genes, grew on SD plates lacking adenine and histidine when both AD–Aft1p and BD–Msn5p were expressed, whereas cells expressing either the AD–Aft1p or BD–Msn5p fusion protein alone did not grow (Figure 3A), indicating that Aft1p and Msn5p interact with each other. Next, we used a yeast two-hybrid assay to determine whether the interaction between Aft1p and Msn5p was affected by the iron status of the cells. We first generated yeast two-hybrid reporter cells in which we could manipulate the iron status. Because regular SD medium is iron replete (Yamaguchi-Iwai et al., 1995, 1996; Philpott et al., 2002; Puig et al., 2005), to achieve the iron-starved condition we disrupted the FET3 gene, whose gene product is required for high-affinity iron uptake (Askwith et al., 1994; Stearman et al., 1996) in PJ69-4A cells, forming PJ69-4Afet3. PJ69-4Afet3 cells expressing both AD–Aft1p and BD–Msn5p fusion proteins were not able to grow on media lacking adenine and histidine, whereas the binding between SV40 large T and p53 was not affected (Figure 3B). However, the interaction between Aft1p and Msn5p in PJ69-4Afet3 was restored by the addition of excess iron (200 µM) to the plates (Figure 3C). These results indicate that Msn5p recognizes Aft1p only in iron-replete conditions. A paralogue of Aft1p, Aft2p, activates its target genes in response to iron starvation, similar to Aft1p (Blaiseau et al., 2001; Rutherford et al., 2001). We therefore examined whether Msn5p also recognizes Aft2p only in iron-rich conditions. Msn5p interacted with Aft2p in PJ69-4A cells and PJ69-4Afet3 cells in the presence, but not in the absence, of excess iron (Figure 3, D–F), suggesting that the activity of Aft2p is regulated by iron via the same mechanism as that of Aft1p. Collectively, these results indicate that Msn5p recognizes both Aft1p and Aft2p in the iron-replete condition.
|
|
; DeVit and Johnston, 1999; Boustany and Cyert, 2002). As we showed above, both Aft1p and Aft2p are recognized by Msn5p in the presence of sufficient iron, and Aft1p is a phosphoprotein (Casas et al., 1997). We therefore searched for serine/threonine residues in the two Msn5p-interacting regions that are conserved in Aft1p and Aft2p as well as in predicted Aft1p orthologues in other fungal species in the Saccharomyces Genome Database. Four serine residues (S170, S210, S224, and S240) in Aft1p(147-270) are conserved between Aft1p and Aft2p (Figure 5A), and S210 and S224 are also highly conserved in other Aft1p orthologues. We replaced these two serine residues with alanines or phospho-mimetic aspartates to create the Aft1p(SA) and Aft1p(SD) mutants, respectively. We also identified a highly conserved sequence in Aft1p(304-498), amino acids 421-435, and mutated four threonine residues within this region (T421, T423, T431, and T435) to alanine to create Aft1p(TA) (Figure 5B). We then assessed the effect of these mutations on the localization of Aft1p in iron-replete cells. Most Aft1p(SA) was localized to the nucleus even in the presence of excess iron. However, individual serine mutations of Aft1p [Aft1p(S170A) and Aft1p(S240A)] did not affect the iron-dependent localization of the protein, indicating that phosphorylation of S210 and/or S224 may be important for the nuclear export of Aft1p (Figure 5C, 3–8). Aft1p(TA) also localized primarily to the nucleus in the presence of excess iron (Figure 5C, 11 and 12), although the distribution was less distinct than with Aft1p(SA). We next generated a combined mutant, Aft1p(STA), in which the two serine (S210 and S224) and four threonine (T421, T423, T431, and T435) residues were replaced with alanine. Aft1p(STA) discretely accumulated in the nucleus even in the presence of excess iron (Figure 5C, 13 and 14). Aft1p with either S210 or S224 mutated to alanine (Aft1pS210A or Aft1pS224A), but not both, was exported to the cytoplasm in iron-replete cells (data not shown). However, the phospho-mimetic mutant of S210 and S224 [Aft1p(SD)] was not exported to the cytoplasm in the iron-depleted condition, suggesting that phosphorylation of S210 and S224 is not enough to create a signal for iron-dependent nuclear export of Aft1p (Figure 5C, 9 and 10). Although we have not examined the effect of single mutations of the four threonines on Aft1p localization, S220 and S224 in Aft1p(147-270) and at least one of the four threonines in Aft1p(304-498) are likely to be involved in creating motifs recognized by Msn5p in the presence of iron.
|
|
The results presented in Figure 4C suggested that two regions of Aft1p, Aft1p(147-270) and Aft1p(304-498), are necessary for the nuclear export of Aft1p. We analyzed interactions between the N-terminal or C-terminal regions of Aft1p by a yeast two-hybrid assay by using PJ69-4A cells (Figure 6D). We did not observe any interaction between Aft1p(1-270) and Aft1p(304-690), leading us to suspect that the sequences intervening between the two Msn5p-interacting regions, amino acids 271-303 [Aft1p(271-303)], may be involved in the interaction between the two domains. Although the addition of Aft1p(271-303) to one fragment had little effect on the interaction between the two fragments, PJ69-4A cells expressing BD-Aft1(1-303) and AD-Aft1p(271-690) were able to grow on –His and –Ade plates. The interaction between the two Aft1p fragments was confirmed to be iron-sensitive, because the interaction between the two domains was not observed in PJ69-4Afet3 cells (Figure 6E). We next examined the involvement of S210 and S224, as well as T421, T423, T431, and T435, in the interaction between Aft1p(1-303) and Aft1p(271-690)(Figure 6F). Although Aft1p(1-303, SA) interacted with Aft1p(271-690), Aft1p(271-690, TA) failed to interact with Aft1p(1-303). These results indicate that at least some of the threonine residues, which are dispensable for the interaction with Msn5p (Figure 6B), do participate in the iron-mediated interaction between the N-terminal and C-terminal regions of Aft1p. Thus, phosphorylation of S210 and S224 may be involved in the interaction with Msn5p (Figure 6, A and C), but not in the interaction between the two fragments of Aft1p.
We examined iron induces intermolecular interaction of Aft1p in cells using coimmunoprecipitation assays (Figure 6F). msn5 cells expressing Aft1p-TAP and Aft1p-HA were cultured in the presence or absence of iron and Aft1p-TAP was immunoprecipitated with IgG beads and the amount of Aft1p-HA that coprecipitated with Aft1p-TAP was assessed. Aft1p-HA was specifically coprecipitated from lysates of cells cultured in the presence of iron, indicating that two Aft1p proteins interact each other in an iron-dependent manner.
Intermolecular Interaction of Aft1p Is Required for Msn5p Recognition and the Mutation Found in Aft1-1up Disrupts the Iron-mediated Intermolecular Interaction and Msn5p Recognition of Aft1p
The results presented above suggest that the iron-dependent intermolecular interaction of Aft1p is involved in its iron-dependent nuclear export. Because Aft1p(271-303) is necessary for the iron-dependent interaction of the N-terminal and C-terminal fragments of Aft1p (Figure 6, D and E), an amino acid(s) in Aft1p (271-303) may be critical for the iron-dependent intermolecular interaction of Aft1p. We have shown that the Aft1-1up mutant (Aft1pC291F) constitutively activates Aft1 target genes, because it localizes exclusively in the nucleus (Yamaguchi-Iwai et al., 2002). Here, we assessed whether Cys291 is necessary for the interaction of the N- and C-terminal regions of Aft1p by using a yeast two-hybrid analysis (Figure 7A). Introduction of the C291F mutation in either Aft1p(1-303) or Aft1p(271-690) weakened the interaction between the two fragments, and the interaction between the two fragments was completely disrupted when the Cys291 in both fragments was mutated to phenylalanine. We next examined whether the C291F mutation of Aft1p affects the iron-dependent intermolecular interaction of the protein (Figure 7B). Aft1pC291F-HA failed to coprecipitate with Aft1pC291F-TAP, whereas Aft1p-HA was able to interact with Aft1p-TAP in the presence of iron. These results confirm that two Aft1p proteins interact each other in the presence of iron and that Cys291 is critical for that intermolecular interaction. Finally, we examined whether the iron-dependent intermolecular interaction of Aft1p is critical for recognition by Msn5p (Figure 7, C and D). Because the interaction between cargo and exportin depends on the GTP-bound form of Ran (Mattaj and Englmeier, 1998), mutants of the yeast homologue of Ran, Gsp1, which lack the GTPase activity (G21V) or specifically bind GDP (T26N), were prepared (Ueta et al., 2003). Lysates of yeast cells expressing Aft1p-HA were incubated with Msn5p-myc in the presence of either GTP-loaded His6-Gsp1G21V or GDP-bound His6-Gsp1T26N, and Msn5p-myc was immunoprecipitated from the mixture. Anti-HA immunoblotting clearly showed that Aft1p-HA interacted with Msn5p in the presence of Ran-GTP (Figure 7C). Moreover, Aft1pC291F cannot interact with Msn5p in the presence of Ran-GTP (Figure 7D). Because the C291F mutation of Aft1p abolishes its ability to interact each other, the intermolecular interaction of Aft1p seems to be critical for the recognition of Aft1p by Msn5p. Aft1-1up may therefore localize in the nucleus because the C291F mutation disrupts the iron-dependent interaction of Aft1p, which in turn blocks its recognition by Msn5p.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Philpott et al., 2002; De Freitas et al., 2003). Change in its nucleocytoplasmic localization in response to iron concentration is a key event in the regulation of iron-dependent transcriptional activation of genes involved in iron metabolism (Yamaguchi-Iwai et al., 2002). Because Pse1p-mediated nuclear import of Aft1p is not regulated by cellular iron status (Ueta et al., 2003), it was of great interest to identify the mechanism underlying nuclear export of the protein. In the present study, we have shown that nuclear export of Aft1p is regulated by the iron status of the cells and that Msn5p is an exportin for Aft1p. The interaction between Aft1p and Msn5p did not occur under conditions of iron starvation, and Aft1p nuclear export was promoted by iron. We therefore propose that the iron-dependent interaction between Aft1p and Msn5p is critical for the iron-regulated nucleocytoplasmic shuttling of Aft1p.
Msn5p was identified as a factor involved in the pheromone response and in calcium signaling (Akada et al., 1996; Matheos et al., 1997) and has since been identified as a nuclear export receptor for many proteins, including transcription factors (Hopper, 1999). The Msn5p-interacting regions within these cargo proteins are large stretches of amino acid sequences (
100 amino acids or more) that do not share any homology (Blondel et al., 1999; DeVit and Johnston, 1999; Boustany and Cyert, 2002; Jaquenoud et al., 2002). Here, we have identified two regions of Aft1p, amino acids 147-270 and 304-498, that are necessary for interacting with Msn5p as well as for nuclear export of Aft1p. Phosphorylation of S210 and S224 within the former region is involved in the Msn5p-mediated nuclear export of Aft1p (Figures 5C and 6, A–C). Serine/threonine phosphorylation within the Msn5p-interacting regions is similarly required for Msn5p recognition of many proteins (Kaffman et al., 1998; DeVit and Johnston, 1999; Jaquenoud et al., 2002). Mig1p, a transcriptional repressor responsible for the repression of many genes in S. cerevisiae in the presence of glucose, is regulated by Msn5p-mediated nuclear export in low glucose conditions (DeVit and Johnston, 1999). Phosphorylation of serine residues within the Msn5p-interacting region of Mig1p by the Snf1p kinase is critical for the glucose-induced nuclear export of Mig1p (DeVit and Johnston, 1999). Snf1p is also involved in the activation of Aft1p in the diauxic shift (the transition between fermentative and oxidative metabolism) (Haurie et al., 2003), but neither S210 nor S224 of Aft1p is an Snf1 phosphorylation site (Dale et al., 1995). Moreover, the activity of Aft1p is regulated by iron in snf1 cells (Haurie et al., 2003). In fact, we observed that iron had no effect on Aft1p phosphorylation (Figure 6C) and that the phospho-mimetic mutations of S210 and S224 did not promote nuclear export of Aft1p in the iron-depleted condition (Figure 5C). Thus, we suspect that S210 and S224 of Aft1p are constitutively phosphorylated in the nucleus, but these phosphoserines are not sufficient for Msn5p-dependent export of Aft1p. We first hypothesized that the phosphorylation of at least one of the threonines, T421, T423, T431, and T435, in the second Msn5p interaction region would also be required for Msn5p recognition. This turned out not to be the case, but our further analysis suggested that at least one of these four threonines is involved in the iron-dependent interaction between the two regions of Aft1p (Figure 6, B and C). We also observed that two Aft1p proteins interacts each other in iron-rich cells, which seems to be a key event in the regulation of Aft1p nuclear export (Figure 6G).
Other metal-sensing transcriptional activators are regulated by metal-dependent intramolecular interactions. Mac1p, a copper-responsive transcriptional activator in S. cerevisiae, is inactivated in the presence of copper through the intramolecular interaction of Cys-rich motifs within the transactivation domain (Jensen and Winge, 1998). Zap1p, a zinc-responsive transcriptional activator in S. cerevisiae, is inactivated in the presence of zinc, and its inactivation is similarly mediated by a zinc-dependent intramolecular interaction (Bird et al., 2003; Wang et al., 2006). Recently, Cuf1p, a copper-sensing transcription factor from Schizosaccharomyces pombe, has also been reported to undergo a copper-induced intramolecular interaction leading to the cytoplasmic retention of Cuf1p (Beaudoin and Labbe, 2006). However, in the case of Aft1p, an intermolecular, but not an intramolecular interaction of Aft1p is critical for its nuclear export and subsequent down-regulation of the expression of genes in the iron regulon. From the results presented here, we propose the following mechanism for the iron-dependent nuclear export of Aft1p. In iron-replete conditions, iron induces the intermolecular interaction of Aft1p. Msn5p recognizes a motif created by the intermolecular interaction of Aft1p and the phosphorylated S210 and S224, and then it exports Aft1p to the cytoplasm. Cys291 is critical for intermolecular interaction of the protein, and its absence in the Aft1-1up mutant (Aft1pC291F) may in part explain why Aft1-1up localizes exclusively in the nucleus and constitutively activates Aft1-target genes.
If intermolecular interaction of Aft1p in iron-replete cells is necessary for the regulation of Aft1p by iron (Figures 6 and 7), what signal induces this intermolecular interaction? It has been hypothesized that Aft1p itself senses iron, which triggers the nucleocytoplasmic shuttling of the protein (Yamaguchi-Iwai et al., 1995; Winge et al., 1998). A recent study demonstrated that the mitochondrial but not the cytoplasmic Fe-S cluster metabolism is important for Aft1p activation, and the binding of the Fe-S cluster directly to Aft1p was questioned (Rutherford et al., 2005). Additionally, an unidentified molecule that is transported from mitochondria to the cytoplasm by the Atm1p ABC transporter has been suggested to regulate Aft1p (Rutherford et al., 2005). Another report indicates that direct binding of glutaredoxin-3 (Grx3p) and -4 (Grx4p) to Aft1p functions in the iron regulation of Aft1p (Ojeda et al., 2006). Glutaredoxin-5 is involved in the Fe-S cluster biogenesis in mitochondria, where it is localized (Rodriguez-Manzaneque et al., 2002), and mitochondrial expression of Grx3p or Grx4p compensates for the defect in grx5 cells (Molina et al., 2004). Indeed, in grx3grx4 cells, Fe incorporation into cytoplasmic Fe-S proteins, as well as iron-dependent nuclear export of Aft1p, are affected (Ojeda et al., 2006; Pujol-Carrion et al., 2006). These observations again raise the possibility that Aft1p may sense iron through its binding to an Fe-S cluster. The 291CXC293 motif of Aft1p is essential for its regulation by iron (Yamaguchi-Iwai et al., 1995), and both Grx3p and Grx4p fail to bind to the Aft1 C291,293F mutant (Ojeda et al., 2006). We showed here that the C291F mutation disrupts the iron-dependent intermolecular interaction of Aft1p (Figure 7B). Thus, it is tempting to speculate that the binding of an Fe-S cluster to the 291CXC293 motif of Aft1p mediates the intermolecular interaction and creates the motif recognized by Msn5p. More work will be needed to clarify the detailed mechanism underlying the iron-induced conformational change that creates the motif recognized by Msn5p.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Address correspondence to: Yuko Yamaguchi-Iwai (yukoiwai{at}kais.kyoto-u.ac.jp) or Kazuhiro Iwai (kiwai{at}med.osaka-cu.ac.jp).
Abbreviations used: AD, Gal4 transactivation domain; BD, Gal4 DNA-binding domain; CIAP, calf intestine alkaline phosphatase; DAPI, 4',6'-diamino-2-phenylindole; FOB, ferrioxamine B; GFP, green fluorescence protein; HA, hemagglutinin; NES, nuclear export signal; NLS, nuclear localization signal; TAP, tandem affinity purification.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Askwith, C. C., de Silva, D., and Kaplan, J. (1996). Molecular biology of iron acquisition in Saccharomyces cerevisiae. Mol. Microbiol 20, 27–34.[CrossRef][Medline]
Askwith, C., Eide, D., Van Ho, A., Bernard, P. S., Li, L., Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994). The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake. Cell 76, 403–410.[CrossRef][Medline]
Beaudoin, J., and Labbe, S. (2006). Copper induces cytoplasmic retention of fission yeast transcription factor cuf1. Eukaryot. Cell 5, 277–292.
Bird, A. J., McCall, K., Kramer, M., Blankman, E., Winge, D. R., and Eide, D. J. (2003). Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function. EMBO J 22, 5137–5146.[CrossRef][Medline]
Blaiseau, P. L., Lesuisse, E., and Camadro, J. M. (2001). Aft2p, a novel iron-regulated transcription activator that modulates, with Aft1p, intracellular iron use and resistance to oxidative stress in yeast. J. Biol. Chem 276, 34221–34226.
Blondel, M., Alepuz, P. M., Huang, L. S., Shaham, S., Ammerer, G., and Peter, M. (1999). Nuclear export of Far1p in response to pheromones requires the export receptor Msn5p/Ste21p. Genes Dev 13, 2284–2300.
Boustany, L. M., and Cyert, M. S. (2002). Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site. Genes Dev 16, 608–619.
Casas, C., Aldea, M., Espinet, C., Gallego, C., Gil, R., and Herrero, E. (1997). The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae. Yeast 13, 621–637.[CrossRef][Medline]
Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, D. G. (1995). Similar substrate recognition motifs for mammalian AMP-activated protein kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1, and mammalian calmodulin-dependent protein kinase I. FEBS Lett 361, 191–195.[CrossRef][Medline]
Dancis, A. (1998). Genetic analysis of iron uptake in the yeast Saccharomyces cerevisiae. J. Pediatr 132, S24–S29.[CrossRef][Medline]
De Freitas, J., Wintz, H., Kim, J. H., Poynton, H., Fox, T., and Vulpe, C. (2003). Yeast, a model organism for iron and copper metabolism studies. Biometals 16, 185–197.[CrossRef][Medline]
DeVit, M. J., and Johnston, M. (1999). The nuclear exportin Msn5 is required for nuclear export of the Mig1 glucose repressor of Saccharomyces cerevisiae. Curr. Biol 9, 1231–1241.[CrossRef][Medline]
Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I. W. (1997). CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90, 1051–1060.[CrossRef][Medline]
Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M., and Nishida, E. (1997). CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308–311.[CrossRef][Medline]
Haurie, V., Boucherie, H., and Sagliocco, F. (2003). The Snf1 protein kinase controls the induction of genes of the iron uptake pathway at the diauxic shift in Saccharomyces cerevisiae. J. Biol. Chem 278, 45391–45396.
Hood, J. K., and Silver, P. A. (1999). In or out? Regulating nuclear transport. Curr. Opin. Cell Biol 11, 241–247.[CrossRef][Medline]
Hopper, A. K. (1999). Nucleocytoplasmic transport: inside out regulation. Curr. Biol 9, R803–R806.[CrossRef][Medline]
Jans, D. A., Xiao, C. Y., and Lam, M. H. (2000). Nuclear targeting signal recognition: a key control point in nuclear transport? Bioessays 22, 532–544.[CrossRef][Medline]
Jaquenoud, M., van Drogen, F., and Peter, M. (2002). Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1). EMBO J 21, 6515–6526.[CrossRef][Medline]
Jensen, L. T., and Winge, D. R. (1998). Identification of a copper-induced intramolecular interaction in the transcription factor Mac1 from Saccharomyces cerevisiae. EMBO J 17, 5400–5408.[CrossRef][Medline]
Kaffman, A., Rank, N. M., O'Neill, E. M., Huang, L. S., and O'Shea, E. K. (1998). The receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus. Nature 396, 482–486.[CrossRef][Medline]
Komeili, A., and O'Shea, E. K. (2000). Nuclear transport and transcription. Curr. Opin. Cell Biol 12, 355–360.[CrossRef][Medline]
Lesuisse, E., Simon-Casteras, M., and Labbe, P. (1998). Siderophore-mediated iron uptake in Saccharomyces cerevisiae: the SIT1 gene encodes a ferrioxamine B permease that belongs to the major facilitator superfamily. Microbiology 144, 3455–3462.
Matheos, D. P., Kingsbury, T. J., Ahsan, U. S., and Cunningham, K. W. (1997). Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. Genes Dev 11, 3445–3458.
Mattaj, I. W., and Englmeier, L. (1998). Nucleocytoplasmic transport: the soluble phase. Annu. Rev. Biochem 67, 265–306.[CrossRef][Medline]
Molina, M. M., Belli, G., de la Torre, M. A., Rodriguez-Manzaneque, M. T., and Herrero, E. (2004). Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins. J. Biol. Chem 279, 51923–51930.
Nakielny, S., and Dreyfuss, G. (1997). Nuclear export of proteins and RNAs. Curr. Opin. Cell Biol 9, 420–429.[CrossRef][Medline]
Ojeda, L., Keller, G., Muhlenhoff, U., Rutherford, J. C., Lill, R., and Winge, D. R. (2006). Role of glutaredoxin-3 and glutaredoxin-4 in the iron regulation of the Aft1 transcriptional activator in Saccharomyces cerevisiae. J. Biol. Chem 281, 17661–17669.
Ossareh-Nazari, B., Bachelerie, F., and Dargemont, C. (1997). Evidence for a role of CRM1 in signal-mediated nuclear protein export. Science 278, 141–144.
Ossareh-Nazari, B., Gwizdek, C., and Dargemont, C. (2001). Protein export from the nucleus. Traffic 2, 684–689.[CrossRef][Medline]
Pemberton, L. F., and Paschal, B. M. (2005). Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6, 187–198.[CrossRef][Medline]
Philpott, C. C., Protchenko, O., Kim, Y. W., Boretsky, Y., and Shakoury-Elizeh, M. (2002). The response to iron deprivation in Saccharomyces cerevisiae: expression of siderophore-based systems of iron uptake. Biochem. Soc. Trans 30, 698–702.[CrossRef][Medline]
Puig, S., Askeland, E., and Thiele, D. J. (2005). Coordinated remodeling of cellular metabolism during iron deficiency through targeted mRNA degradation. Cell 120, 99–110.[CrossRef][Medline]
Pujol-Carrion, N., Belli, G., Herrero, E., Nogues, A., and de la Torre-Ruiz, M. A. (2006). Glutaredoxins Grx3 and Grx4 regulate nuclear localisation of Aft1 and the oxidative stress response in Saccharomyces cerevisiae. J. Cell Sci 119, 4554–4564.
Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol 17, 1030–1032.[CrossRef][Medline]
Rodriguez-Manzaneque, M. T., Tamarit, J., Belli, G., Ros, J., and Herrero, E. (2002). Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes. Mol. Biol. Cell 13, 1109–1121.
Rutherford, J. C., Jaron, S., Ray, E., Brown, P. O., and Winge, D. R. (2001). A second iron-regulatory system in yeast independent of Aft1p. Proc. Natl. Acad. Sci. USA 98, 14322–14327.
Rutherford, J. C., Ojeda, L., Balk, J., Muhlenhoff, U., Lill, R., and Winge, D. R. (2005). Activation of the iron regulon by the yeast Aft1/Aft2 transcription factors depends on mitochondrial but not cytosolic iron-sulfur protein biogenesis. J. Biol. Chem 280, 10135–10140.
Stade, K., Ford, C. S., Guthrie, C., and Weis, K. (1997). Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90, 1041–1050.[CrossRef][Medline]
Stearman, R., Yuan, D. S., Yamaguchi-Iwai, Y., Klausner, R. D., and Dancis, A. (1996). A permease-oxidase complex involved in high-affinity iron uptake in yeast. Science 271, 1552–1557.[Abstract]
Strom, A. C., and Weis, K. (2001). Importin-beta-like nuclear transport receptors. Genome Biol 2, REVIEWS3008.
Taura, T., Krebber, H., and Silver, P. A. (1998). A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export. Proc. Natl. Acad. Sci. USA 95, 7427–7432.
Ueta, R., Fukunaka, A., and Yamaguchi-Iwai, Y. (2003). Pse1p mediates the nuclear import of the iron-responsive transcription factor Aft1p in Saccharomyces cerevisiae. J. Biol. Chem 278, 50120–50127.
Wang, Z., Feng, L. S., Matskevich, V., Venkataraman, K., Parasuram, P., and Laity, J. H. (2006). Solution structure of a Zap1 zinc-responsive domain provides insights into metalloregulatory transcriptional repression in Saccharomyces cerevisiae. J. Mol. Biol 357, 1167–1183.[CrossRef][Medline]
Wen, W., Meinkoth, J. L., Tsien, R. Y., and Taylor, S. S. (1995). Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463–473.[CrossRef][Medline]
Winge, D. R., Jensen, L. T., and Srinivasan, C. (1998). Metal-ion regulation of gene expression in yeast. Curr. Opin. Chem. Biol 2, 216–221.[CrossRef][Medline]
Yamaguchi-Iwai, Y., Dancis, A., and Klausner, R. D. (1995). AFT 1, a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae. EMBO J 14, 1231–1239.[Medline]
Yamaguchi-Iwai, Y., Stearman, R., Dancis, A., and Klausner, R. D. (1996). Iron-regulated DNA binding by the AFT1 protein controls the iron regulon in yeast. EMBO J 15, 3377–3384.[Medline]
Yamaguchi-Iwai, Y., Ueta, R., Fukunaka, A., and Sasaki, R. (2002). Subcellular localization of Aft1 transcription factor responds to iron status in Saccharomyces cerevisiae. J. Biol. Chem 277, 18914–18918.
Yun, C. W., Ferea, T., Rashford, J., Ardon, O., Brown, P. O., Botstein, D., Kaplan, J., and Philpott, C. C. (2000). Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake. J. Biol. Chem 275, 10709–10715.
This article has been cited by other articles:
|
|
 |
|
  J. Towpik, D. Graczyk, A. Gajda, O. Lefebvre, and M. Boguta Derepression of RNA Polymerase III Transcription by Phosphorylation and Nuclear Export of Its Negative Regulator, Maf1 J. Biol. Chem., June 20, 2008; 283(25): 17168 - 17174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kumanovics, O. S. Chen, L. Li, D. Bagley, E. M. Adkins, H. Lin, N. N. Dingra, C. E. Outten, G. Keller, D. Winge, et al. Identification of FRA1 and FRA2 as Genes Involved in Regulating the Yeast Iron Regulon in Response to Decreased Mitochondrial Iron-Sulfur Cluster Synthesis J. Biol. Chem., April 18, 2008; 283(16): 10276 - 10286. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Philpott and O. Protchenko Response to Iron Deprivation in Saccharomyces cerevisiae Eukaryot. Cell, January 1, 2008; 7(1): 20 - 27. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
