Expression of Both Isu1 and Isu2 Are Increased in ssq1Δ
As an initial step toward our goal of understanding the basis of the up-regulation of levels of Isu in the absence of Ssq1, we sought to determine the relative contributions made by Isu1 and Isu2 to the total Isu levels. First, we assessed the reactivity of antibodies raised against purified Isu1 to the 83% identical Isu2. When equivalent amounts of purified Isu1 and Isu2 were subjected to immunoblot analysis, the signals obtained were indistinguishable (A, two left lanes). We conclude that these antibodies recognize the two proteins equally well. The ratio of the signals obtained when these antibodies were used to analyze extracts from cells expressing only Isu1 or Isu2 (isu2Δ and isu1Δ, respectively) was ~7:1. Supporting the idea that Isu1 is normally more abundant than Isu2, the signal obtained using extracts from wild-type cells was very similar to that from isu2Δ cells. Because our interest was in the up-regulation that occurs in cells lacking Ssq1, we also compared Isu levels in extracts from ssq1Δ and isu2Δ ssq1Δ. In both cases the signals were more than 15-fold higher than in extracts from cells expressing Ssq1. We conclude that Isu1 is the predominant ISU protein in wild-type cells, and its levels dramatically increase when Ssq1 is absent.
Figure 1. Comparison of ISU1 and ISU2 protein and mRNA levels. (A) Expression of Isu1 and Isu2. Purified Isu1pstrep or Isu2pstrep, 5 ng of both, (left lanes) or 0.1 OD600 units of whole cell lysates from the indicated strains (right lanes) were subjected to electrophoresis (more ...)
Next we asked whether ISU1
mRNA levels are increased in ssq1
Δ cells. Such an increase would be consistent with previous observations that ISU1
mRNA levels increase when Aft1 is activated (Garland et al., 1999
) and that the iron regulon is up-regulated in cells lacking Ssq1 (Knight et al., 1998
). To determine directly whether ISU
mRNA levels increase when Ssq1 is absent, we assessed ISU
mRNA levels in four strains: wild type, isu1
Δ, and ssq1
Δ. Unlike ISU1
proteins, the mRNAs can be distinguished, because of differences in size and divergence of nucleotide sequence (B). ISU1
mRNA levels were very similar in wild-type and isu2
Δ cells, as were the levels of ISU2
mRNA in wild-type and isu1
Δ cells. However, in an SSQ1
deletion strain, both Isu1 and Isu2 mRNAs levels were increased on the order of 2–3-fold, relative to the wild-type strain. This 2–3-fold increase in ISU
mRNA levels is not sufficient to explain the 15–20-fold higher level of Isu in cells lacking Ssq1 than wild-type cells.
Deletion of AFT1 Suppresses the Severe Growth Defect of ssq1Δ
Because the modest increase in ISU
mRNA levels is insufficient to account for the high level of Isu in ssq1
Δ, we wanted to further explore the basis of this regulation. However, a more comprehensive examination was hampered both by the severely compromised growth of ssq1
Δ cells (Schilke et al., 1996
) and by the propensity of the ssq1
Δ strain to accumulate suppressor mutations. Therefore, we sought situations in which ssq1
Δ cells grew more vigorously, whereas Isu up-regulation was maintained. We isolated insertion mutations in AFT1
as suppressors of the growth defect of ssq1
Δ cells (see Materials and Methods
) and subsequently determined that a complete deletion of AFT1
resulted in substantial rescue of the growth defect caused by deletion of SSQ1
(A). As AFT1
is specifically needed to up-regulate iron transporters of the plasma and mitochondrial membranes (Yamaguchi-Iwai et al., 1995
; Rutherford et al., 2003
), such suppression is consistent with the idea that slow growth of ssq1
Δ could partially be rescued by the depletion of iron from growth medium (Knight et al., 1998
). To test the idea in our strain background that iron overload is in part responsible for the poor growth of Δssq1
cells, we added the iron chelator BPS to growth medium. ssq1
Δ cells were able to form colonies in the presence of BPS, although growth was compromised compared with wild-type cells (B). Also consistent with the idea that iron overload is deleterious, deletion of AFT2,
which does not result in reduction of iron levels, did not suppress the growth defect of ssq1
Δ cells (data not shown).
Figure 2. Effect of absence of Aft1 and Aft2 on ssq1Δ cells. (A) Serial dilutions at 1:10 of the indicated strains were spotted onto glucose-rich medium, and the plate was incubated for 3 d at 30°C. WT, wild type. (B) Dilutions at 1:10 of the indicated (more ...)
We then asked if ISU mRNA and protein was up-regulated in aft1Δ ssq1Δ cells. Similar levels of both mRNA and protein were found whether or not Aft1 was present (, C and D). Because Aft2, the paralog of Aft1, also binds iron regulatory elements, we tested expression levels of ISU mRNA in an ssq1Δ aft2Δ strain. Isu1 and Isu2 mRNA levels were both up-regulated, consistent with the idea that either Aft1 or Aft2 is capable of activating ISU1 and ISU2. However, most relevant for this report, Isu up-regulation was maintained in aft1Δ ssq1Δ cells, making the AFT1 deletion background appropriate for our studies.
Contributions of Transcriptional and Posttranscriptional Regulation of Isu
To more thoroughly assess the contribution of posttranscriptional regulation to Isu levels, we worked toward establishing conditions under which the amounts of ISU
mRNA levels were the same in cells lacking or expressing Ssq1. First, we addressed the issue of transcriptional regulation of ISU1
, the more highly expressed of the paralogs (). The ISU1
promoter has a site that fits the consensus for an Aft1/2-binding site (Garland et al., 1999
; Rutherford et al., 2003
). We introduced a mutation changing the core consensus sequence from CACCC to CAGGG, generating the promoter mutant we designate ISU1
*. Analogous mutations in other Aft1/2 target gene promoters destroy the functionality of the iron regulatory response element (Yamaguchi-Iwai et al., 1996
). Indeed, the increase in levels of ISU1
mRNAs seen in ssq1
Δ cells was obviated when the promoter mutations were present (, compare lanes 4 and 5, top panel), indicating that Aft1/2 transcription factors are responsible for the up-regulation of Isu1 mRNA.
Figure 3. Isu is regulated at the posttranscriptional level. Equivalent amounts of total mRNA (top panels) or cell extracts (bottom panels) from the indicated yeast strains lacking AFT1 were subjected to separation by electrophoresis, blotted to membranes, and (more ...)
ISU protein levels were also compared. The levels of ISU protein were 10–15-fold higher in the absence, compared with the presence, of SSQ1 (, lanes 1–3 compared with 4–6). Most importantly, Isu1 levels were on the order of 10-fold higher even when ISU mRNA levels were the same, that is with ISU1 under the control of the ISU1* promoter and in the absence of ISU2 (, compare lanes 3 and 6). We conclude that substantial up-regulation of Isu1 occurs at the posttranscriptional level when Ssq1 is absent.
Difference in the Rate of Isu1 Degradation in the Absence of Ssq1
To begin to understand the basis of the posttranscriptional up-regulation of ISU protein, we next determined the rate of synthesis and degradation of ISU1 protein. Four independently obtained strains ssq1Δ aft1Δ isu2Δ ISU1* and two isogenic aft1Δ isu2Δ ISU1* control strains were analyzed. Cells were pulse-labeled for 2 min with [35S]methionine and [35S]cysteine. The total amount of radioactivity incorporated by the two strains was comparable, as expected, because of their similar growth rates. Extracts prepared from the strains were subjected to immunoprecipitation with Isu-specific antibodies. The rate of synthesis of Isu1 was statistically indistinguishable in cells containing or lacking Ssq1 (A, inset).
Figure 4. Synthesis and degradation of Isu1 in the presence and absence of SSQ1. Cells were pulse-labeled for 2 min at 30°C by addition of 35S-labeled amino acids, and samples were removed and subjected to immunoprecipitation and analysis as described in (more ...)
Because there was no significant difference in the rate of synthesis of Isu1 between the two strains, we next examined the rate of Isu1 degradation. Cells were pulse-labeled and then “chased” with an excess of nonradiolabeled amino acids to prevent further incorporation of radioactivity. Extracts prepared from samples collected over a period of 4 h were subjected to immunoprecipitation, and the radioactivity was quantified. The half-life of Isu1 from the two strains differed by about fourfold, with Isu1 in cells expressing Ssq1 having a half-life of 29 min and in cells lacking Ssq1 a half-life of 111 min (A). As a control, the half-life of the other Hsp70 of the mitochondrial matrix, Ssc1, was also determined. The half-life of Ssc1 was similar in aft1Δ isu2 Δ ISU1* and ssq1Δ aft1Δ isu2 Δ ISU1*, 112 min and 105 min, respectively (B). We conclude that there is a significant difference in the degradation rate of Isu1 in the two strains, leading to higher levels of Isu1 in cells lacking Ssq1.
These results are consistent with the idea that the increase in stability of Isu1 is a response to an alteration in the process of Fe-S cluster biogenesis. However, it is also possible that the increase in stability of Isu is simply a consequence of its increased abundance. To test this idea, we utilized an ISU1 gene placed under the control of the tetR promoter. Using this construct, Isu1 could be expressed at the higher level typically found in ssq1Δ cells, even when Ssq1 was present (C, inset). The rate of degradation of overexpressed Isu1 was only slightly slower in such cells compared with the rate in the cells having normal levels of Isu1, 28 min compared with 21 min (C). We conclude that the rate of Isu1 degradation is not dependent upon its concentration in the cell.
Biological Importance of Isu Up-Regulation
The experiments described above establish that the levels of Isu are up-regulated when Ssq1 is absent. However, they do not answer the question of whether these higher levels are biologically important. To address this issue, we again utilized the tetR-ISU1 construct. As discussed above, Isu1 is present at levels near that found in ssq1Δ cells when expression is driven from the tetR promoter (, right lanes). Cells grew with similar doubling times (2 h) whether Isu1 expression was driven by the endogenous promoter or by the tetR promoter.
Figure 5. Expression of Isu1 in the absence of Ssq1. Cell extracts were prepared from strains having the aft1Δ isu2Δ mutation, in addition to the indicated genetic alterations. As indicated by the line, four independent transformants were analyzed. (more ...)
To test the effect of lower levels of Isu1 on growth, we took advantage of the fact that addition of doxycycline represses transcription from the tetR promoter. Four independently derived transformants carrying the ssq1Δ mutation and expressing ISU1 from the tetR promoter were analyzed. In the presence of drug, expression of Isu1 was dramatically lowered in all four (, lanes 2–5), with levels ranging from very similar to that found in the control cells containing SSQ1 to about threefold higher (lanes 3 and 2, respectively). All four grew more slowly in the presence of drug when Isu1 expression is reduced (compare lanes 2–5, and 7). However, their growth rates differed. The higher the level of Isu, the more rapid the growth rate. For example, cells having levels similar to that of the control strains expressing Isu1 from its own promoter had a doubling time of ~5 h compared with 2 h (compare lanes 1 and 3); cells having threefold higher levels grew more rapidly, with a doubling time of ~3.9 h (compare lanes 1 and 2), but still almost twice as slowly as control cells. Thus, we conclude that in the absence of Ssq1 higher levels of Isu1 protein are necessary for robust growth.
Specificity of Isu Regulation
Having determined that up-regulation of Isu in the absence of the Hsp70 Ssq1 occurs at both the transcriptional and posttranslational levels, we tested the specificity of the response. First, we asked whether other components of the Fe-S cluster biogenesis pathway were also up-regulated in the absence of Ssq1. The levels of eight additional, known or putative, components of the pathway (Nfs1, Yfh1, Yah1, Mge1, Jac1, Isa1, Isa2, and Nfu1) were compared in extracts from wild-type and ssq1Δ mitochondria. None of the proteins tested differed in levels by more than twofold (). Thus, Isu is atypical, if not unique, among components of the Fe-S cluster biogenesis pathway in regards to up-regulation in response to the absence of Ssq1.
Figure 6. Level of components of Fe-S cluster biogenesis system in the absence of Ssq1. Equivalent amounts of mitochondrial extracts prepared from wild-type and ssq1Δ cells were separated by SDS-PAGE and probed with polyclonal antibodies specific for the (more ...)
Next we addressed the effect of reduced activity of components of the Fe-S cluster biogenesis pathway other than Ssq1 on levels of Isu. We utilized deletion mutants for testing the nonessential components Yfh1 and Grx5. To test the essential components, Jac1 and Nfs1, we used the partial loss of function mutants, nfs1L191S
, a temperature-sensitive (ts) mutant (Li et al., 1999
) and jac1LKDDEQ
, which although having no obvious growth defect, encodes a protein partially defective in binding to Isu (Andrew et al., 2006
). Substantially higher levels of Isu were observed in grx5
Δ and yfh1
Δ, as well as jac1LKDDEQ
, but not in nfs1L191S
(A). Because the difference between the jac1LKDDEQ
was striking, we considered the possibility that the NFS1
mutant had less of an effect on Fe-S cluster biogenesis than the JAC1
Figure 7. Effect of reduced activity of components of the Fe-S biogenesis system on Isu levels. (A) Cell lysates, prepared from indicated strains, were separated by SDS-PAGE and subjected to immunoblot analysis with Isu-specific and, as a loading control, Mge1-specific (more ...)
The degree of up-regulation of the “iron regulon” is an indication of the severity of the defect in Fe-S cluster biogenesis. We therefore monitored the response by testing expression from the promoter of FET3, which encodes the high-affinity plasma membrane iron transporter whose expression is driven by Aft1 and Aft2, utilizing a FET3:lacZ fusion. β-Galactosidase activity was threefold higher in nfs1L191S mutant compared with the control strain, but the jac1LKDDEQ mutant and its control strain had very similar activities (B). Thus, all available data indicate that the NFS1 mutant has greater effects on Fe-S cluster biogenesis than the JAC1 mutant.
To determine whether the increase in Isu levels in jac1LKDDEQ cells is due to an increase in stability compared with cells expressing wild-type Jac1, we carried out a pulse-chase analysis of protein degradation. The half-life of Isu1 from the two strains differed by ~5.4-fold, with Isu1 in cells expressing wild-type Jac1 having a half-life of 36 min and in cells expressing Jac1LKDDEQ, a half-life of 197 min (C). As expected, the level of Isu1 mRNA was similar in both strains (data not shown). Thus, Isu1 is regulated at the level of protein degradation in cells having reduced Ssq1 or Jac1 activity.
Dependence of Isu Up-Regulation on Nfs1
To extend the comparison between Jac1 and Nfs1, we placed the expression of these proteins under the control of the GAL1 promoter, allowing us to monitor Isu levels after repression of either Jac1 or Nfs1 upon transfer from galactose- to glucose-based medium. In the case of Jac1, the levels of Isu1 began increasing when Jac1 levels reached 25% of normal, rising to ~15-fold higher 6 h later (A). Nfs1 depletion showed a very different pattern. About 12 h after Nfs1 dropped to below 25% of normal levels, Isu levels began to rise, but only about fourfold, and dropped to about twofold by the end of the experiment (A). In sum, a decrease in Nsf1 activity did not result in a substantial increase in Isu levels.
Figure 8. Effect of Nfs1 depletion on Isu expression. (A) GAL-JAC1 (top) and GAL-NFS1 (bottom) were grown in galactose-based medium and then shifted to glucose-based medium at zero time. Because expression of both Jac1 and Nfs1 were expressed above normal levels (more ...)
Of the five components tested for the effect of their activity on the expression of Isu, only Nfs1 depletion failed to show a 10-fold or greater increase in levels. Therefore, we decided to ask whether Nfs1 was required for the increase in Isu levels to occur. To this end we combined the jac1LKDDEQ mutation with the Gal:Nfs1 repression system. In the galactose-based medium, conditions under which expression of Nfs1 was not repressed, Isu levels were high, as expected. However, after inhibition of Nfs1 synthesis the level of Isu dropped (B). Our results are consistent with Nfs1 being necessary for the increase in Isu levels.