Cys-27 and Cys-64 in domain I are required for mitochondrial import of Ccs1
The import of Ccs1 into mitochondria depends on the disulfide relay of the IMS (Mesecke et al., 2005
; Reddehase et al., 2009
). For the twin CX9
C and twin CX3
C substrates of Mia40 the cysteines are critical for efficient import. We therefore decided to identify the cysteine(s) in Ccs1 that influence its localization by analyzing the mitochondrial import of Ccs1 cysteine-to-serine mutants. First, we established a protocol to monitor the mitochondrial import of wild-type Ccs1 (). To this end, we incubated radioactively labeled Ccs1 with isolated mitochondria. At the end of the import reaction the sample was split into four parts and analyzed either directly or after treatment with proteinase K (PK). The latter treatment either was applied to intact mitochondria or was preceded by hypoosmotic swelling of mitochondria to generate mitoplasts (mitochondria lacking their outer membrane) or by mitochondrial lysis with Triton X-100. A band migrating at the expected size of Ccs1 was detected when intact mitochondria were treated with PK, indicating import of Ccs1 into mitochondria (, lane 3). On hypoosmotic swelling and subsequent PK treatment this signal disappeared, implying import of Ccs1 into the IMS (, lane 4). Notably, in some experiments we observed that the band representing Ccs1 migrated slightly faster on SDS–PAGE after PK treatment compared with untreated samples. In these cases a small part of the Ccs1 protein might still be exposed to the exterior after the import reaction. This exposed part would then be truncated upon digestion by PK. However, the main part of the protein was inaccessible to protease. The import of twin CX3
C and twin CX9
C substrates of Mia40 exhibits a marked dependence on reductants such as reduced glutathione (GSH; Bien et al., 2010
). When testing the import of Ccs1 we found a similar dependence of Ccs1 import (). At low concentrations of GSH (5 mM) import of Ccs1 proceeded faster than import in the absence of reductant. However, at higher concentrations of GSH (15 mM) Ccs1 import was strongly decreased.
We next investigated the import of the Ccs1 cysteine mutants (). We generated double mutants of the two cysteine pairs that were previously shown to contribute to copper binding and transfer (C17/C20 and C229/C231; ) and single mutants of the remaining three cysteines. The Ccs1 double-cysteine mutants Ccs1C17S/C20S and Ccs1C229S/C231S, as well as Ccs1C159S, were imported with comparable efficiency to that of wild-type Ccs1. However, Ccs1C27S and Ccs1C64S exhibited strongly diminished import, indicating that these cysteines are critical for the mitochondrial localization of Ccs1.
The cysteines at positions 27 and 64 are both situated in domain I of Ccs1 (). A closer analysis of the structures of this domain (Lamb et al., 1999
) revealed that C27 and C64 face each other and, at least in one of the structures, covalently connect two antiparallel α-helices (). Thus the structure of domain I resembles the structures of typical Mia40 substrates. We therefore asked whether domain I (corresponding to residues 2–74 of Ccs1) on its own is imported into mitochondria and whether it can serve as a targeting signal for a nonmitochondrial protein. Applying our import assay, we first demonstrated that domain I can be imported into isolated mitochondria, although with lower efficiency than full-length Ccs1 (, lanes 1–3). Mutation of either C27 or C64 abolished the import of domain I into mitochondria, confirming their relevance for the import process (). Moreover, wild-type domain I fused to the cytosolic protein dihydrofolate reductase (DHFR) allowed the import of this chimeric protein into mitochondria, whereas the C27S and C64S mutants again failed to be transported into mitochondria (, lanes 4–6). In summary, we found that domain I alone is sufficient for C27- and C64-dependent targeting of Ccs1 to mitochondria.
In vitro C27 and C64 of Ccs1 can be oxidized by Mia40
Cellular depletion of Mia40 results in lowered amounts of mitochondrial Ccs1 (Reddehase et al., 2009
). We verified this finding by immunoblot analyses of mitochondria isolated from strains with increased or lowered Mia40 levels (). Notably, the mitochondrial levels of Ccs1 did not appear to be as sensitive toward depletion of Mia40 as the levels of the twin CX3
C protein Tim10.
FIGURE 2: A disulfide bond between Cys-27 and Cys-64 of Ccs1 can be formed by Mia40 in vitro. (A) Protein levels in mitochondria isolated from strains with varying Mia40 levels. The yeast strain GalL–Mia40 was used to regulate the protein levels of Mia40. (more ...)
During mitochondrial import Ccs1 also forms an intermediate with Mia40 that is connected by a disulfide bond (Reddehase et al., 2009
). Because C27 and C64 are critical for Ccs1 import, we next tested whether these cysteines are involved in intermolecular disulfide bond formation with Mia40 (). We imported radioactively labeled Ccs1 into mitochondria, stopped thiol–disulfide exchange reactions by treatment with N
-ethylmaleimide (NEM), and then subjected Mia40 to immunoprecipitation. When analyzing the precipitate from the experiment with wild-type Ccs1 by nonreducing SDS–PAGE and autoradiography we indeed identified a band at a size of ~90 kDa corresponding to the combined size of Mia40 and Ccs1 (, lane 3). When analyzing the C27S and C64S mutants for an interaction with Mia40, we found that the C27S variant still interacted with Mia40, although to a lesser extent than the wild-type protein (, lane 6). However, the interactions between Mia40 and the C64S mutant, as well as those between Mia40 and the C27S/C64S mutant, were completely abolished, indicating that Mia40 presumably interacts with Cys-64 of Ccs1 during import into mitochondria (, lanes 9 and 12). These results are in agreement with a similar experiment performed by Groß et al., (2011
, in this issue of MBoC
). Notably, the amounts of the Mia40–Ccs1 complex detected by this method were significantly lower than the amounts of the Mia40–Tim9 complex observed in a control experiment (, compare lanes 3 and 15).
The interactions of twin CX3
C and twin CX9
C substrates with Mia40 result in the formation of intramolecular disulfides in these substrates. We therefore assessed whether Mia40 also can introduce a disulfide between C27 and C64 (). To this end, we applied an in vitro assay that we had previously established to monitor the Mia40-mediated oxidation of small twin CX9
C proteins (Bien et al., 2010
). In this assay a reduced and radioactively labeled protein is incubated with either buffer, purified oxidized wild-type Mia40 (Mia40-WT), or a purified Mia40 active-site mutant (Mia40-SPS). At certain time points the reaction is stopped by trichloroacetic acid (TCA) precipitation, which prevents all further thiol–disulfide exchange reactions. Subsequently, free (reduced) thiols but not oxidized disulfides are modified with the alkylating agent mm-PEG24. This derivatization results in an increase in the molecular weight of the protein, which can be detected by a changed migration behavior on SDS–PAGE. Thus these alkylation shift experiments enable us to determine the redox state of a given protein. When applying the assay to a Ccs1 variant in which all cysteines except C27 and C64 were mutated (Ccs1C17S/C20S/C159S/C229S/C231S
) we found that the C27–C64 disulfide was formed in the presence of Mia40-WT, although with significantly slower kinetics than with the in vitro oxidation of twin CX9
C proteins (Bien et al., 2010
). We also found that during the reaction Mia40-WT and Ccs1 formed an intermolecular disulfide bond that was absent when we analyzed the reaction on a reducing gel (, compare lanes 3–8 with lanes 11–16). Instead, a semioxidized form of Ccs1 appeared (i.e., a form in which only one cysteine can be modified with mm-PEG24). Of importance, in the absence of functional Mia40 no oxidation of the two cysteines occurred (). Taken together, the results demonstrate that Ccs1 interacts with Mia40 most likely through C64 and that Mia40 can oxidize Ccs1 in vitro.
In vivo mitochondrial Ccs1 contains a structural disulfide bond between C27 and C64
Next we asked whether C27 and C64 form a disulfide bond in vivo. To address this question, we relied on in vivo alkylation shift experiments (). First, we rapidly lysed cells by treatment with TCA, thus “freezing” the in vivo redox state of Ccs1. Then we modified thiols with the alkylating agent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) to induce changes in migration on SDS–PAGE comparable to those depicted in and analyzed the proteins by SDS–PAGE and immunoblotting against Ccs1.
FIGURE 3: In vivo Cys-27 and Cys-64 form a structural disulfide bond in IMS-localized Ccs1. (A) Scheme depicting the setup of the in vivo redox state measurements. (B) Migration standard for AMS alkylation experiments. Cells expressing different cysteine-to-serine (more ...)
For a detailed analysis of the Ccs1 redox state in vivo we relied on a series of yeast strains expressing different Ccs1 cysteine mutants in a CCS1-deletion background. To this end, we generated constructs that encode CCS1 gene variants under the control of its endogenous promoter and terminator (pRS), as well as under the control of an overexpression promoter (pYX; see Supplemental Figure S1 for expression levels).
We used these strains first to generate an AMS-migration standard for Ccs1 (). We lysed cells from the different Ccs1 mutant strains, incubated the protein samples at 96°C with the reductant dithiothreitol (DTT) to open all disulfide bonds, and, after TCA precipitation to remove excess DTT, modified cysteine residues with AMS. The individual size shifts of AMS-treated Ccs1 variants correlated with the number of cysteine residues (; see added AMS molecules, and compare with lane 8).
Next we assessed the in vivo redox state of the wild-type Ccs1 (). At steady state Ccs1 was present in two fractions: a completely reduced form (modified with seven AMS molecules) and one that migrated at an apparent size that corresponds to a modification with five AMS molecules, indicating the presence of one disulfide bond in this form (, lane 2). The disulfide bond in this semioxidized form of Ccs1 appears to be very stable (i.e., structural) because it could be opened only upon DTT treatment at 96°C but not at 20°C or by DTT treatment of intact cells (, compare lanes 3–5). Because C17/C20 and C229/C231 are localized to CXXC and CXC motifs, respectively, which usually form unstable disulfides, we reasoned that C27 and C64 might form this structural disulfide bond. We therefore determined the redox state of Ccs1C27S/C64S in vivo (, lanes 6–10). Ccs1C27S/C64S was completely reduced (, lane 9), indicating that the stable disulfide in the wild type was indeed formed between C27 and C64. Notably, our results demonstrate that in vivo all other cysteines of Ccs1 (C17, C20, C159, C229, C231) are in the reduced state, which for C17/C20 and C229/C231 is in accordance with their role in copper binding and transfer.
Because wild-type Ccs1 exists at steady state in two different redox forms we wondered whether mitochondrial Ccs1 might have a redox state different from that of cytosolic Ccs1. To perform this experiment in vivo, we generated yeast cells in which Ccs1 exclusively localizes to the IMS. To this end, the CCS1-deletion strain was transformed with plasmids encoding IMS-targeted Ccs1 variants. These plasmids encoded an extended bipartite mitochondrial targeting signal of cytochrome b2 fused to Ccs1 (b2–Ccs1). This signal consists of a matrix-targeting signal followed by a hydrophobic sorting sequence ensuring transfer into the inner membrane, and an additional IMS-localized domain that prevents accidental matrix import. After import into mitochondria a part of the bipartite targeting signal is removed, and the mature b2–Ccs1 protein localizes to the IMS. Using the alkylation shift assay we found that all wild-type b2–Ccs1 molecules contained the stable C27–C64 disulfide bond (, lane 2). b2–Ccs1C27S/C64S migrated at the same position (, lane 9), confirming that in wild-type, IMS-localized Ccs1, C27 and C64 are oxidized (because they could not be modified with AMS). Thus our results indicate that at least a fraction of the cytosolic Ccs1 does not contain the C27–C64 disulfide (, lane 2).
We repeated this redox state determination multiple times and observed that the ratio between the completely reduced and the semioxidized fraction varied significantly in whole cells but not in mitochondria (Supplemental Figure S2, A-C). Such redox state changes at steady state are frequently observed also in other redox systems and might be due, for example, to differing growth states, cell densities, nutrient supplies, and oxygen tensions (Appenzeller-Herzog and Ellgaard, 2008
; Appenzeller-Herzog et al., 2008
). Indeed, we observed differences in the redox state of Ccs1 by changes in its expression level and by varying the oxygen tension (Supplemental Figure S2, A, B, and D). When comparing the redox state of Ccs1 at endogenous and overexpressed levels ( and Supplemental Figure S2, A and B) we found that in both cases the protein was in part semioxidized, but Ccs1 present at endogenous levels appeared to be overall in a more oxidized state. This finding might indicate that upon overexpression of Ccs1 the oxidation system in the cytosol becomes limiting. Similarly, in cells exposed to 20% oxygen a larger portion of C27 and C64 was present in the oxidized state than in cells cultured under hypoxic conditions (Supplemental Figure S2D). Taken together, the results demonstrate that C27 and C64 form a disulfide bond in the mitochondrial fraction of Ccs1, whereas a varying fraction of cytosolic Ccs1 is completely reduced.
Cysteine mutations in domain I affect the cellular distribution of Ccs1
Next we assessed whether the cysteine residues at positions 27 and 64 are also required for import under in vivo conditions by analyzing their cellular distribution. First, we compared the expression levels of the Ccs1 cysteine mutants expressed from the endogenous CCS1 promoter in whole cells (). All cells contained similar amounts of Ccs1 except for the strains expressing Ccs1C27S, Ccs1C64S, and Ccs1C27S/C64S. We assume that these three mutants are less stable compared with the wild-type Ccs1 and thus become rapidly degraded. This is also supported by circular dichroism spectra of the purified domain I of both the wild type and the C27S/C64S mutant of Ccs1, as the latter variant exhibits less secondary structure compared with the wild type (Supplemental Figure S3).
FIGURE 4: Mutations of Cys-27 and Cys-64 result in decreased mitochondrial levels of Ccs1. (A) Ccs1 protein levels in Δccs1 cells expressing different Ccs1 variants under the control of the endogenous promoter (pRS). Cells were grown to mid-log phase in (more ...)
We then isolated mitochondria from all strains and treated them with low amounts of PK to remove any residual proteins still attached to the outside of the mitochondria. We confirmed with control Western blots against the IMS protein Mia40 that mitochondria remained intact during the procedure (, bottom). Notably, we found that mitochondria isolated from the cells expressing Ccs1C27S, Ccs1C64S, and Ccs1C27S/C64S contained only minute amounts of Ccs1 (). To exclude that these minute amounts of mitochondrial Ccs1C27S, Ccs1C64S, and Ccs1C27S/C64S result only from the low cellular levels of Ccs1, we compared strains expressing the wild type and Ccs1C27S/C64S in more detail (). We found that the strain containing Ccs1C27S/C64S harbored only ~10% of the Ccs1 found in the wild type. However, the mitochondrial Ccs1 levels in the former strain were well below 2% compared with those in the wild type, indicating that the cellular distribution of Ccs1C27S/C64S to mitochondria is indeed impaired.
To further strengthen this notion, we repeated the experiment using cells expressing the different Ccs1 variants from an overexpression plasmid. The protein levels in these cells were ~10–20 times higher than with wild-type cells (Supplemental Figure S1). Moreover, the amounts of Ccs1C27S, Ccs1C64S, and Ccs1C27S/C64S were also significantly higher in these strains than in the strains expressing Ccs1 under the control of the endogenous promoter (Supplemental Figures S1 and S4, A and D). When comparing total Ccs1 levels with mitochondrial Ccs1 amounts () we clearly confirmed that Ccs1C27S, Ccs1C64S, and Ccs1C27S/C64S were lacking from the mitochondrial fraction. Moreover, the distribution of Ccs1 between mitochondria and the remainder of the cell was not substantially altered compared with the distribution in cells containing endogenous levels of Ccs1 (; also compare , with ). An exception was the C159S mutant of Ccs1, which appeared to become enriched in the mitochondrial fraction upon overexpression. Taken together, these results support that also in vivo C27 and C64 contribute to the accumulation of Ccs1 in mitochondria.
Cysteine mutations in domain I do not affect the enzyme activity of Ccs1
We were also interested in the effect of cysteine mutations in Ccs1 on its activity. In yeast Ccs1 is essential for the activation of Sod1, and thus Sod1 activity can be used as a measure for the enzymatic activity of Ccs1. We applied a gel-based Sod activity assay on lysates of cells and of isolated mitochondria from yeast strains expressing different Ccs1 cysteine mutants in a CCS1
-deletion background (). When analyzing whole cells we confirmed previous findings (Schmidt et al., 1999
) that the mutation of C229 and C231 in Ccs1 results in the complete loss of Sod1 activity (). Deletion of the remaining cysteines, however, did not affect Sod1 activity in whole-cell lysates (). This was in strong contrast with the situation in mitochondria, in which C27 and C64 were essential for the accumulation of Sod1 activity (). This was due to the import defect of these Ccs1 mutants into mitochondria because fusion of the variants to a b2
-targeting sequence restored mitochondrial Sod1 activity ().
FIGURE 5: Levels of active mitochondrial Sod1 are lower in strains expressing the C27S and C64S cysteine mutants of Ccs1. (A) Sod1 protein and activity levels in Δccs1 cells expressing different Ccs1 variants under the control of the endogenous promoter (more ...)
Mutations in amino acids forming the potential IMS-targeting signal result in the same phenotype as the C27S and C64S mutants of Ccs1
The small twin CX3
C and twin CX9
C substrates of Mia40 contain an internal mitochondrial targeting signal, a so-called mitochondria IMS–sorting signal (MISS) or intermembrane space–targeting signal (ITS; Milenkovic et al., 2009
; Sideris et al., 2009
). Such a signal has not been defined for Ccs1. Closer analysis of the Ccs1 sequence and structure revealed that amino acids I24, I57, and L61 might potentially form part of such a sequence, as they are hydrophobic residues localized to the α-helix presumably interacting with Mia40 (). To investigate the influence of these residues on the cellular distribution, mitochondrial import, and redox state of Ccs1, we generated and analyzed I24E and L61E mutants (). We found that these Ccs1 variants were not imported into isolated mitochondria () and that they were present only at low levels in whole cells and in minute amounts in mitochondria (). Although they were in principle capable of activating Sod1 in whole cells, mitochondria contained lower levels of active Sod1 (). Thus both Ccs1 variants behaved like Ccs1C27S
and might indeed constitute a part of the MISS/ITS of Ccs1.
FIGURE 6: MISS/ITS mutants in Ccs1 behave like the C27S and C64S mutants of Ccs1. (A) Scheme of domain I of Ccs1 and location of the amino acids forming the putative MISS/ITS. Locations of amino acids forming the potential MISS/ITS are indicated in the structure (more ...)