PROPUESTA PEDAGÓGICA

The results obtained with the single cysteine variants of Mia40 suggest that the first, second, third and the sixth cysteine residues in Mia40 play important roles for the function of Mia40 in the disulfide-relay system in the IMS. To get more insight in their function, the results obtained with the single cysteine mutants of Mia40 were extended by studies using selected double cysteine to serine mutants of Mia40.

3.4.2.1. Double cysteine mutants of Mia40, apart from for the Mia40C4/5S, show defects in cell growth and the biogenesis of small IMS proteins

Firstly, the complementation of the MIA40 deletion with the Mia40 double cysteine mutants, indicated in Fig.30, was tested. The Mia40C4/5S variant complemented fully the deletion of the wild type MIA40, which is in line with the results acquired from the Mia40C4S and the Mia40C5S mutants (Fig.30A). This indicates that the fourth and the fifth cysteine residues in Mia40 are not crucial for the Mia40 function and thus for the viability of yeast cells. In agreement with the results obtained for the Mia40C2S mutant, the Mia40C1/2S mutant failed to restore cell viability. The Mia40C5/6S mutant also did not complement the deletion of the wild type MIA40, pointing out that the sixth cysteine residue in Mia40 is essential when the fifth cysteine residue is absent. Surprisingly, the mutation of the third cysteine residue alone in Mia40 results in lethality, but an additional mutation of the sixth cysteine residue allows growth of yeast cells though with strongly reduced rates (Mia40C3/6S mutant). This finding reveals that the third cysteine residue is not absolutely essential for the function of Mia40. Similar effects on the cell growth were observed with the GAL-MIA40 Figure 29. Analysis of the Mia40 interaction with Erv1 in mitochondria harbouring the single cysteine mutants of Mia40. Different amounts of mitochondria isolated from cells expressing Mia40WT and Mia40 mutant proteins were used (25 µg for Mia40WT, 50 µg for Mia40C1S, Mia40C2S, Mia40C4S and Mia40C5S, 100

µg for Mia40C6S and 250 µg for Mia40C3S). Mitochondria were incubated for 20 min at 250_{C with 80 mM} iodoacetamide. After lysis of mitochondria, the proteins were resolved under non- reducing conditions by SDS-PAGE, transferred to nitrocellulose membrane and decorated with Erv1-specific antibodies.

strains expressing the double cysteine variants of Mia40 after depletion of endogenous Mia40 (Fig.30B). In summary, the results of the Mia40 double mutants confirm that the first three cysteine residues are important for the function of Mia40, though only the second cysteine residue appears to be strictly essential.

Figure 30. Analysis of the growth phenotypes of strains expressing the double cysteine mutants of Mia40. (A) Plasmids containing selected double cysteine mutants of Mia40 were introduced into ∆mia40 strain harboring on an URA plasmid the wild-type MIA40 gene and analyzed for complementation by growth on 5- fluoroorotic acid containing medium. Empty vector and plasmid carrying WT MIA40 gene were used as controls. (B) The GAL-MIA40 strain carrying the MIA40 gene under control of the GAL10 promoter was transformed with plasmids encoding the double cysteine mutants of Mia40.The growth characteristics of the transformants were checked in a drop dilution test. The endogenous levels of Mia40 protein were downregulated by cell growth on medium containing 2 % glucose for three days at 30o_C.

Next, to study the effects of the double cysteine mutations on the molecular function of Mia40, mitochondria were isolated from the GAL-MIA40 strains depleted of endogenous Mia40 and expressing the different double cysteine variants with a C-terminal His6-tag. The

expression levels of most of the Mia40 mutant proteins were comparable to the wild-type Mia40 levels, only the Mia40C5/6S mutant show slightly reduced levels, possibly due to higher protein instability (Fig.31A). Both the endogenous levels and the in vitro import rates of Tim13, the substrate of Mia40-dependent pathway, were considerably reduced in mitochondria of the Mia40C1/2S, the Mia40C3/6S and the Mia40C5/6S mutants (Fig.31B, C). In contrast, Mia40C4/5S variant showed no major effects in the biogenesis of the small cysteine-rich IMS proteins. The expression levels of the proteins of the outer membrane (Tom40), inner membrane (Tim23) or the matrix (AcoI) were not affected in any of the Mia40 double cysteine mutants. Similarly, the import of the IMS protein cytochrome c heme lyase (CCHL), which is imported independently of Mia40, was not impaired in these mutants. To summarize, the specific effects on the biogenesis of the small IMS proteins in the double cysteine mutants of Mia40 are in agreement with their growth phenotypes.

Results

3.4.2.2. Interactions of Mia40 with Tim13 and with Erv1 are affected in most of the double cysteine mutants of Mia40

Finally, the interactions of Mia40 double cysteine mutants with the substrate Tim13 and with Erv1 were analyzed. Consistent with the import results, the formation of the Tim13- Mia40 complex was not detected in the Mia40C1/2S and Mia40C5/6S variants (Fig.32A). In the Mia40C3/6S mutant this complex was formed, although in comparison to the wild type Mia40 a significant reduction was observed. The Mia40C4/5S variant showed no alteration in Tim13-Mia40 complex formation. The results obtained for the interaction of Mia40 with its oxidase Erv1 were comparable to the ones concerning the formation of the Tim13-Mia40 complex. The Erv1-Mia40 complex was absent in the Mia40C1/2S and the Mia40C5/6S mutants and present in the Mia40C4/5S variant (Fig.32B). In the Mia40C3/6S mutant the interaction with Erv1 could be detected but was strongly reduced. Taken together, the findings suggest that the observed defects in the cell growth and in the biogenesis of the small IMS proteins in the double cysteine mutants of Mia40 are due to the failure in the formation of the crucial interactions of Mia40 with its substrates and with Erv1.

Figure 31. Endogenous levels of mitochondrial proteins and import of precursor proteins in mitochondria containing the double cysteine mutants of Mia40. (A) The GAL-MIA40 strain was transformed with plasmids encoding Mia40 with double Cys to Ser mutations and depleted of endogenous Mia40 for 27h. Mitochondria from those strains were isolated and analyzed for the expression of the mutant Mia40 proteins by SDS- PAGE and immunoblotting. (B) Levels of the indicated proteins were analyzed by immunoblotting of mitochondria described in (A). (C) The indicated radiolabeled preproteins were imported into mitochondria described in (A) and analyzed by SDS-PAGE and autoradiography.

Figure 32. Analysis of the interaction of Mia40 with Tim13 and Erv1 in mitochondria harbouring the double cysteine mutants of Mia40. (A) Radiolabeled precursor of Tim13 was imported for 20 min at 120_{C into} mitochondria isolated from cells expressing Mia40WT and the double cysteine mutants. After lysis with 1% sodium dodecylsulfate, the His6-tagged proteins were isolated via NiNTA affinity chromatography and analyzed by non-reducing SDS-PAGE and autoradiography. (B) Different amounts of mitochondria (25 µg for Mia40WT and Mia40C4/5S, 100 µg for Mia40C1/2S, 200 µg for Mia40C3/6S and Mia40C5/6S) were incubated for 20 min at 250_{C with 80 mM iodoacetamide. Then mitochondria were solubilized and the probes were loaded on a non-} reducing SDS-PAGE gel. After transfer to a nitrocellulose membrane, the Mia40-Erv1 complex was detected with Erv1-specific antibodies.

In conclusion, the double mutation of the fourth and the fifth cysteine residues in Mia40 led to no significant defects in the yeast cell viability and function, indicating that these residues do not crucially contribute to the Mia40 activity or stability. The replacement of the third and the sixth cysteine residues with serine residues at the same time resulted in strong impairment of the cell growth and the mitochondrial function. The simultaneous mutation of the fifth and the sixth cysteine residues in Mia40 showed even more severe defects. The results of these double cysteine mutants (Mia40C3/6S, Mia40C5/6S) as well as the findings of the Mia40C3S and the Mia40C6S mutants suggest that the third and the sixth cysteine residues play very important but not essential roles in the function or stability of Mia40 in the mitochondrial IMS. The failure to restore cell viability in the ∆mia40 context and the biochemical data obtained from the Mia40C2S and the Mia40C1/2S mutants indicate that the second cysteine residue in the Mia40 motif is essential for the Mia40 function. The first cysteine residue also contributes greatly to the Mia40 activity, since the Mia40C1S mutant showed great defects in the cell growth and in the biogenesis of small IMS proteins. The observed viability of this Mia40 mutant strain might be explained by the remaining ability of the Mia40C1S mutant protein to interact with Erv1, in contrast to the Mia40C2S protein. In summary, the first three and the sixth cysteine residues in Mia40 motif play a significant role for the function of Mia40.

4.3. Characterization of the redox states of Mia40 and the Mia40 cysteine mutants