SDH assembly in S. cerevisiae: focus on the functional role of SDH6

Yeast is a well recognised model for the study of human mitochondrial pathologies thanks its exceptionally ability to survive without a functional mitochondrial respiratory metabolism, provided that a fermentable carbon source is made available. Succinate dehydrogenase (SDH, complex II) is a conserved mitochondrial enzyme of the inner membrane that catalyzes the oxidation of succinate to fumarate during TCA cycle, using at the same time the reducing equivalents in the electron transport chain (ETC). Despite the extensive knowledge of the structural and catalytic properties of the complex, only two assembly factors specific for the SDH complex has been recently found: SDHAF1 and SDHAF2. The SDHAF1 gene was identified in humans as linked to infantile leukoencephalopathy. A yeast strain deleted in SDH6, the SDHAF1 ortholog, was OXPHOS incompetent, due to a severe and specific reduction of SDH activity. However, the Km value for succinate was similar in wild-type and in the null mutant, suggesting that defective SDH activity was caused by reduced number of enzyme units rather than by qualitative alterations of complex II. We have given more insights into the knowledge of SDH6 through genetic and biochemical studies performed in S. cerevisiae. Affinity purification analyses have shown that Sdh6p is not a physical interactor of subunit 1 and 2 of SDH complex. Furthermore Sdh6p is not part of complex II or other molecular complexes, as shown in BN-PAGE analyses. Moreover, Sdh6p is a rate limiting factor of SDH assembly: indeed despite the overexpression of all four SDH subunits in the null mutant the SDH activity is not restored unlike what occurred in the wild type strain suggesting the pivotal role of this protein in the complex II assembly. The results obtained in this work suggested that SDH6 plays a different role from that played by SDH5, the yeast ortholog of SDHAF2. Indeed the overexpression of SDH6 did not rescue the OXPHOS defect of Δsdh5 and viceversa. Therefore the two SDH specific assembly factors are not interchangeable. The presence of a LYR motif in the Sdh6 protein prompted us to investigate a putative role in the insertion of Fe-S clusters in complex II. Isd11p, a protein involved in the iron-sulfur biogenesis, shares a significant sequence similarity with Sdh6p and contains a LYR motif. We tried to rescue the OXPHOS deficient phenotype of sdh6 mutant by overexpressing ISD11 but the results obtained indicated that the two proteins do not share similar functions. To further investigate the functional role of SDH6 a search for multicopy suppressors was performed. This analysis identified YAP1 and YAP2, two transcription factors involved in several cellular processes. Although YAP1 and YAP2 are able to rescue the OXPHOS defect of sdh6 mutant, their action is not exerted by increasing the SDH activity. Their suppression mechanism seems to be independent of complex II. Thus Sdh6p might have an additional role besides the SDH assembly: if this is the case the OXPHOS negative phenotype of sdh6 null mutant would be due to both a reduced SDH activity and to a lack of some other putative function. The observation that several sdh mutants with a 30-40% of residual SDH activity are able to grow on oxidative carbon sources much better than Δsdh6 strengthens this hypothesis. In order to understand the mechanism of suppression exerted by YAP1 and YAP2 we focused on two main functions linked to these genes: the oxidative stress and the iron metabolism. All together these analyses favor the hypothesis that YAP1/YAP2 suppression mechanism could be mainly ascribed to their role in iron metabolism/homeostasis. Moreover when the effects of iron supplementation on the oxidative growth of the Δsdh6 strain were tested a rescue of the OXPHOS phenotype was observed without any increase of SDH activity. The results obtained support the view that the second role of Sdh6p could be related to iron metabolism/homeostasis.