RESUMO
The mitochondrial translocase Bcs1 is required for the correct assembly of complex III of the mitochondrial respiratory chain. Because of its importance, Bcs1 was recently proposed as a target for antifungal agents. The function of this AAA (ATPase Associated with diverse cellular Activities) protein has been extensively characterized in Saccharomyces cerevisiae. This yeast as well as previously studied mammals each encode only one homolog. In contrast, the pathogenic mold Aspergillus fumigatus encodes three putative Bcs1 homologs, none of which have been characterized to date. To study the role of these three homologs in A. fumigatus, conditional and deletion mutants of the respective genes AFUA_3G13000 (bcs1A), AFUA_4G01260 (bcs1B), and AFUA_2G14760 (bcs1C) were generated. A deletion or downregulation of bcs1A resulted in drastically reduced growth and sporulation rates and in a significantly altered susceptibility to azole antifungals. In contrast, mutants lacking Bcs1B or Bcs1C did not show any phenotypes differing from the wild type. Salicylhydroxamic acid-an inhibitor of the alternative oxidase that allows the respiratory chain to bypass complex III in some species-caused a complete growth arrest of the bcs1A deletion mutant. In a Galleria mellonella infection model, the deletion of bcs1A resulted in significantly decreased virulence. Only Bcs1A was able to partially complement a deletion of BCS1 in S. cerevisiae. The subcellular localization of Bcs1B and Bcs1C outside of mitochondria suggests that these Bcs1 homologs exert cellular functions different from that of Bcs1. Our data demonstrate that Bcs1A is the sole Bcs1 ortholog in A. fumigatus.
RESUMO
The mitochondrial AAA ATPase Msp1 is well known for extraction of mislocalized tail-anchored ER proteins from the mitochondrial outer membrane. Here, we analyzed the extraction of precursors blocking the import pore in the outer membrane. We demonstrate strong genetic interactions of Msp1 and the proteasome with components of the TOM complex, the main translocase in the outer membrane. Msp1 and the proteasome both contribute to the removal of arrested precursor proteins that specifically accumulate in these mutants. The proteasome activity is essential for the removal as proteasome inhibitors block extraction. Furthermore, the proteasomal subunit Rpn10 copurified with Msp1. The human Msp1 homologue has been implicated in neurodegenerative diseases, and we show that the lack of the Caenorhabditis elegans Msp1 homologue triggers an import stress response in the worm, which indicates a conserved role in metazoa. In summary, our results suggest a role of Msp1 as an adaptor for the proteasome that drives the extraction of arrested and mislocalized proteins at the mitochondrial outer membrane.