Only One of Three Bcs1 Homologs in Aspergillus fumigatus Confers Respiratory Growth.

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.

α- and ß-1,3-Glucan Synthesis and Remodeling.

Glucans are characteristic and major constituents of fungal cell walls. Depending on the species, different glucan polysaccharides can be found. These differ in the linkage of the D-glucose monomers which can be either in α- or ß-conformation and form 1,3, 1,4 or 1,6 O-glycosidic bonds. The linkages and polymer lengths define the physical properties of the glucan macromolecules, which may form a scaffold for other cell wall structures and influence the rigidity and elasticity of the wall. ß-1,3-glucan is essential for the viability of many fungal pathogens. Therefore, the ß-1,3-glucan synthase complex represents an excellent and primary target structure for antifungal drugs. Fungal cell wall ß-glucan is also an important pathogen-associated molecular pattern (PAMP). To hide from innate immunity, many fungal pathogens depend on the synthesis of cell wall α-glucan, which functions as a stealth molecule to mask the ß-glucans itself or links other masking structures to the cell wall. Here, we review the current knowledge about the biosynthetic machineries that synthesize ß-1,3-glucan, ß-1,6-glucan, and α-1,3-glucan. We summarize the discovery of the synthases, major regulatory traits, and the impact of glucan synthesis deficiencies on the fungal organisms. Despite all efforts, many aspects of glucan synthesis remain yet unresolved, keeping research directed toward cell wall biogenesis an exciting and continuously challenging topic.

Msp1 cooperates with the proteasome for extraction of arrested mitochondrial import intermediates.

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.

Structure of the Bcs1 AAA-ATPase suggests an airlock-like translocation mechanism for folded proteins.

Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly elusive. Here, we present the structure of Saccharomyces cerevisiae Bcs1, an AAA-ATPase of the inner mitochondrial membrane. Bcs1 facilitates the translocation of the Rieske protein, Rip1, which requires folding and incorporation of a 2Fe-2S cluster before translocation and subsequent integration into the bc1 complex. Surprisingly, Bcs1 assembles into exclusively heptameric homo-oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together they form two aqueous vestibules, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane, with both separated by the seal-forming middle domain. On the basis of this unique architecture, we propose an airlock-like translocation mechanism for folded Rip1.

Azole-induced cell wall carbohydrate patches kill Aspergillus fumigatus.

Azole antifungals inhibit the fungal ergosterol biosynthesis pathway, resulting in either growth inhibition or killing of the pathogen, depending on the species. Here we report that azoles have an initial growth-inhibitory (fungistatic) activity against the pathogen Aspergillus fumigatus that can be separated from the succeeding fungicidal effects. At a later stage, the cell wall salvage system is induced. This correlates with successive cell integrity loss and death of hyphal compartments. Time-lapse fluorescence microscopy reveals excessive synthesis of cell wall carbohydrates at defined spots along the hyphae, leading to formation of membrane invaginations and eventually rupture of the plasma membrane. Inhibition of ß-1,3-glucan synthesis reduces the formation of cell wall carbohydrate patches and delays cell integrity failure and fungal death. We propose that azole antifungals exert their fungicidal activity by triggering synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus. The elucidated mechanism may be potentially exploited as a novel approach for azole susceptibility testing.

Mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus: therapeutic and evolutionary implications.

Mitochondria within eukaryotic cells continuously fuse and divide. This phenomenon is called mitochondrial dynamics and crucial for mitochondrial function and integrity. We performed a comprehensive analysis of mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus. Phenotypic characterization of respective mutants revealed the general essentiality of mitochondrial fusion for mitochondrial genome maintenance and the mold's viability. Surprisingly, it turned out that the mitochondrial rhomboid protease Pcp1 and its processing product, s-Mgm,1 which are crucial for fusion in yeast, are dispensable for fusion, mtDNA maintenance and viability in A. fumigatus. In contrast, mitochondrial fission mutants show drastically reduced growth and sporulation rates and increased heat susceptibility. However, reliable inheritance of mitochondria to newly formed conidia is ensured. Strikingly, mitochondrial fission mutants show a significant and growth condition-dependent increase in azole resistance. Parallel disruption of fusion in a fission mutant partially rescues growth and sporulation defects and further increases the azole resistance phenotype. Taken together, our results indicate an emerging dispensability of the mitochondrial rhomboid protease function in mitochondrial fusion, the suitability of mitochondrial fusion machinery as antifungal target and the involvement of mitochondrial dynamics in azole susceptibility.

Bcs1, a AAA protein of the mitochondria with a role in the biogenesis of the respiratory chain.

The family of AAA+ proteins in eukaryotes has many members in various cellular compartments with a broad spectrum of functions in protein unfolding and degradation. The mitochondrial AAA protein Bcs1 plays an unusual role in protein translocation. It is involved in the topogenesis of the Rieske protein, Rip1, and thereby in the biogenesis of the cytochrome bc(1) complex of the mitochondrial respiratory chain. Bcs1 mediates the export of the folded FeS domain of Rip1 across the mitochondrial inner membrane and the insertion of its transmembrane segment into an assembly intermediate of the cytochrome bc(1) complex. We discuss structural elements of the Bcs1 protein compared to other AAA proteins in an attempt to understand the mechanism of its function. In this context, we discuss human diseases caused by mutations in Bcs1 that lead to different properties of the protein and subsequently to different symptoms.

A pathway of protein translocation in mitochondria mediated by the AAA-ATPase Bcs1.

The AAA+ family in eukaryotes has many members in various cellular compartments with a role in protein unfolding and degradation. We show that the mitochondrial AAA-ATPase Bcs1 has an unusual function in protein translocation. Bcs1 mediates topogenesis of the Rieske protein, Rip1, a component of respiratory chains in bacteria, mitochondria, and chloroplasts. The oligomeric AAA-ATPase Bcs1 is involved in export of the folded Fe-S domain of Rip1 across the inner membrane and insertion of its transmembrane segment into an assembly intermediate of the cytochrome bc(1) complex, thus revealing an unexpected mechanistical concept of protein translocation across membranes. Furthermore, we describe structural elements of Rip1 required for recognition and export by as well as ATP-dependent lateral release from the AAA-ATPase. In bacteria and chloroplasts Rip1 uses the Tat machinery for topogenesis; however, mitochondria have lost this machinery during evolution and a member of the AAA-ATPase family has taken over its function.

Mitochondrial disulfide bond formation is driven by intersubunit electron transfer in Erv1 and proofread by glutathione.

The disulfide relay system in the intermembrane space of mitochondria is of crucial importance for mitochondrial biogenesis. Major players in this pathway are the oxidoreductase Mia40 that oxidizes substrates and the sulfhydryl oxidase Erv1 that reoxidizes Mia40. To analyze in detail the mechanism of this oxidative pathway and the interplay of its components, we reconstituted the complete process in vitro using purified cytochrome c, Erv1, Mia40, and Cox19. Here, we demonstrate that Erv1 dimerizes noncovalently and that the subunits of this homodimer cooperate in intersubunit electron exchange. Moreover, we show that Mia40 promotes complete oxidation of the substrate Cox19. The efficient formation of disulfide bonds is hampered by the formation of long-lived, partially oxidized intermediates. The generation of these side products is efficiently counteracted by reduced glutathione. Thus, our findings suggest a role for a glutathione-dependent proofreading during oxidative protein folding by the mitochondrial disulfide relay.