Macromolecular organization of ATP synthase and complex I in whole mitochondria.

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

The Ustilago maydis Cys2His2-type zinc finger transcription factor Mzr1 regulates fungal gene expression during the biotrophic growth stage.

The smut fungus Ustilago maydis establishes a biotrophic relationship with its host plant maize to progress through sexual development. Here, we report the identification and characterization of the Cys(2)His(2)-type zinc finger protein Mzr1 that functions as a transcriptional activator during host colonization. Expression of the U. maydis mig2 cluster genes is tightly linked to this phase. Upon conditional overexpression, Mzr1 confers induction of a subset of mig2 genes during vegetative growth and this requires the same promoter elements that confer inducible expression in planta. Furthermore, expression of the mig2-4 and mig2-5 genes during biotrophic growth is strongly reduced in cells deleted in mzr1. DNA-array analysis led to the identification of additional Mzr1-induced genes. Some of these genes show a mig2-like plant-specific expression pattern and Mzr1 is responsible for their high-level expression during pathogenesis. Mzr1 function requires the b-dependently regulated Cys(2)His(2)-type cell cycle regulator Biz1, indicating that two stage-specific regulators mediate gene expression during host colonization. In spite of a role as transcriptional activator during biotrophic growth, mzr1 is not essential for pathogenesis; however, conditional overexpression interfered with proliferation during vegetative growth and mating ability, caused a cell separation defect, and triggered filamentous growth. We discuss the implications of these findings.

Intramembrane proteolysis of Mgm1 by the mitochondrial rhomboid protease is highly promiscuous regarding the sequence of the cleaved hydrophobic segment.

Rhomboids are a family of intramembrane serine proteases that are conserved in bacteria, archaea, and eukaryotes. They are required for numerous fundamental cellular functions such as quorum sensing, cell signaling, and mitochondrial dynamics. Mitochondrial rhomboids form an evolutionarily distinct class of rhomboids. It is largely unclear how their activity is controlled and which substrate determinants are responsible for recognition and cleavage. We investigated these requirements for the mitochondrial rhomboid protease Pcp1 and its substrate Mgm1. In contrast to several other rhomboid proteases, Pcp1 does not require helix-breaking amino acids in the cleaved hydrophobic region of Mgm1, termed 'rhomboid cleavage region' (RCR). Even transmembrane segments of inner membrane proteins that are normally not processed by Pcp1 become cleavable when put in place of the authentic RCR of Mgm1. We further show that mutational alterations of a highly negatively charged region located C-terminally to the RCR led to a strong processing defect. Moreover, we show that the determinants required for Mgm1 processing by mitochondrial rhomboid protease are conserved during evolution, as PARL (the human ortholog of Pcp1) showed similar substrate requirements. These results suggest a surprising promiscuity of the mitochondrial rhomboid protease regarding the sequence requirements of the cleaved hydrophobic segment. We propose a working hypothesis on how the mitochondrial rhomboid protease can, despite this promiscuity, achieve a high specificity in recognizing Mgm1. This hypothesis relates to the exceptional biogenesis pathway of Mgm1.