A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria.

Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites.

Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood. We found that the yeast membrane protein Yet3, the homolog of human BAP31, is localized to multiple ER contact sites. We show that Yet3 interacts with all the enzymes of the post-squalene ergosterol biosynthesis pathway and recruits them to create sterol-rich domains. Increasing sterol levels at ER contacts causes its depletion from the plasma membrane leading to a compensatory reaction and altered cell metabolism. Our data shows that Yet3 provides on-demand sterols at contacts thus shaping organellar structure and function. A molecular understanding of this protein's functions gives new insights into the role of BAP31 in development and pathology.

The multifaceted mitochondrial OXA insertase.

Most mitochondrial proteins are synthesized in the cytosol and transported into mitochondria by protein translocases. Yet, mitochondria contain their own genome and gene expression system, which generates proteins that are inserted in the inner membrane by the oxidase assembly (OXA) insertase. OXA contributes to targeting proteins from both genetic origins. Recent data provides insights into how OXA cooperates with the mitochondrial ribosome during synthesis of mitochondrial-encoded proteins. A picture of OXA emerges in which it coordinates insertion of OXPHOS core subunits and their assembly into protein complexes but also participates in the biogenesis of select imported proteins. These functions position the OXA as a multifunctional protein insertase that facilitates protein transport, assembly, and stability at the inner membrane.

Rcf proteins and their differential specificity for respiratory chain complexes: A unique role for Rcf2 on oxygen sensitive supercomplexes?

The respiratory chain, embedded in the inner mitochondrial membrane, is organized as a network of individual complexes, as well as large supercomplex structures. In the yeast S. cerevisiae, these supercomplexes consist of a dimeric cytochrome bc1-complex adjoined by one or two copies of cytochrome c oxidase. The formation of these complexes is a dynamic process and is regulated by various factors in order to adapt to environmental and metabolic changes. These adaptions occur at the level of enzyme regulation, complex assembly, as well as altered nuclear and mitochondrial transcription and translation. Members of the Rcf protein family (Rcf1, Rcf2 and Rcf3) are required for respiratory complex biogenesis and act mainly by regulating the assembly and enzyme activity of complex IV within supercomplexes. Rcf1 functions in the assembly process via the COX3 module, whereas Rcf2 and Rcf3 are responsible for enzymatic regulation. In this study, we have extended this knowledge to show that Rcf2 and Rcf3 can also associate with newly synthesized mitochondrial encoded proteins, such as Cox3, and therefore contribute to complex IV assembly. Since the Rcf proteins have overlapping regions of sequence similarities, we engineered novel fusion proteins of Rcf1 and Rcf3, with parts of Rcf2, to probe which of the Rcf protein domains can be attributed to their functions. The fusion proteins could compensate for the individual phenotypes of the complexIV assembly defect and the lack of complex IV regulation. Finally, the role of Rcf proteins for defined species of respiratory chain complexes in a hypoxic model was investigated, uncovering a unique association of Rcf2 with the hypoxic III2IV supercomplex. We therefore suggest an involvement of Rcf2 for adaption of the respiratory chain to altering oxygen levels.