An Improved Method to Isolate Mitochondrial Contact Sites.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

The UbiB family member Cqd1 forms a novel membrane contact site in mitochondria.

Mitochondria are essential organelles of eukaryotic cells and are characterized by their unique and complex membrane system. They are confined from the cytosol by an envelope consisting of two membranes. Signals, metabolites, proteins and lipids have to be transferred across these membranes via proteinaceous contact sites to keep mitochondria functional. In the present study, we identified a novel mitochondrial contact site in Saccharomyces cerevisiae that is formed by the inner membrane protein Cqd1 and the outer membrane proteins Por1 and Om14. Similar to what is found for the mitochondrial porin Por1, Cqd1 is highly conserved, suggesting that this complex is conserved in form and function from yeast to human. Cqd1 is a member of the UbiB protein kinase-like family (also called aarF domain-containing kinases). It was recently shown that Cqd1, in cooperation with Cqd2, controls the cellular distribution of coenzyme Q by a yet unknown mechanism. Our data suggest that Cqd1 is additionally involved in phospholipid homeostasis. Moreover, overexpression of CQD1 and CQD2 causes tethering of mitochondria to the endoplasmic reticulum, which might explain the ability of Cqd2 to rescue ERMES deletion phenotypes.

Quantification of ion-pairing effects on the nucleophilic reactivities of benzoyl- and phenyl-substituted carbanions in dimethylsulfoxide.

Second-order rate constants for the reactions of acceptor-substituted phenacyl (PhCOCH(-) Acc) and benzyl anions (PhCH(-) Acc) with diarylcarbenium ions and quinone methides (reference electrophiles) have been determined in dimethylsulfoxide (DMSO) solution at 20 °C. By studying the kinetics in the presence of variable concentrations of potassium, sodium and lithium salts (up to 10(-2)  mol L(-1) ), the influence of ion-pairing on the reaction rates was examined. As the concentration of K(+) did not have any influence on the rate constants at carbanion concentrations in the range of 10(-4) -10(-3)  mol L(-1) , the acquired rate constants could be assigned to the reactivities of the free carbanions. The counter ion effects increase, however, in the series K(+)