Coenzyme Q is a redox-active lipid that functions as an electron carrier in the mitochondrial respiratory chain. Q-biosynthesis in Saccharomyces cerevisiae requires at least nine proteins (Coq1p-Coq9p). The molecular function of Coq8p is still unknown; however, lack of Q and the concomitant accumulation of the intermediate 3-hexaprenyl-4-hydroxybenzoic acid in the absence of Coq8p suggest an essential role in Q-biosynthesis. Localization studies identify Coq8p as a soluble mitochondrial protein, with characteristics of a protein of the matrix or associated with the inner mitochondrial membrane. Coq8p forms homomeric structure(s) as revealed by two-hybrid analysis and tandem affinity purification. Two-dimensional (2D)-Blue Native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis suggests that Coq8p - together with Coq2p and Coq10p - is predominantly associated with a complex of about 500 kDa, whereas Coq3p, Coq5p and Coq9p are mainly organized in a 1.3 MDa Q-biosynthesis complex that is not associated with the complex III and IV supracomplexes of the respiratory chain. Loss of Coq8p is accompanied by destabilization of Coq3p, but not of Coq9p from the 1.3 MDa Q-biosynthesis complex. This effect cannot be reversed by Q(6) supplementation. The detection of Coq3p isoforms by 2D-isoelectric focusing is in line with the proposed function of Coq8p as a kinase, with Coq3p as a target.

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The activity of yeast pyruvate dehydrogenase complex is regulated by reversible phosphorylation. Recently we identified two enzymes that are involved in the phosphorylation (Pkp1p) and dephosphorylation (Ppp1p) of Pda1p, the ␣-subunit of the pyruvate dehydrogenase complex. Here we provide evidence that two additional mitochondrial proteins, Pkp2p (Ygl059wp) and Ppp2p (Ycr079wp), are engaged in the regulation of this complex by affecting the phosphorylation state of Pda1p. Our data indicate complementary activities of the kinases and a redundant function for the phosphatases. Both proteins are associated with the complex. We propose a model for the role of the regulatory enzymes and the phosphorylation state of Pda1p in the assembly process of the pyruvate dehydrogenase complex.

Fluorescent cellular biomarkers play a prominent role in biosciences. Most of the available biomarkers have some drawbacks due to either physical and optical or cytotoxic properties. In view of this, we investigated the potential of green fluorescent nanodiamonds as biomarkers in living cells. Nanodiamonds were functionalized by attaching antibodies that target intracellular structures such as actin filaments and mitochondria. Then, the nanodiamond conjugates were transfected into HeLa cells. Transfections were mediated by 4(th)-generation dendrimers, cationic liposomes and protamine sulfate. Using fluorescence microscopy, we confirmed successful transfections of the nanodiamonds into HeLa cells. Nanodiamond fluorescence could be easily differentiated from cellular autofluorescence. Furthermore, nanodiamonds could be targeted selectively to intracellular structures. Therefore, nanodiamonds are a promising tool for intracellular assays.

Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX). It was suggested that Sco1p aids copper delivery to the catalytic center of COX. Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p. In addition we provide evidence that Sco1p can form homomeric complexes. Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His-239, Cys-148 or Cys-152. These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper. ß