Using the technique of blue native gel electrophoresis, the oligomeric state of the yeast mitochondrial F 1 F 0 -ATP synthase was analysed. Solubilization of mitochondrial membranes with low detergent to protein ratios led to the identification of the dimeric state of the ATP synthase. Analysis of the subunit composition of the dimer, in comparison with the monomer, revealed the presence of three additional small proteins. These dimer-specific subunits of the ATP synthase were identified as the recently described subunit e/Tim11 (Su e/Tim11), the putative subunit g homolog (Su g) and a new component termed subunit k (Su k). Although, as shown here, these three proteins are not required for the formation of enzymatically active ATP synthase, Su e/Tim11 and Su g are essential for the formation of the dimeric state. Su e/Tim11 appears to play a central role in this dimerization process. The dimer-specific subunits are associated with the membrane bound F 0 -sector. The F 0 -sector may thereby be involved in the dimerization of two monomeric F 1 F 0 -ATP synthase complexes. We speculate that the F 1 F 0 -ATP synthase of yeast, like the other complexes of oxidative phosphorylation, form supracomplexes to optimize transduction of energy and to enhance the stability of the complex in the membrane.

We propose that transient activation of c-Myc drives keratinocytes from the stem to the transit-amplifying compartment and thereby stimulates proliferation and differentiation along the epidermal and sebaceous lineages. The ability, demonstrated here for the first time, to manipulate exit from the stem cell compartment in vivo will facilitate further investigations of the relationship between stem cells and cancer.

Activation of Myc (c-Myc) causes epidermal cells to exit the stem cell compartment and differentiate into sebocytes and interfollicular epidermis at the expense of the hair lineages. To investigate how Myc exerts these effects we analysed the transcription of more than 10,000 genes following Myc activation in the basal layer of mouse epidermis for 1 or 4 days. The major classes of induced genes were involved in synthesis and processing of RNA and proteins, in cell proliferation and in differentiation. More than 40% of the downregulated genes encoded cell adhesion and cytoskeleton proteins. Repression of these genes resulted in profound changes in the adhesive and motile behaviour of keratinocytes. Myc activation inhibited cell motility and wound healing, correlating with decreased expression of a large number of extracellular matrix proteins. Cell adhesion and spreading were also impaired,and this correlated with decreased expression of the α6β4 integrin,decreased formation of hemidesmosomes and decreased assembly of the actomyosin cytoskeleton. We propose that Myc stimulates exit from the stem cell compartment by reducing adhesive interactions with the local microenvironment or niche, and that the failure of hair differentiation reflects an inability of keratinocytes to migrate along the outer root sheath to receive hair inductive stimuli.

The inner membrane of mitochondria is one of the protein's richest cellular membranes. The biogenesis of the respiratory chain and ATP-synthase complexes present in this membrane is an intricate process requiring the coordinated function of various membrane-bound proteins including protein translocases and assembly factors. It is therefore not surprising that a distinct quality control system is present in this membrane that selectively removes nonassembled polypeptides and prevents their possibly deleterious accumulation in the membrane. The key components of this system are two AAA proteases, membrane-embedded ATP-dependent proteolytic complexes, which expose their catalytic sites at opposite membrane surfaces. Other components include the prohibitin complex with apparently chaperone-like properties and a regulatory function during proteolysis and a recently identified ATP-binding cassette (ABC) transporter that exports peptides derived from the degradation of membrane proteins from the matrix to the intermembrane space. All of these components are highly conserved during evolution and appear to be ubiquitously present in mitochondria of eukaryotic cells, indicating important cellular functions. This review will summarize our current understanding of this proteolytic system and, in particular, focus on the mechanisms guiding the degradation of membrane proteins by AAA proteases.

We report here the identification of the novel subunit of the mitochondrial F1F0‐ATPase from Saccharomyces cerevisiae, ATPase subunit e. Yeast ATPase subunit e displays significant similarities in both amino acid sequence, properties (hydropathy and predicted coiled‐coil structure) and orientation in the inner membrane, with previously identified mammalian ATPase subunit e proteins. Estimation of its native molecular mass and ability to be co‐immunoprecipitated with α subunit of the F1‐ATPase, demonstrate that subunit e is a subunit of the F1F0‐ATPase. Stable expression of subunit e requires the presence of the mitochondrially encoded subunits of the F0‐ATPase. Subunit e had been previously identified as Tim11 and was proposed to be involved in the process of sorting of proteins to the mitochondrial inner membrane.

No abstract

In this study, the angiogenetic effect of sintered 45S5 Bioglass Ò was quantitatively assessed for the first time in the arteriovenous loop (AVL) model. An AVL was created by interposition of a venous graft from the contralateral side between the femoral artery and vein in the medial thigh of eight rats. The loop was placed in a Teflon isolation chamber and was embedded in a sintered 45S5 Bioglass Ò granula matrix filled with fibrin gel. Specimens were investigated 3 weeks postoperatively by means of microcomputed tomography, histological, and morphometrical techniques. All animals tolerated the operations well. At 3 weeks, both microcomputed tomography and histology demonstrated a dense network of newly formed vessels originating from the AVL. All constructs were filled with cell-rich, highly vascularized connective tissue around the vascular axis. Analysis of vessel diameter revealed constant small vessel diameters, indicating immature new vessel sprouts. This study shows for the first time axial vascularization of a sintered 45S5 Bioglass Ò granula matrix. After 3 weeks, the newly generated vascular network already interfused most parts of the scaffolds and showed signs of immaturity. The intrinsic type of vascularization allows transplantation of the entire construct using the AVL pedicle.

Proteins of the mitochondrial inner membrane display a wide variety of orientations, many spanning the membrane more than once. Some of these proteins are synthesized with NH 2 -terminal cleavable targeting sequences (presequences) whereas others are targeted to mitochondria via internal signals. Here we report that two distinct mitochondrial targeting signals can be present in precursors of inner membrane proteins, an NH 2 -terminal one and a second, internal one. Using cytochrome c 1 as a model protein, we demonstrate that these two mitochondrial targeting signals operate independently of each other. The internal targeting signal, consisting of a transmembrane segment and a stretch of positively charged amino acid residues directly following it, initially directs the translocation of the preprotein into the intermembrane space. It then inserts into the inner membrane from the intermembrane space side in a ⌬-dependent manner and thereby determines the orientation the protein attains in the inner membrane. Analysis of a number of other presequence-containing protein of the inner membrane suggest that they too contain such internal targeting signals.