Background: Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking.

The TIM10 chaperone facilitates the insertion of hydrophobic proteins at the mitochondrial inner membrane. Here we report the novel molecular mechanism of TIM10 assembly. This process crucially depends on oxidative folding in mitochondria and involves: (i) import of the subunits in a Cys-reduced and unfolded state; (ii) folding to an assembly-competent structure maintained by intramolecular disulfide bonding of their four conserved cysteines; and (iii) assembly of the oxidized zinc-devoid subunits to the functional complex. We show that intramolecular disulfide bonding occurs in vivo, whereas intermolecular disulfides observed in vitro are abortive intermediates in the assembly pathway. This novel mechanism of compartment-specific redox-regulated assembly is crucial for the formation of a functional TIM10 chaperone.Cysteine has unique biological functions by using its sulfhydryl (ϪSH) group in the active site of an enzyme, in chelating metals, or as the active site of disulfide reshuffling. For example, in the case of the molecular chaperone, Hsp33 activity is regulated by a redox switch with its inactive form reduced and zinc-coordinated and its active form turned on by oxidation and disulfide formation (1). The transcription factor OxyR is similarly activated through the formation of a disulfide bond and inactivated by enzymatic reduction with glutaredoxin (2). Disulfide bond formation in general is an essential step in the folding of many proteins, and it is catalyzed in vivo by the dsb system in the bacterial periplasm (3) and the functionally related PDI/Ero1 (4) system in the ER of eukaryotic cells. Although a mitochondrial intermembrane space sulfhydryl oxidase, Erv1p, has been identified (5), there has been no report suggesting disulfide bond formation in mitochondria. Here we demonstrate that the mitochondrial intermembrane space can allow oxidative folding events, challenging the commonly accepted notion that this compartment is in complete redox equilibrium with the reducing cytosol. We show that substrates for this oxidation event are Tim9 and Tim10, the subunits of the TIM10 chaperone that mediates hydrophobic protein insertion at the inner mitochondrial membrane (6 -9). This complex binds to the hydrophobic segments of the precursor (10) at an early import stage as the precursor emerges from the outer membrane protein import channel (translocase of the outer membrane, TOM 1 complex). Subsequently, the precursor is carried across the intermembrane space and passed onto the TIM22 membrane-embedded complex that facilitates insertion (11-13) through a twin pore involving two voltage-dependent steps (14).As all of the TIM subunits are imported themselves from the cytosol, correct assembly to their respective complex is essential for their function. Tim9 and Tim10 partner each other specifically to form the TIM10 complex, but the structural basis and the mechanism of this assembly process remain unclear. Although the "twin CX3C" motif common to all of the small Tim proteins is thought to be important for...

The tet-regulatable promoter system is commonly used for genetic studies in many eukaryotic organisms. The promoter is regulated using doxycycline. There are no obvious phenotypic effects observed when doxycycline is added to the growth medium of yeast to control expression from the promoter. It is widely accepted that doxycycline is innocuous to yeast. Global genetic studies are now commonplace and the tetOsystem is being used in transcriptome studies. Hence, we wanted to ensure that the absence of phenotypic effects, on addition of doxycycline to the growth medium, is mirrored in transcriptome data. We have demonstrated that doxycycline has no significant effect on global transcription levels and will continue to use the tetOregulatable promoter system for genetic studies.

This study aimed to correlate the onset of functional deficits in diabetic neuropathy with changes in gene expression in rat dorsal root ganglia (DRG). After 1, 4, or 8 weeks of streptozotocin-induced diabetes, sensory and motor nerve conduction velocities (NCV) were measured as an indicator of neuropathy and changes in gene expression were measured using Affymetrix oligonucleotide microarrays. No significant changes in NCV were found after 1 week of diabetes, but after 4 and 8 weeks, there was a significant reduction in both sensory and motor NCV. Global gene expression changes in diabetic rat DRG were evident from principal component analysis of microarray data after 1, 4, and 8 weeks. Expression changes in individual genes were relatively small in line with a gradual degenerative neuropathy indirectly resulting from diabetes. Sets of differentially expressed genes have been identified and quantitative reverse transcriptase-polymerase chain reaction has been used to confirm the microarray data for several genes. Gene ontology overrepresentation analysis was performed on the microarray data to identify biologic processes altered in diabetic DRG. The genes identified in this study may be responsible for causing the functional deficits and suggest pathways/processes that require further investigation as possible targets for therapeutic intervention.

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