Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22.

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.

Ligand-enhanced expression and in-cell assay of human peroxisome proliferator-activated receptor alpha ligand binding domain.

A human peroxisome proliferator-activated receptor alpha ligand binding domain (PPAR alpha LBD)-maltose binding protein fusion construct was expressed in Escherichia coli. A codon optimized DNA sequence encoding human PPAR alpha LBD (aa196-468) was synthesized and ligated into the pDEST17 E. coli expression vector downstream of a MBP solubility fusion tag and an intermittent TEV protease cleavage site. Following auto-induction at 28 degrees C, PPAR alpha LBD protein was purified to electrophoretic homogeneity by a nickel affinity chromatographic step, on-column TEV protease cleavage followed by Sephacryl S200 size exclusion chromatography. The recombinant protein displayed cross-reactivity with goat anti-(human PPAR alpha) polyclonal antibody and was identified as human PPAR alpha by trypic peptide mass finger-printing. The addition of a PPAR alpha specific ligand (fenofibric acid, GW7647 or GW590735) to the growth media significantly stabilized the PPAR alpha LBD structure and enhanced the expression of soluble protein. In-cell ligand binding was examined by monitoring the enhancement of PPAR alpha LBD expression as a function of the concentration of ligand in the growth media. The efficient expression and in-cell assay of the reported PPAR alpha LBD construct make it amenable to high through-put screening assays in drug discovery programs.

Structure, topology and function of the translocase of the outer membrane of mitochondria.

Proteins destined for the mitochondria required the evolution of specific and efficient molecular machinery for protein import. The subunits of the import translocases of the inner membrane (TIM) appear homologous and conserved amongst species, however the components of the translocase of the outer membrane (TOM) show extensive differences between species. Recently, bioinformatic and structural analysis of Tom20, an important receptor subunit of the TOM complex, suggests that this protein complex arose from different ancestors for plants compared to animals and fungi, but has subsequently converged to provide similar functions and analogous structures. Here we review the current knowledge of the TOM complex, the function and structure of the various subunits that make up this molecular machine.

Oligosaccharide recognition and binding to the carbohydrate binding module of AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) contains a carbohydrate-binding module (beta1-CBM) that is conserved from yeast to mammals. Beta1-CBM has been shown to localize AMPK to glycogen in intact cells and in vitro. Here we use Nuclear Magnetic Resonance spectroscopy to investigate oligosaccharide binding to 15N labelled beta1-CBM. We find that beta1-CBM shows greatest affinity to carbohydrates of greater than five glucose units joined via alpha,1-->4 glycosidic linkages with a single, but not multiple, glucose units in an alpha,1-->6 branch. The near identical chemical shift profile for all oligosaccharides whether cyclic or linear suggest a similar binding conformation and confirms the presence of a single carbohydrate-binding site.