Philip K. Hammen

MITOCHONDRIAL LEADER SEQUENCES : STRUCTURAL SIMILARITIES AND SEQUENCE DIFFERENCES

While having essentially no amino acid sequence homology, the mitochondrial leader sequences of different pre-proteins carry out the same function of targeting the protein to mitochondria. Among the common attributes that have been noted for leader sequences are a net positive charge and the ability to form amphiphilic alpha-helices. The aim of the research described here was to determine the relative importance of these two attributes in the leader sequence of rat liver mitochondrial aldehyde dehydrogenase. Through site-directed mutagenesis, arginine residues were systematically replaced by glutamine. It was found that individual arginines could be replaced without loss of import competence, so the total charge of the leader sequence was not required for the pre-protein to be imported. However, when two arginines were replaced simultaneously, especially Arg3 and Arg10, the pre-protein lost the ability to be imported. This ability was restored by modifying the leader sequence to increase dramatically its helix-forming potential. This leader sequence did not contain a positive-charged side-chain until Arg11. Therefore, it has been shown that positive charge is not required in the first ten residues provided the sequence could form a relatively more stable alpha-helix. The ability to form an amphiphilic alpha-helix remains the essential factor in determining whether or not a leader sequence can carry out its import function.

Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin, Griffonia simplicifolia lectin II (GSII)

Site-directed mutagenesis previously identified the residues responsible for the biological activity of the plant defense legume lectin, Griffonia simplicifolia lectin II (GSII) [Proc. Natl. Acad. Sci. USA 95, (1998) 15123-15128]. However, these results were inconclusive as to whether these residues function as direct defense determinants through carbohydrate binding, or whether substantial changes of the protein structure had occurred in mutated proteins, with this structural disruption actually causing the loss of biochemical and biological functions. Evidence shown here supports the former explanation: circular dichroism and fluorescence spectra showed that mutations at carbohydrate-binding residues of GSII do not render it dysfunctional because of substantial secondary or tertiary structure modifications; and trypsin treatment confirmed that rGSII structural integrity is retained in these mutants. Reduced biochemical stability was observed through papain digestion and urea denaturation in mutant versions that had lost carbohydrate-binding ability, and this was correlated with lower Ca(2+) content. Accordingly, the re-addition of Ca(2+) to demetalized proteins could recover resistance to papain in the carbohydrate-binding mutant, but not in the non-binding mutant. Thus, both carbohydrate binding (presumably to targets in the insect gut) and biochemical stability to proteolytic degradation in situ indeed contribute to anti-insect activity, and these activities are Ca(2+)-dependent.

Structure of the cytosolic domain of TOM5, a mitochondrial import protein.

TOM5 is a small outer mitochondrial membrane protein in Saccharomyces cerevisiae and is part of a multi‐protein translocator complex, which mediates protein import into mitochondria. Presently, nothing is known about the conformational preferences of TOM5 or other mitochondrial import proteins. In this report, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy are used to determine the conformational preferences of the cytosolic domain of TOM5. The CD spectra show evidence of a helical structure that is invariant with pH. NOESY data revealed that TOM5 forms a stable helical core between E11 and R15 with a less structurally rigid helix extending to the C‐terminus.

Timing and structural consideration for the processing of mitochondrial matrix space proteins by the mitochondrial processing peptidase (MPP)

Most mitochondrial matrix space proteins are synthesized as a precursor protein, and the N‐terminal extension of amino acids that served as the leader sequence is removed after import by the action of a metalloprotease called mitochondrial processing peptidase (MPP). The crystal structure of MPP has been solved very recently, and it has been shown that synthetic leader peptides bind with MPP in an extended conformation. However, it is not known how MPP recognizes hundreds of leader peptides with different primary and secondary structures or when during import the leader is removed. Here we took advantage of the fact that the structure of the leader from rat liver aldehyde dehydrogenase has been determined by 2D‐NMR to possess two helical portions separated by a three amino acid (RGP) linker. When the linker was deleted, the leader formed one long continuous helix that can target a protein to the matrix space but is not removed by the action of MPP. Repeats of two and three leaders were fused to the precursor protein to determine the stage of import at which processing occurs, if MPP could function as an endo peptidase, and if it would process if the cleavage site was part of a helix. Native or linker deleted constructs were used. Import into isolated yeast mitochondria or processing with recombinantly expressed MPP was performed. It was concluded that processing did not occur as the precursor was just entering the matrix space, but most likely coincided with the folding of the protein. Further, finding that hydrolysis could not take place if the processing site was part of a stable helix is consistent with the crystal structure of MPP. Lastly, it was found that MPP could function at sites as far as 108 residues from the N terminus of the precursor protein, but its ability to process decreases exponentially as the distance increases.