| Abstrakt: |
The LEU4 gene of Saccharomyces cerevisiae is the major structural gene involved in the production of alpha-isopropylmalate synthase. It was recently proposed that LEU4 should be capable of encoding two forms of alpha-isopropylmalate synthase, based mainly on the observation that two of four major transcription start sites are located downstream from the ATG at the beginning of the LEU4 open reading frame (Beltzer, J. P., Chang, L. L., Hinkkanen, A. E., and Kohlhaw, G. B. (1986) J. Biol. Chem. 261, 5160-5167). The two forms with molecular weights of 68,000 and 65,000, respectively, would differ only in the N-terminal region, and only the larger of the two forms would be imported into the mitochondria. We have now constructed LEU4'-'lacZ translational fusion plasmids that contain either a normal LEU4' portion (expected to express both the long and the short forms of the fusion protein) or a modified LEU4' portion in which productive translation is possible only from the second in-frame AUG (expected to express only the short form of the fusion protein). beta-Galactosidase measurements and immunoblotting of crude mitochondrial and cytoplasmic fractions of yeast cells transformed with the fusion plasmids indicate that two forms of hybrid protein are produced and that only the larger form is targeted to the mitochondria. The distinguishing feature of the targeting sequence appears to be its ability to form an 18-residue long amphiphilic helix. Expression of the native short form of LEU4-encoded alpha-isopropylmalate synthase behind a strong yeast promoter has enabled us to show that the short form is functional in leucine biosynthesis, is inhibited by leucine with an apparent inhibitor constant of approximately 0.4 mM, and exists as a cytoplasmic dimer. |
