Yeast LEU4: One gene, two proteins, and multiple controls

James P Beltzer, Purdue University

Abstract

The nucleotide sequence of the yeast LEU4 gene and flanking regions has been determined. The open reading frame of 1857 nucleotides specifies a protein of 619 residues. The derived amino acid sequence and calculated molecular weight (68,416) agree well with earlier findings using highly purified $\alpha$-isopropylmalate synthase. Two of the four major transcription start sites mapped downstream from the ATG of the open reading frame. A second in-frame ATG is located at position +91 of the open reading frame. Differential transcription-translation would enable the LEU4 gene to encode two proteins with molecular weights of 68,000 and 65,000 differing only at their N-terminal, and only the larger protein would be imported into the mitochondria. LEU4$\sp\prime$-$\sp\prime lacZ$ translational fusion plasmids were constructed. One construct mimics the native LEU4 situation while a second contains a modified LEU4 such that productive translation is possible only from the +91 ATG. $\beta$-Galactosidase measurements and immunoblotting of crude mitochrondrial and cytoplasmic fractions of yeast harboring these plasmids demonstrated that two forms of hybrid protein are produced and that only the larger form is targeted to the mitochondria. Targeting information is apparently present in the N-terminal region of the long form which, unlike the short form, would be predicted to form an 18 residue long amphiphilic helix. Expression of the LEU4-encoded short form of $\alpha$-isopropylmalate synthase behind a strong yeast promoter demonstrated that it is functional and is present as a cytoplasmic dimer. The 5$\sp\prime$ noncoding region of LEU4 revealed sequence identities with the 5$\sp\prime$-TGACTC-3$\sp\prime$ element shown to be required for the general control of amino acid biosynthetic genes, and the G + C rich palindromic region implicated in the "leucine specific" regulation of LEU2. To examine the regulation of LEU4, a LEU4$\sp\prime$-$\sp\prime lacZ$ fusion was constructed in a plasmid maintained at essentially one copy per cell. This plasmid was expressed in yeast strains in which either the general control system or the "leucine specific" regulatory system (or both) had been disabled by mutation. In this report it is demonstrated that LEU4 expression can be regulated by both general control and "leucine specific" regulatory systems. When cells were grown in the presence of leucine, isoleucine, and valine, the $\beta$-galactosidase activity was repressed 2-5 fold in all strains examined, suggesting that LEU4 expression is also regulated by "multivalent repression".

Degree

Ph.D.

Advisors

Kohlhaw, Purdue University.

Subject Area

Biochemistry

COinS