Functional characterization of the INO2 gene of Saccharomyces cerevisiae. A positive regulator of phospholipid biosynthesis.

D.M Nikoloff,S.A Henry

doi:10.1016/s0021-9258(17)37299-x

Abstract

The INO2 locus encodes a novel product showing structural similarity to the basic helix-loop-helix (b-HLH) family of regulatory proteins (Nikoloff, D.M., McGraw, P., and Henry, S.A. (1992) Nucleic Acids Res. 20, 3253). The ino2 mutants exhibit pleiotropic defects in phospholipid metabolism including inability to derepress the biosynthetic enzyme inositol-1-phosphate synthase. Localization of mutations in ino2 strains has demonstrated that the b-HLH domain is required for biological activity and is sensitive to perturbation, thereby establishing a correlation between the structure and function of Ino2p. Defects in the b-HLH domain of Ino2p resulted in reduced DNA binding activity. In addition, the absence of a specific DNA-protein complex correlated with a reduction or loss of INO1 transcription. Studies using Ino2p-specific antibody revealed that Ino2p participates in the formation of specific DNA-protein complexes. Ino2p-dependent binding activity overlapped with a region of the INO1 promoter that contains two potential HLH consensus binding sites. Furthermore, Ino2p showed single base pair discrimination in a putative binding site, establishing a relationship between Ino2p and its target binding site.

Highlights

Localizationof mutationsin in02 strains has demonstratedthathe b-HLHdomain is requiredfor biological activity and is sensitive to perturbation, thereby establishing a correlation between the structure and functioonf Ino2p.Defects in the b-HLHdomain of Ino2p resulted in reduced DNA binding activity
Extracts preparefrdom in02and in& mutants both fail to form a DNA-protein complex that is present in extracts prepared from a wild type strain when incubated with a 105-base pair DNA fragment from the INOlproaddition,the absence of aspecific DNA-protein complex moter [10].Cloning and sequencingof the IN02 generevealed correlated with a reduction or loss of INOl transcrip- a structural similaritybetween IN02 and thebasic helix-looption
The proteincomplexes.Ino2p-dependentbinding activity b-HLH family of regulatory proteins shares a common strucoverlapped with a regiofnthe INOl promoter that con- ture consisting of a 68-amino acid HLH domainthat is respontains two potential HLH consensus binding sites

Summary

A POSITIVE REGULATOR OF PHOSPHOLIPID BIOSYNTHESIS*

(Receivedfor publication, October 8, 1993, and in revised form, December 6, 1993). From the Department of Biological Sciences, Carnegie Mellon University,Pittsburgh, Pennsylvania 15213. The in and in mutants show phospholipidmetabolismincludinginability to dere- reduced levels of transcription for the co-regulated phosphopress the biosynthetic enzyme inositol-1-phosphastyen- lipid structural genes, such asZNOl, encoding inositol-l-phosthase. A branched pathway originating correlation between the structure andfunction of Ino2p These from phosphatidic acid gives rise to two of the major phospho- findings are consistent with models that propose a common lipids, phosphatidylinositol and phosphatidylcholine. The ac- mechanism of action for all b-HLH proteins and makea comtivities of many of t h e biosynthetic enzymes of this pathway in pelling argument for t h e functional significance of t h e b-HLH yeast are subject to transcriptional control in response to the motif in Ino2p in the transcriptional regulatiofnphospholipid availability of the phospholipid precursors inositol and choline synthesis in yeast. (1).Studies of strains harboring mutationsin regulatory genes have identified regulatory factors that mediate both positive

MATERIALS AND METHODS

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DISCUSSION