Isocitrate lyase of Ashbya gossypii – transcriptional regulation and peroxisomal localization

• Published in: FEBS letters 1999-02, Vol.444 (1), p.15-21
• Main Authors: Maeting, Ines, Schmidt, Georg, Sahm, Hermann, Revuelta, José Luis, Stierhof, York-Dieter, Stahmann, K.-Peter
• Format: Article
• Language: English
• Subjects: Acetates - metabolism; Acetates - pharmacology; Amino Acid Sequence; Ashbya gossypii; Centrifugation, Density Gradient; Cloning, Molecular; Ethanol - metabolism; Ethanol - pharmacology; Gene Expression Regulation, Enzymologic - drug effects; Genetic Complementation Test; Glucose - metabolism; Glucose - pharmacology; Glycerol - metabolism; Glycerol - pharmacology; Immunoelectron microscopy; Isocitrate lyase; Isocitrate Lyase - chemistry; Isocitrate Lyase - genetics; Isocitrate Lyase - metabolism; Microbodies - enzymology; Microbodies - ultrastructure; Microscopy, Immunoelectron; Molecular Sequence Data; Molecular Weight; Mutagenesis, Insertional - genetics; Peroxisome; Promoter Regions, Genetic - genetics; RNA, Messenger - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomycetales - enzymology; Saccharomycetales - metabolism; Saccharomycetales - ultrastructure; Sequence Alignment; Soybean Oil - metabolism; Soybean Oil - pharmacology
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(99)00017-4

The isocitrate lyase-encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62 584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C-terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C 2 compounds ethanol and acetate, and fully induced by soybean oil.