Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans

Abstract

The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creAd-30 strain showed that the creA gene is not involved in this process.

The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes

“…Characterisation of this phenomenon has shown that the first step involves reversible inactivation of the enzyme by phosphorylation, while in the second stage final degradation of the protein occurs (Lopez-Boado et al 1988;Ordiz et al 1995). However, preliminary studies of A. nidulans (De Lucas et al 1994b) suggested that proteolysis of the enzyme is the only mechanism involved in the glucose-induced inactivation of isocitrate lyase, although this hypothesis has to be confirmed by additional experiments.…”

“…Enzyme assays and Western blotting Crude extracts were prepared by the method described by De Lucas et al (1994b). Protein concentration was determined by the method of Lowry et al (1951) using the BioRad DC protein assay (Bio-Rad Laboratories, USA) and bovine serum albumin as the standard.…”

“…Cells were lysed in extraction buffer (50 mM sodium phosphate, pH 7.0, 5 mM EDTA, 10 mM β-mercaptoethanol, 25 µg phenylmethyl sulphonyl fluoride ml -1 ) using the method described by De Lucas et al (1994b). Crude extracts were centrifuged at 10000×g at 4°C for 15 min in a microcentrifuge and the clear supernatants obtained (400 µl) were mixed with 800 µl of immunoprecipitation buffer (10 mM sodium phosphate buffer, pH 7.0, 190 mM NaCl, 5 mM EDTA, 1.25% v/v Triton X-100).…”

“…The glucose-induced inactivation of isocitrate lyase has been reported in several fungi including the phycomycete Phycomyces blakesleeanus (Rua et al 1989) and the ascomycetes Saccharomyces cerevisiae (LopezBoado et al 1987) and A. nidulans (De Lucas et al 1994b). In S. cerevisiae the inactivation of isocitrate lyase has been extensively analysed and occurs by a two-stage…”

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy

“…Crude extracts were prepared as described before (De Lucas et al 1994). Protein concentration was determined according to Bradford (1976) using the BioRad Protein Assay Kit (BioRad, Munich, Germany) and bovine serum albumin (BSA) as standard.…”