Aim. The study of the mechanisms of fructose-1,6-bisphosphatase degradation in methylotrophic yeasts Pichia pastoris. Methods. Methods of determination the specific activity of fructose-1,6-bisphosphatase in the wild type and mutant strains of methylotrophic yeast P. pastoris after shifting cells from the medium with methanol into the medium with glucose were used. The study of fructose-1,6-bisphosphatase protein degradetion was performed by Western blot analysis. Results. The changes of the specific activity of fructose-1,6-bisphosphatase in the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of P. pastoris in short-term and long-term induction with methanol, and with or without the addition of the MG132 (proteasome degradation inhibitor) was investigated. Degradation of fructose‑1,6‑bisphosphatase by the Western blot analysis in GS200, SMD1163 and Δgss1 strains was studied. Conclusions. It was shown that the duration of cell incubation on methanol has no particular effect on the inactivation of the enzyme. The effect of the proteasome inhibitor MG132 was insignificant. Catabolic inactivation of cytosolic and peroxisomal enzymes is damaged in the Δgss1 mutant as glucose signaling is impaired. Fructose-1,6-bisphosphatase degrades by a vacuolar pathway, regardless of the duration of methanol induction, which correlates with the activity data of this enzyme.
Keywords: fructose-1,6-bisphosphatase, yeasts, Pichia pastoris, methanol, autophagy.
Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris
