Registration Number
0220U102557,
0116U002209
, R & D reports
Title
Genetic and biochemical aspects of the regulation of some catabolic and anabolic processes in microorganisms: alcoholic fermentation, catabolism of methanol, biosynthesis of flavins, glycerol, hydrogen and glutathione
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Head
Sybirnyi Andrii A.,
Registration Date
25-03-2020
Organization
Institute of Cell Biology
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Today, the mechanisms of degradation of cytosolic proteins of their own, as well as of recombinant foreign proteins of biotechnological significance with cytosolic localization in methylotrophic yeast, remain unclear. Since autophagy dysfunction is associated with cancer, neurodegeneration, microbial infection and aging, the study of various aspects of autophagy on a model object (methylotrophic yeast) and extrapolation of the data to other eukaryotic organisms is important for medicine.
Fructose-1,6-bisphosphatase (FBP) was selected to study the possible degradation pathways of cytosolic enzymes. In an environment with glucose, this enzyme is rapidly inactivated and degraded and is therefore a convenient model protein for research. Recombinant K. pastoris yeast methylotrophic yeast strains have been engineered to express Fbp1 K. pastoris genes fused to the green fluorescent protein GFP and simultaneously express the gene encoding red fluorescence protein RFP to PTS1 signaling. These strains were grown on glycerol medium and then transferred to glucose medium to stimulate FBP degradation in K. pastoris. Vacuum dye FM 4-64 was also used to label the vacuoles. Fluorescence microscopy data clearly demonstrated that Fbp1 degrades in vacuoles by autophagy. At the same time, Fbp1 degradation was damaged in a strain with blocked vacuolar proteinases, indicating a significant role of vacuoles in Fbp1 degradation. Fluorescence microscopy confirmed our findings that Fbp1 in K. pastoris degrades in a vacuolar manner.
A new method for the selection of K. phaffii mutants with damage to genes associated with the process of autophagic cytosolic protein degradation due to the use of K. lactis β-galactosidase fused with GFP is proposed.
This method was used to obtain insertion mutants that slow down the process of β-galactosidase degradation after transfer to glucose.
Product Description
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Vasylyshyn Roksolana V.
Dmytruk Olena V.
Kurylenko Olena O.
Semkiv Marta V.
Fedorenko Victor O.
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2020-04-01
