THE MECHANISM OF FORMALDEHYDE DEHYDROHENASE DEGRADATION IN METHYLOTROPHIC YEAST KOMAGATAELLA PHAFFII
Yanchuk L1,2 Dmytruk O2 Dmytruk K2 Sibirny A.2,3
1Ivan Franko National University of Lviv,
Department of Microbiology, Faculty of Biology
2Institute of Cell Biology, National Academy of Science of Ukraine,
Department of Molecular Genetics and Biotechnology
3University of Rzeszow, Department of Biotechnology and Microbiology, Rzeszow, Poland
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. Autophagy dysfunction is associated with cancer, neurodegeneration, microbial infection and aging, therefore, the study of various aspects of autophagy on a model object (methylotrophic yeast) and extrapolation of the obtained data to other eukaryotic organisms may be important for medicine.
Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. During shift of methanol grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase (FLD), formate dehydrogenase (FDH), fructose-1,6-bisphosphatase (FBP)) enzymes of methanol metabolism is observed. Inactivation of peroxisomal enzymes occurs due to the autophagic degradation (pexophagy) whereas mechanisms of inactivation of cytosolic enzymes remain unknown. We aimed to study the mechanisms of FLD degradation in methylotrophic yeasts Komagataella phaffii.
The changes of the specific activity of FLD in the wild type strain GS200 and strain defected in autophagy pathway SMD1163 of K. phaffii in short-term and long-term induction with methanol, and with or without the addition of the MG132 (proteasome degradation inhibitor) was investigated. To confirm FLD degradation pathway the recombinant strains with GFP-labeled Fld of K. phaffii were constructed on the background of GS200 and SMD1163. Degradation of Fld by the Western blot analysis in GS200 and SMD1163 strains with GFP-labeled Fld was studied. The fluorescent microscopy analysis of the constructed strains was made. It was shown that the effect of the proteasome inhibitor MG132 was insignificant. FLD degrades by a vacuolar pathway, regardless of the duration of methanol induction, which correlates with the activity data of this enzyme.