Fructose‐1,6‐bisphosphatase degradation in the methylotrophic yeast Komagataella phaffii occurs in autophagy pathway

Abstract

Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. After shift of methanol‐grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase, formate dehydrogenase, and fructose‐1,6‐bisphosphatase [FBP]) along with peroxisomal enzymes of methanol metabolism is observed. Mechanisms of inactivation of cytosolic enzymes remain unknown. To study the mechanism of FBP inactivation, the changes in its specific activity of the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of Komagataella phaffii with or without the addition of the MG132 (proteasome degradation inhibitor) were investigated after shift of methanol‐grown cells in glucose medium. Western blot analysis showed that inactivation of FBP in GS200 occurred due to protein degradation whereas inactivation in the strains SMD1163 and gss1Δ was negligible in such conditions. The effect of the proteasome inhibitor MG132 on FBP inactivation was insignificant. To confirm FBP degradation pathway, the recombinant strains with GFP‐labeled Fbp1 of K. phaffii and red fluorescent protein‐labeled peroxisomes were constructed on the background of GS200 and SMD1163. The fluorescent microscopy analysis of the constructed strains was performed using the vacuolar membrane dye FM4‐64. Microscopic data confirmed that Fbp1 degrades by autophagy pathway in K. phaffii. K. phaffii transformants, which express heterologous β‐galactosidase under FLD promoter, have been constructed.

The Komagatella phaffii ACG1 gene, encoding β‐1,6‐N‐acetylglucosaminyltransferase, is involved in the autophagy of cytosolic and peroxisomal proteins

“…Previously, we developed a system for the isolation of the mutants defective in cytosolic enzyme degradation in K. phaffii (Dmytruk et al, 2021). In brief, K. phaffii strains GS200 and SMD1163 were transformed with vector pFLD-LAC (Figure 1a) to produce recombinant strains GS200/LAC and SMD1163/LAC with the expression of the K. lactis gene LAC4 coding for β-galactosidase, fused with a GFP under control of the methanol-induced promoter of the K. phaffii gene FLD1 (encoding formaldehyde dehydrogenase).…”

“…Cell-free extracts were obtained as described earlier (Dmytruk et al, 2021) with modifications: instead of 50 mM Tris-HCl buffer, pH 7.5, 50 mM potassium phosphate buffer, pH 7.5 was used.…”

“…For baker's yeast Saccharomyces cerevisiae , it was shown that the cytosolic protein fructose‐1,6‐bisphosphatase can be degraded by either the proteasomes or by autophagy, depending on the time of glucose starvation that preceded the glucose addition that was an inducer of inactivation and degradation of fructose‐1,6‐bisphosphatase (Hung et al, 2004). For K. phaffii , we have shown that degradation of fructose‐1,6‐bisphosphatase occurs by autophagy regardless of the preceding period of glucose starvation (Dmytruk et al, 2021). We aimed to identify genes specifically involved in the process of autophagic degradation of cytosolic proteins in K. phaffii .…”

“…For K. phaffii, we have shown that degradation of fructose-1,6bisphosphatase occurs by autophagy regardless of the preceding period of glucose starvation (Dmytruk et al, 2021). We aimed to identify genes specifically involved in the process of autophagic degradation of cytosolic proteins in K. phaffii.…”

“…It will allow in the future to construct more efficient producers of heterologous proteins with a lower rate of protein degradation. For this purpose, in our previous study, we have constructed the K. phaffii strain which expresses the LAC4 gene of Kluyveromyces lactis , coding for β‐galactosidase, and developed approaches for selection of the mutants defective in degradation of this protein (Dmytruk et al, 2021). In this paper, we present the properties of one such mutant.…”