Deletion of Gcw13 represses autophagy in Pichia pastoris cells grown in methanol medium with sufficient amino acids
The purpose of this article is to study the underlying cause of the induction of autophagy in Pichia pastoris cells grown in amino acid-rich methanol medium during methanol adaptation.
Autophagy was induced in P. pastoris GS115 when cells were grown in amino acid-rich methanol medium. Transcriptome analysis revealed that genes involved in amino acid biosynthesis were upregulated. The deletion of Gcw13, a GPI-anchored protein that plays a role in the endocytosis of the general amino acid permease Gap1, resulted in the inhibition of autophagy, the activation of TORC1 and an increase in the uptake of glutamine and asparagine in methanol-grown cells.
Our results demonstrated that the autophagy induced in P. pastoris cells grown in amino acid-rich methanol medium was nitrogen source independent and may be due to a Gcw13-dependent decrease in amino acid uptake during methanol adaptation.
KeywordsPichia pstoris Autophagy Amino acid starvation TORC1 Gcw13
This work was supported by the National Natural Science Foundation of China (No. 31170031). The funding source had no role in this study.
Supplementary Figure 1—The effect of ATG1 deletion on methanol-grown GS115. (a) Identification of ATG1 deletion using viability assay. atg1Δ strain loss viability under amino acid starvation. (b) The effect of ATG1 deletion on the growth. (c) The effect of ATG1 deletion on the expression of other ATG genes.
Supplementary Figure 2—Depletion of glutamine and asparagine in BMMY medium. P. pastoris cells were grown in BMMY medium for 12 h. The concentration of glutamine and asparagine in the medium was determined using the method reported previously (Zou et al. 2018). Data are presented as mean ± SD, n = 3. ***, p < 0.001.
Supplementary Table 1—Strains used in this study.
Supplementary Table 2—Primers used in this study.
Supplementary Table 3—Differentially expressed genes between methanol-grown and glycerol-grown GS115.
Supplementary Table 4—Genes involved in amino acid biosynthesis pathway.
Supplementary Table 5—Gene ontology enrichment results.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflict of interest.
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