The Study Of Pichia Pastoris Casein Kinase 2 In Regulating Autophagy

Autophagy refers to a collection of multiple processes that transport cytoplasmic components（including organelles）to lysosomes/vacuoles for degradation,and is an evolutionarily conserved pathway.In addition to non-selective autophagy induced by nutrient deprivation,selective autophagy specifically targets various organelles and protein aggregates and exerts various physiological functions in eukaryotic cells.Yeast casein kinase 2（CK2）consists of two catalytic subunits（Cka1 and Cka2）and two regulatory subunits（Ckb1 and Ckb2）.CK2 is highly conserved in eukaryotes and can affect many different cellular processes by phosphorylation of hundreds of proteins such as cell polarity,stress response,transcription and translation,etc.Earlier studies have found that in Pichia pastoris,peroxisomes depend on Atg30for degradation through selective autophagy pathways.Peroxisome receptors and mislocalized peroxisome proteins in the cytoplasm are degraded independent of Atg30.To study the key proteins involved in this pathway,we randomly inserted the p REMI plasmid into the yeast genome to screen mutants and performed the following experiments:1.Screen the proteins that affect the degradation of cytoplasmic Pot1 by randomly inserting yeast mutantsPot1（Thiolase）is a matrix protein that is localized to peroxisomes and relies on its receptor Pex7 to enter peroxisomes through the PTS2 pathway.In strains lacking PEX7,Pot1 is located in the cytoplasm and cannot be transported to peroxisomes.Using the p REMI plasmid,a mutant library based on theΔpex7::Pot1-GFP strain was constructed.By inducing autophagy and observing under a fluorescence microscope,mutant strains whose cytoplasmic Pot1 could not enter the vacuole and degraded were screened out.The yeast flanking genome where the p REMI plasmid is located was sequenced to identify genes that affect the degradation of cytoplasmic Pot1.Using this method to screen strain No.148,the degradation of cytoplasmic Pot1-GFP was delayed.After identification,the results showed that a p REMI fragment was inserted into the CKB1 gene of strain No.148.2.Explore the effect of knockout of CKB1 on the degradation of cytoplasmic localized PTS1 and PTS2 proteinsFox2（hydratase-dehydrogenase-epimerase）is a matrix protein located in peroxisomes,which relies on its receptor Pex5 to enter peroxisomes through the PTS1pathway.In strains lacking PEX5,Fox2 is localized in the cytoplasm.The homologous recombination method was used to knock out CKB1 inΔpex7::Pot1-GFP andΔpex5::GFP-Fox2.The mutant strains further induced autophagy,and the degradation of Pot1-GFP and GFP-Fox2 was observed under a fluorescence microscope.The results showed that cytoplasmic Pot1-GFP and GFP-Fox2 could not enter the vacuole for degradation at 0 h-12 h,but entered the vacuole for degradation at24 h,indicating that CK2 was involved in the degradation of cytoplasmic localized peroxisome proteins.3.Exploring the effect of knockout of CK2 regulatory subunits on the growth of Pichia pastorisTo explore whether the knockout of CK2 regulatory subunits affects the growth of Pichia pastoris,theΔckb1 andΔckb2 strains were obtained by homologous recombination.The growth curves of PPY12（WT）,Δckb1,Δckb2,andΔatg1 in YPD were found the knockout of CKB1 and CKB2 would inhibit the growth of Pichia pastoris,and the knockout effect of CKB2 was more significant.4.Exploring the effect of knockout of CK2 regulatory subunits on peroxisome autophagyTo explore the effect of knockout of CKB1 or CKB2 on pexophagy,homologous recombination was used to knock out CKB1 or CKB2 in PPY12::GFP-Fox2,PPY12::Pot1-GFP,respectively.After obtaining the mutant strain,pexophagy was induced,and the degradation of GFP-Fox2 and Pot1-GFP was observed at the cell level at 0 h,6 h,12 h,and 24 h.GFP-Fox2 and Pot1-GFP in PPY12 entered the vacuole and degraded at the 6th hour after autophagy started.InΔckb1 andΔckb2,GFP-Fox2 and Pot1-GFP entered the vacuole and degraded at the 12 h,and there was still some undegraded at the 24h.InΔatg1,GFP-Fox2 and Pot1-GFP are not degraded due to the deletion of the autophagy core protein Atg1.At the biochemical level,the GFP-Cleavage experiment was used to analyze pexophagy.The results showed that,compared with PPY12,the degradation rate of GFP-Fox2 or Pot1-GFP in CKB1 or CKB2 knockout strains was slower.The above results indicate that CK2 is involved in pexophagy.5.Explore the effect of knockout of CK2 regulatory subunits on mitophagyTo explore the effect of CKB1 or CKB2 knockout on mitophagy,Tom20-GFP was transferred into PPY12,Δatg1,Δckb1,andΔckb2.After obtaining the successfully transformed strain,mitophagy is induced.At the cellular level,mitophagy was determined by fluorescence observation.At the 36th hour of mitophagy,Tom20-GFP in PPY12 entered the vacuole and degraded,while inΔatg1,Δckb1,andΔckb2,Tom20-GFP could not enter the vacuole and degraded.Western blot was performed at the biochemical level,and the mitophagy was analyzed by GFP-Cleavage experiment.The results showed that in PPY12,with the increase of autophagy time,Tom20-GFP continued to decrease and Free-GFP continued to increase.Tom20-GFP was not degraded inΔatg1,Δckb1,andΔckb2.The above results indicate that the knockout of CK2 regulatory subunit blocks mitophagy.6.Explore the impact of knockout of CK2 regulatory subunits on the Cvt（cytosol to the vacuole pathway,Cvt）pathwayTo explore the influence of CK2 on the Cvt pathway,we cloned the Ape I promoter,Ape I coding region,and GFP sequence and ligated it to the vector p MY72.The successfully constructed plasmid was linearized into PPY12,Δatg1,Δckb1,andΔckb2.The successfully transformed strain induces the Cvt pathway.At the cellular level,the Ape I maturation is observed by fluorescence.At the 3 h of the Cvt pathway,Ape I-GFP matures in PPY12,Δckb1 andΔckb2 and is mainly located in the vacuole,inΔatg1 Ape I-GFP cannot mature and accumulates in punctate distribution.At the biochemical level,Western blot was used to analyze whether pr Ape I was converted into mature Ape I.The results showed that the maturation of Ape I-GFP can be detected in PPY12,Δckb1,andΔckb2.InΔatg1,the Cvt pathway is inhibited due to the deletion of the autophagy core protein Atg1.The above results indicate that the knockout of the CK2 regulatory subunit does not affect the Cvt pathway.7.Explore the impact of knockout of CK2 regulatory subunits on the non-selective autophagy pathwayIn order to explore the effect of CK2 on the non-selective autophagy pathway,we cloned the Atg8 promoter,Atg8 coding region,and GFP sequence and ligated it to the vector p MY72.The successfully constructed plasmid was linearized into PPY12,Δatg1andΔckb1.The successfully transformed strain induces a non-selective autophagy pathway.Western blot was performed at the biochemical level,and non-selective autophagy was analyzed by GFP-Cleavage experiment.The results showed that:in PPY12,Δckb1,with the increase of autophagy time,GFP-Atg8 continued to decrease,and Free-GFP continued to increase.GFP-Atg8 is not degraded inΔatg1.The above results indicate that the knockout of the CK2 regulatory subunit does not affect the non-selective autophagy pathway.8.Explore the molecular mechanism of the knockout of CK2 regulatory subunits affecting autophagyIn Pichia pastoris,CKB1 is involved in the degradation of cytoplasmic peroxisome matrix protein;CKB1 and CKB2 are involved in pexophagy;CKB1 or CKB2 knockout will block mitophagy;the knockout of CKB1 or CKB2 does not affect the Cvt pathway.We constructed the vector P_CKB1-CKB1-GFP-p MY72 and transferred them into Pichia pastoris PPY12 and SMD1163 respectively,and prepared to use GFP-Trap for Co-IP experiments to screen the proteins that interact with CKB1-GFP,and then study the mechanism of CK2 regulating selective autophagy in Pichia pastoris.According to literature reports,CK2 regulates mitophagy through phosphorylation of mitophagy receptors in Saccharomyces cerevisiae and Mammalia,but does not affect macroautophagy and other forms of selective autophagy.The innovation of this research work is:CK2 not only participates in mitophagy in Pichia pastoris but also participates in other forms of selective autophagy,such as cytoplasmic peroxisome protein autophagy,pexophagy.The research of this experiment is of great significance for expanding the function of CK2 in the autophagy pathway.