Mechanism Of Cytoplasmic Localization Of Peroxisome Proteins By Selective Autophagy

Autophagy refers to the process of degrading certain components of the cytoplasm,such as damaged or redundant organelles,proteins,into lysosomes or vacuoles.Under conditions of energy deprivation,autophagy is activated to regulate cellular homeostasis by modulating various biological metabolic pathways to counteract adverse factors.Currently,more than 40 self-crossing genes(ATGs)have been identified.Peroxisomes are important organelles in cells whose main function is to catalyze the elimination of fatty acid ?-oxidation and free radicals.After induction by glucose or ethanol,excess peroxisomes in Pichia pastoris degrade in vacuoles by selective autophagy.This process is also known as pexophagy.Pexophagy in Pichia pastoris is dependent on its receptor Atg30.There are two pathways for peroxisome matrix proteins to enter peroxisomes: the PTS1 and PTS2 pathways.The thiolase(Pot1)protein is a peroxisome matrix protein with a PTS2 signal that enters the peroxisome via the PTS2 pathway.The receptors for Pot1 are Pex7 and Pex20.When PEX7 is knocked out,Pot1 cannot enter the peroxisome and is localized in the cytoplasm.After induction with oleic acid medium,Pot1 proliferated in a large amount,and autophagy occurred after transfer to SD-N medium.Previous work in the laboratory found that the cytoplasmic localization of Pot1 is degraded by new independent autophagy,independent of Atg30.So how does the cytoplasmic localization of Pot1 degrade? We linked Pot1 to GFP to express green fluorescent protein.We can observe the position of green fluorescence under fluorescence microscope to judge the degradation of Pot1.1.Cytoplasm-localized Pot1 enters vacuole degradation by selective cellular autophagyAtg1 is a core protein involved in autophagy.It was found that Pot1 could not enter vacuolar degradation in the strain of ?pex7 ?atg1::Pot1-GFP,but Pot1 entered the vacuole at 10 h in ?pex7::Pot1-GFP strain,indicating cytoplasm-localized Pot1 is degraded by vacuolar entry into the vacuole.Atg11 is an important protein for selective autophagy,so we knocked out ATG11 in the ?pex7::Pot1-GFP strain to see if it would affect the degradation of Pot1 cytoplasmic localization.Fluorescence microscopy showed that when the ?pex7?atg11::Pot1-GFP strain was transfected into SD-N medium for 10 h,the cytoplasm-localized Pot1 could not enter the vacuolization degradation,but it entered the vacuolar degradation at 24 h,indicating that it was in ?pex7 ?atg11::Pot1-GFP strain.Pot1 cytoplasmic localization is delayed by vacuolar degradation.Then,it was verified at the protein level whether Atg11 is involved in the degradation of cytoplasmic Pot1.Detection of the degradation of Pot1-GFP by the GFP-Cleavage assay also confirmed this result.Therefore,the cytoplasmic localization of Pot1 is involved in vacuolar degradation through selective cellular autophagy.2.Screening for key genes involved in Pot1 degradation in the cytoplasm by random insertion mutationIn order to identify other key genes involved in the cytoplasmic Pot1 selective autophagy degradation pathway,restriction endonuclease-mediated integration was used to linearize the p REMI plasmid with bleomycin and to integrate by electroporation.In the genome of ?pex7::Pot1-GFP strain,random mutation of the gene was caused,and then the monoclonal strain was cultured to induce autophagy for10 h and 24 h,and the location of cytoplasmic localized Pot1 was observed.Pot1 could not be screened at 10 h and 24 h.Enter the strain of vacuolar degradation.Selected mutant binding molecular cloning methods and bioinformatics methods were used to identify the assay.More than 6,000 Pichia pastoris mutants were identified by high-throughput screening experiments and 15 mutant strains affecting cytoplasmic Pot1 degradation were screened.The insertion site of the p REMI plasmid in the genome of these mutants was determined by molecular biological methods,and some genes related to autophagy,including ATG2,ATG3,ATG9,ATG14,ATG101,VPS15 and the like,were identified.In addition,some genes that delay cytoplasmic Pot1 degradation,such as IMDH,RXT3,CK2,and Tubulin folding factor D(referred to as TFD),were also screened.Whether these proteins are involved in autophagy in yeast and whether they specifically participate in the degradation of cytoplasmic Pot1 remains to be further studied.3.Select functional verification of the gene to delay the degradation of Pot1-GFPIn order to verify the function of the TFD or IMDH gene and to exclude interference from other causes,we knocked out the TFD or IMDH gene by homologous recombination in the ?pex7::Pot1-GFP strain,and induced autophagy for10 h and 24 h,respectively.Observe the degradation of Pot1 into the vacuole.The results showed that the degradation of Pot1 was delayed in the mutant strains that knocked out the TFD or IMDH gene,indicating that these two genes are indeed involved in the degradation process of cytoplasmic Pot1.4.By co-immunoprecipitation(Co-IP screening and interaction with Pot1-GFP)To identify more genes involved in the cytoplasmic Pot1 degradation process,we screened for interaction with Pot1-GFP by immunoprecipitation using GFP-Trap.The?pex7 ?atg1::Pot1-GFP strain was induced by oleic acid and then transferred to SD-N for 4 hours to collect cells and purify the protein.After SDS-PAGE electrophoresis,mass spectrometry was performed.By mass spectrometry,we screened several proteins that might interact with Pot1,such as Vps29,Vps41 and Gpa2.The Gpa2 protein contains the WD40 domain.It is known that the receptor P1 of Pot1 is also a domain protein containing WD40.After knocking out PEX7,Gpa2 acts as an autophagy receptor and binds to Pot1 located in the cytoplasm.Pot1 was transferred to vacuolar degradation.In order to verify the function of Gpa2,a plasmid for knocking out GPA2 was constructed,and related work was being carried out.