Functional Analysis Of Autophagy-related Protein Atg5 In Nuclear Programmed Death Of Tetrahymena Thermophila

Autophagy is a highly conserved catabolic process in eukaryotes.In order to maintain cell physiological homeostasis and respond to starvation or other external stress,redundant cell components or harmful substances can be degraded through the fusion pathway of lysosomes and autophagosomes.Autophagy has two types,i.e.non-selective autophagy and selective autophagy.According to the different degradation substrates,selective autophagy is subdivided into mitophagy,reticulophagy,nucleophagy and so on.The programmed nuclear death?PND?of the parental macronucleus?pa MAC?is a unique type of nuclear autophagy during the sexual reproduction of the Tetrahymena thermophila.A variety of autophagy-related proteins may be involved in regulating the PND.In this study,autophagy-related gene ATG5 was identified with T.thermophila,and the function of the ATG5 gene in the PND process was analyzed.The main results are as follows:1.Bioinformatics analysis of Autophagy-related gene ATG5 Based on the amino acid sequences of human Atg5 and Saccharomyces cerevisiae Atg5,homologous sequence alignment were performed in the T.thermophila genome database?http://www.ciliate.org?.Gene TTHERM₀0494030 was found to be similar to humans and yeast,and both have APG5 conserved structure area.Therefore,TTHERM₀0494030 was predicted as an ATG5 gene in T.thermophila.Phylogenetic analysis showed that the Tt Atg5 and Pt Atg5 of had similar evolutionary status.The potential interacting protein of Tt Atg5 was predicted,Atg5 may interact with many core autophagy proteins including Atg8 and Vps34.Atg8 and Vps34 are important proteins in the PND process of T.thermophila.Further analysis of the ATG5 expression profile revealed that ATG5 was expressed during growth,starvation and sexual reproduction.And the peak expression of ATG5 appeared at 10 h during the sexual reproduction.This point in time corresponds to the PNDstage.This result suggests that Tt Atg5 may be involved in regulation the PND process.2.Tt Atg5 is involved in regulation the PND To analyzes the function of Tt Atg5 during the PND process,the HA-tagged vector p XS75-ATG5 was constructed and transformed into T.thermophila.The mutant strains were screened for paromomycin stress.Immunofluorescence stainning showed that HA-Atg5 localized in the cytoplasm at 2 h,4 h,and 6 h of sexual reproduction.At 8 h,Atg5 began to localize to the pa MAC,until the pa MAC disappeared.Overexpressed HA-Atg5 did not affect the normal acidification of pa MAC and fusion between lysosomes and parental macronucleus membrane,but delayed degradation of the pa MAC.Furthermore,RNAi-ATG5 strains were constructed.The degradation of meiotic products of micronucleus was blocked in 19.4% of RNAi-ATG5 cells at 8 h during the sexual reproduction.In the RNAi-ATG5 cells,the pa MAC could not migrate to the bottom of the cell in 12.7% of cells at 10 h.Meanwhile,the pa MAC could not be acidified and degraded normally.At 36 h,there were still undegraded pa MAC in 22%of cells.The results indicated that Atg5 is involved in the programmed death of meiotic products of micronucleus and pa MAC.3.Tt Atg5 regulates the degradation of pa MAC by mediating the localization and lipidation of Atg8.2 Atg8 is a key factor in extension of autophagosome membrane and recognition of degraded cargoes.Recruitment and localization of Atg8 depends on the Atg12-Atg5-Atg16 complex.Co-immunoprecipitation and mass spectrometry analysis revealed that Atg5 interacted with Atg8.2.Therefore,the HA-tagged vector p XS75-ATG8.2 was constructed.Immunofluorescence localization was analyzed,in the paired cells of wild type and OE-ATG8.2,Atg8.2 was recruited near the pa MAC and gradually localized to the parental macronucleus membrane.In the paired cells of RNAi-ATG5 and OE-ATG8.2,recruitment and localization of Atg8.2were affected during the PND process.To further study the effect of Atg5 knockdown on function of Atg8.2 during PND,the expression levels ofHA-Atg8.2 and HA-Atg8.2-PE proteins were detected by Western Blot.The results showed that after knockdown of Atg5,lipidation of Atg8.2 was significantly affected.The above results indicate that Atg5 is involved in regulating the degradation of the pa MAC by affecting the localization and lipidation of Atg8.2 during PND.In summary,Tt ATG5 was identified in this study,and Atg5 was localized to the pa MAC during PND.Overexpression of ATG5 delayed the degradation of the pa MAC.Knockdown of ATG5 results in hindered degradation of micronucleus meiosis.At the same time,the migration,acidification and degradation of the pa MAC during the PND process are hindered.Further analysis confirmed that there is an interaction between Atg5 and Atg8.2.ATG5 regulates the lipidation of Atg8.2 and further affects its location on the pa MAC.