Functional Analysis Of Autophagy-Related Protein Atg18 In Nuclear Programmed Degradation Of Tetrahymena Thermophila

Autophagy is an evolutionarily conserved lysosomal degradation pathway in all eukaryotes.It is characterized by the formation of double-membrane autophagosomes in the pre-autophagosome structure(PAS).In this process,different autophagy-related proteins(Atg proteins)are recruited in an orderly manner to perform regulatory functions.Autophagy can be divided into selective autophagy and non-selective autophagy.In Tetrahymena thermophila,parental macronucleus(pa MAC)programmed nuclear death(PND)is a typical type of nuclear selective autophagy during sexual reproduction.A variety of Atg proteins are involved in the regulation of the PND process.In this study,two homologous genes,TtATG18.1 and TtATG18.2,were identified in the Tetrahymena Genome Database(TGD)with human and yeast ATG18sequences,and the functions of these two genes in the pa MAC PND process were analyzed.The main results are as follows:1.TtATG18.1 and TtATG18.2 bioinformatics analysis:Based on the human and yeast Atg18 amino acid sequence,the homologous sequence alignment in the TGD database found that TTHERM?00614790and TTHERM?00577340 are homologous to human and yeast Atg18proteins,and both have WD40 Structural domain.The TTHERM?00614790 and TTHERM?00577340 genes are predicted to be Tetrahymena autophagy-related gene ATG18,named TtATG18.1 and TtATG18.2,respectively.Further analysis found that TtATG18.1 and TtATG18.2 have a conserved LRRG motif similar to yeast.Molecular evolution analysis showed that Tt Atg18.1 is closely related to Tt Atg18.2,and Tt Atg18.2 is relatively closely related to human WIPI3.The protein interaction prediction results showed that Tt Atg18.1,Tt Atg18.2 protein interacts with Atg5,Atg8,Atg3 and other proteins.Through gene expression profile analysis,Tt Atg18.1 and Tt Atg18.2 are expressed in the growth period,starvation period,and sexual reproduction period.The expression of Tt Atg18.1 and Tt Atg18.2 increased significantly during 8h-10 h of sexual reproduction,and this period is the parental macronuclear PND period.This result suggests that Tetrahymena Tt Atg18.1 and Tt Atg18.2 may be involved in regulation of the pa MAC PND process.2.TtATG18.1 participates in the parental macronuclear PND process during sexual reproduction:In order to study the function of Tt Atg18.1,the overexpression plasmid p XS-ATG18.1 fused with HA tag was first constructed and transferred into Tetrahymena via gene gun.Using different concentrations of paromomycin to screen and obtain ATG18.1 overexpression mutant strains.In the starvation phase,Tt Atg18.1 is located in the cytoplasm,but there are a lot of cavities in the cell.In the early stage of sexual reproduction,Tt Atg18.1 localizes in the cytoplasm,starts to localize around pa MAC at 8 h,and then localizes throughout pa MAC until the pa MAC degradation signal disappears.The overexpression of TtATG18.1 was induced by Cd²⁺,and nuclear development statistics showed that overexpression of TtATG18.1promoted the degradation of the parental macronucleus.The above results indicated that Tt Atg18.1 participated in the regulation of the parental macronuclear PND.3.TtATG18.2 participates in the regulation of parental macronucleus PND and the development of new small nuclei:In order to study the function of Tt Atg18.2,an overexpression plasmid p XS-ATG18.2 fused with HA tag was constructed and transferred into Tetrahymena via gene gun,Different concentrations of paromomycin were screened to obtain TtATG18.2 overexpression mutant strains.In the starvation phase,Tt Atg18.2 is located in the cytoplasm,but there are a lot of cavities in the cytoplasm.In the early stage of sexual reproduction,Tt Atg18.2 was localized in the cytoplasm,located on pa MAC and one of the new small nuclei to be degraded for 8 h,and then the degradation signal of the new small nucleus disappeared with the degradation of pa MAC.The degradation of pa MAC is delayed after the overexpression of TtATG18.2 induced by Cd²⁺,indicating that Tt Atg18.2 is involved in the regulation of pa MAC PND.In order to further analyze the function of Tt Atg18.2 in the PND process,an RNAi-ATG18.2 mutant cell line was constructed.After knocking down TtATG18.2,pa MAC could not be acidified normally,and the cells developed to 36 h,and there was still pa MAC undegraded in 18%knockdown cells.The above results further confirmed that TtATG18.2 participated in the regulation of pro-pa MAC PND,and it was dissolved with pa MAC and autophagy Enzyme fusion related.In addition,the deletion of Tt Atg18.2 hindered the degradation of new small nuclei in 14%of paired cells at the later stage of isolation.This result indicates that Tt Atg18.2 is not only involved in pa MAC PND,but also related to the degradation of new small nuclei.These results indicate that TtATG18.2 is involved in the regulation of parental large nuclear PND and the degradation of new small nuclei.In summary,Tetrahymena TtATG18.1 and TtATG18.2 are both involved in the regulation of parental macronuclear PND during sexual reproduction.In addition,TtATG18.2 is also related to the degradation of new small nuclei.