| Autophagy is an important catabolic process for degrading biomacromolecules or organelles by lysosomes to maintain cell homeostasis in the cells.Autophagy-related proteins precisely regulate this process.Different autophagy-related proteins play different roles in the process.The programmed nuclear death(PND)of the parental macronucleus is a unique type of nuclear autophagy during the sexual reproduction of the Tetrahymena thermophila.Different autophagy-related proteins may be involved in the regulation of PND.In this study,two homologous genes Tt ATG4.1 and Tt ATG4.4 were identified from T.thermophila,and functions of these two genes were analyzed.The main results were as follows:1.Bioinformatics analysis of Tt ATG4.1 and Tt ATG4.4: Four C54 peptidase family proteins,Tt Atg4.1,Tt Atg4.2,Tt Atg4.3 and Tt Atg4.4,were identified by homologous sequence alignment in the Tetrahymena Genome Database(http://www.ciliate.org).As cysteine proteases,four autophagy-related proteins contained a conserved catalytically active site Cys and a highly conserved GWGCM motif.The clustering analysis of Atg4 proteins showed consistent with that of the evolution of species.The four homologous proteins of might evolved perform different functions in the process of PND.Tt ATG4.1 was specifically expressed in sexual reproduction and praked at 8 h,while Tt ATG4.4 had the highest expression at 12 h of sexual reproductionwhich coincided with the time of PND.These resultssuggested that Tt Atg4.1 and Tt Atg4.4 may be involved in the regulation of PND.2.Tt ATG4.1 participates in the degradation of the parental macronucleus during the PND process To analyze the function of Tt Atg4.1,the HA-tagged vector p XS75-ATG4.1 was constructed and transformed into T.thermophila.The mutant strains were screened under paromomycin stress.Immunofluorescence stainning showed that HA-Atg4.1 localized in the cytoplasm and the degraded parental macronucleus.Overexpressed HA-Atg4.1 did not affect the normal acidification of parental macronucleus and fusion between autophagy lysosome and parental macronucleus membrane,but promoted degradation of the parental macronucleus.Furthermore,RNAi-ATG4.1 strains were created.ATG4.1 knockdown resulted in abnormal degradation of the parental macronucleus and delayed cellular development.In this process,autolysosome seemed to gradually "gnaw" parental macronuclear endosome.Localization of Atg8 was also abnormal.3.Tt ATG4.4 is involved in the regulation of micronucleus of meiotic and the degradation of the parental macronucleus Tt ATG4.4 was highly expressed at 2 h and 12 h in sexual reproduction.RNAi-ATG4.4 mutant strains were created.ATG4.4 knockdown inhibited meiosis of micronucleus and sexual reproduction was aborted.The HA-tagged vector p XS75-ATG4.4 was created and transformed into T.thermophila.Overexpression of ATG4.4 strains were obtained by paromomycin screening.Immunofluorescence staining indicated that HA-Atg4.4 localized on the parental macronucleus.Overexpressed HA-Atg4.4 affected degradation of meiotic micronuclei.The parental macronucleus loosely expanded and failed to move to the posterior of cell during anlagen stage.The results indicated that Atg4.4 not only involved in regulation of meiotic micronucleus,but also regulation of the degradation of the parental macronucleus during PND.In this study,the autophagy-related genes Tt ATG4.1 and Tt ATG4.4 of T.thermophila were identified for the first time.Both autophagy-related genes were involved in the degradation of the parental macronucleus in PND,and the Tt ATG4.4 also affected micronucleus meiosis by regulating the autophagy pathway.Atg4.1 and Atg4.4 have different functions in PND.The identification of these autophagy-related genes was useful for further understanding the degradation mechanism of the PND in Tetrahymena. |
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