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RNAseq and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16

ABSTRACT: Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. The process is driven by many autophagy-related (ATG) proteins, including the core members ATG9 and ATG16. Here, we applied RNAseq and tandem mass tag (TMT) proteomic approaches to identify differentially expressed genes (DEGs) and proteins (DEPs) in ATG9‾, ATG16‾ and ATG9‾/16‾ strains in comparison to AX2 wild-type cells. In total, we identified 332 (279 up and 53 down), 639 (487 up and 152 down) and 260 (114 up and 146 down) DEGs and 124 (83 up and 41 down), 431 (238 up and 193 down) and 677 (347 up and 330 down) DEPs in ATG9‾, ATG16‾ and ATG9‾/16‾ strains, respectively. Thus, in the single knock-out strains, the number of DEGs was higher than the number of DEPs while in the double knock-out strain the number of DEPs was higher. Comparison of RNAseq and proteomic data further revealed, that only a small proportion of the transcriptional changes were reflected on the protein level. This suggests that most changes in protein abundance were not caused by transcriptional changes but were rather due to changes in protein homeostasis. Gene ontology (GO) analysis revealed an enrichment of DEPs involved in lipid metabolism and oxidative phosphorylation. Furthermore, we found increased expression of the anti-oxidant enzymes glutathione reductase (gsr) and catalase A (catA) in ATG16‾ and ATG9‾/16‾ cells, respectively, indicating adaptation to excess reactive oxygen species (ROS). Our study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells and adds to our knowledge of functions of these proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Dictyostelium Discoideum (slime Mold)

SUBMITTER: Qiuhong Xiong

LAB HEAD: Qiuhong Xiong

PROVIDER: PXD023730 | Pride | 2021-06-15

REPOSITORIES: Pride

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Project description:Ananas comosus var. bracteatus has high ornamental value and widespread application because of its chimeric leaves. However, little is known about the molecular mechanism regulating this characteristic. Here, comparative transcriptomic and proteomic analyses of the white parts (Whs) and green parts (Grs) of the chimeric leaves were performed to identify differentially expressed genes (DEGs) and differentially expressed proteins (DEPs). In total, 1,685 DEGs, including 712 up- and 973 down-regulated ones, and 5,428 DEPs, including 1,018 up- and 795 down-regulated ones, were identified between the Whs and Grs. Comparisons with the GO and KEGG annotations revealed that the DEGs were involved mostly in carbon fixation, porphyrin and chlorophyll metabolism and oxidative phosphorylation. The DEPs were mainly involved in ribosomes, photosynthesis, photosynthesis antennas, and porphyrin and chlorophyll metabolism. Combined analysis showed that nine proteins related to chlorophyll biosynthesis, photosynthetic pigments, and photosynthesis were unchanged at mRNA level but suppressed at protein level. These results indicated that the albino phenotype of the Whs was caused by the proteomic-level suppression of key enzymes involved in the chlorophyll biosynthesis pathway and that translational and post-translational regulation may play important roles in both the biosynthesis of photosynthetic pigments and photosynthesis. Biological signiﬁcance: Leaves of Ananas comosus var. bracteatus serve as the best materials for the study of albino mechanism. Because the chemic trait of A. comosus var. bracteatus is unstable and the molecular mechanism of the albino cells was poorly understood, we performed comparative analyses both at the transcriptome and proteome levels. This work revealed suppressed proteomic-level and translational and post-translational regulation contribute to the albino phenotype formation. Our results provide better information concerning the molecular mechanism within the chimeric leaves of A. comosus var. bracteatus.

Dataset Information

RNAseq and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16

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