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

Qiuhong Xiong,Ning Song,Ludwig Eichinger,Prerana Wagle,Gernot Glöckner,Ping Li,Roman Konertz,Changxin Wu,Sarah Fischer

doi:10.1186/s12864-021-07756-2

Abstract

BackgroundAutophagy 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. However, the functions of these two core ATG proteins still need further elucidation. Here, we applied RNAseq and tandem mass tag (TMT) proteomic approaches to identify differentially expressed genes (DEGs) and proteins (DEPs) in Dictyostelium discoideum ATG9‾, ATG16‾ and ATG9‾/16‾ strains in comparison to AX2 wild-type cells.ResultIn 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. 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).ConclusionsOur study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells. Our results suggest, that most changes in protein abundance were not caused by transcriptional changes, but were rather due to changes in protein homeostasis. In particular, knock-out of atg9 and/or atg16 appears to cause dysregulation of lipid metabolism and oxidative phosphorylation.

Highlights

Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes
The analysis revealed for fold changes (FC) ≥ 2.0 and for pvalues ≤ 0.01 versus ≤ 0.05 only a marginal decrease for the number of up-regulated genes for the ATG9 ̅ strain, while no reduction in the number of differentially regulated genes was seen for all other comparisons
If we look at the percentages of common differentially upregulated genes, we find that ATG9 ̅ cells share 75 % of the up-regulated genes with ATG16 ̅ cells, and, vice versa, ATG16 ̅ cells 43 % of the up-regulated genes with ATG9 ̅ cells

Summary

Introduction

Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. Autophagy is an evolutionary ancient mechanism for the recycling of cellular material In this process, portions of the cytosol are engulfed within a newly formed double-membrane vesicle, the autophagosome, and delivered to the lysosome for degradation [1]. ATG9, the only transmembrane core autophagy protein, cycles between different organelle compartments via vesicular transport pathways and delivers membrane lipids to the autophagosome formation site in response to induction of autophagy [7]. ATG16 acts in the heterotetrameric ATG16/ATG12 ~ 5 complex, which determines the ATG8 lipidation sites and facilitates transfer of ATG8 from ATG3 to phosphatidylethanolamine (PE) [6, 8] Apart from their autophagic functions, most of the ATGs are involved in different autophagy-independent processes [9]. For ATG16 several autophagy-independent functions, as for example in antigen presentation, in hormone secretion, and in plasma membrane repair have been reported [6]

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Conclusion