Mitochondrial Proteostatic Stress Induces Aggrephagy to Triage Unimported Proteins

We report the results of overexpression of the wildtype and mutant ANT1 (SLC25A4) gene in human embryonic kidney 293T (HEK293T) cells from the cytomegalovirus (CMV) promoter. Libraries were prepared from three independent replicates per condition using stranded RNA sequencing and run at 2 x 75 read length at a targeted output of 60 million paired-end reads per sample. We found only a limited number of nuclear genes whose expression is altered by wildtype ANT1-overexpression. Among the most upregulated genes is the zinc-finger proteins Egr1, a transcriptional factor with diverse cellular functions, including the repression of genes encoding mitochondrial inner membrane protein. The upregulation of EGR1 likely serves as a retrograde mechanism to reduce protein loading on the IMM and to alleviate mPOS. Overexpression of the triple and quadruple mutant alleles altered the expression of 206 and 560 genes, respectively, with 32 commonly up-regulated in both. These include EGR1, ZCCHC12 and SFPQ, which were also upregulated in cells overexpressing the wild-type ANT1. The 32 up-regulated genes are involved in functions including RNA-processing, autophagy/intracellular structure/trafficking, transcriptional control, ribosomal biogenesis/translational control, chaperones/proteasomal function and mitochondrial biogenesis. Upstream regulator analysis subsequently revealed 42 pathways predicted to be either activated or inhibited by these genes, including PPARGC1A, ATF4, Heat Shock Factor 1 (HSF1), mechanistic Target of Rapamycin (mTOR), Myc, EGFR and HMGA1. Overall design: Examination of effects of overexpression of wildtype and mutant ANT1 alleles in HEK293T cells

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Liu Y, Wang X, Coyne LP, Yang Y, Qi Y, Middleton FA, Chen XJ