Fig. 1: Loss of mitochondrial protein import motor components induces mitophagy in human cells and C. elegans.

a Experimental scheme of a genome-wide CRISPR/Cas9 screen to identify genes that induce mitophagy when knocked-out. HeLa FlpIn cells expressing the mitophagy reporter mt-mKEIMA and PRKN (synonym PARKIN) were infected with a lentiviral particle library. Cells exhibiting induced mitophagy (high mt-mKEIMA 561 nm/405 nm ratio) were sorted and analyzed by next-generation sequencing. b Scatter plot presenting MAGeCK algorithm-based enrichment of targeted genes and determined robust ranking aggregation value of this gene in positive selection (sorted versus total population). Data shown in Supplementary Data 1. c Depiction of the mitochondrial import machinery and mitochondrial protein processing. Gene depletions identified in b as mitophagy inducers were labeled. d Validation of mitophagy-inducing gene knock-outs from b that are part of the matrix import machinery. Indicated genes were knocked-out individually by two gRNAs and mitophagy induction monitored by flow cytometry analysis of mt-mKEIMA. The gray line indicates the upper value of negative controls. e Experimental scheme to assess mitophagy in C. elegans using the autophagosomal marker LGG-1::mCherry (red) and mitochondrial matrix marker SIR2.2::GFP (green). f Quantification of co-localization of green mitochondria with red LGG-1 upon RNAi knock-down of components of the PAM complex in C. elegans. Bar graphs indicate median values ±s.d. (n = 27 control worms compared to n = 26 grpel1/2, n = 9 tim-16, n = 5 tim-44 and n = 16 dnj-21 worms, p-values were calculated by two-sided unpaired t test). See also Supplementary Fig. 1. mt mitochondrial.