Fig. 4: PAM complex sequestration mediates import defects upon protein misfolding to induce mitophagy. | Nature Communications

Fig. 4: PAM complex sequestration mediates import defects upon protein misfolding to induce mitophagy.

From: Protein import motor complex reacts to mitochondrial misfolding by reducing protein import and activating mitophagy

Fig. 4

a Experimental scheme for the analysis of insoluble mitochondrial proteins by quantitative proteomics. b HeLa FlpIn TRex PARKIN cells treated for 96 h with LONP1 siRNA or with GTPP for 6 h. Log2 fold changes of insoluble mitochondrial protein fractions upon mitochondrial proteostasis perturbation compared to control conditions shown as density plots. Dotted line indicates the median of all identified proteins. Average of n = 3 replicates used and soluble PAM components labeled. c Representative immunoblots of mitochondrial soluble and insoluble protein fractions after GTPP or CCCP treatment shown for n = 3. Equal input amounts shown by TOMM40 staining of total (mitochondria). MW = molecular weight. d Experimental design of TurboID-TIMM44 proximity proteomics. e HeLa FlpIn TRex TIMM44-TurboID with 24 h dox, 20 min biotin treatment. Volcano plot presentation of proteins in close proximity of TIMM44-TurboID compared to control. Dotted line indicates significance p = 0.05 by two-sided, unpaired t-testing. f TIMM44-TurboID interactions with the translocon and PAM16 during protein folding stress induced by LONP1 RNAi for 96 h or GTPP treatment for 6 h, normalized to controls and interactome data from e. Data represented as mean ±s.d. for n = 3. Statistical significance determined by two-sided unpaired t testing. g Proposed model: (I) Under basal conditions, the PAM complex associates with the TIMM translocon and allows protein import. PINK1 is partially imported, leading to its degradation and prevention of mitophagy. (II) Reduced mitochondrial protein import (e.g., genetic or pharmacological perturbation) leads to PINK1 stabilization and activation on the OMM to induce mitophagy. IMM depolarization is not required for this process. (III) This mechanism is also activated during mitochondrial protein misfolding stress, in which PAM components become insoluble, lose their interaction with the translocon, and protein import is reduced. ∆Ψ = Mitochondrial membrane potential.

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