The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo

Abstract

The accumulation of damaged mitochondria has been proposed as a key factor in aging and the pathogenesis of many common age-related diseases, including Parkinson disease (PD). Recently, in vitro studies of the PD-related proteins Parkin and PINK1 have found that these factors act in a common pathway to promote the selective autophagic degradation of damaged mitochondria (mitophagy). However, whether Parkin and PINK1 promote mitophagy under normal physiological conditions in vivo is unknown. To address this question, we used a proteomic approach in Drosophila to compare the rates of mitochondrial protein turnover in parkin mutants, PINK1 mutants, and control flies. We found that parkin null mutants showed a significant overall slowing of mitochondrial protein turnover, similar to but less severe than the slowing seen in autophagy-deficient Atg7 mutants, consistent with the model that Parkin acts upstream of Atg7 to promote mitophagy. By contrast, the turnover of many mitochondrial respiratory chain (RC) subunits showed greater impairment in parkin than Atg7 mutants, and RC turnover was also selectively impaired in PINK1 mutants. Our findings show that the PINK1-Parkin pathway promotes mitophagy in vivo and, unexpectedly, also promotes selective turnover of mitochondrial RC subunits. Failure to degrade damaged RC proteins could account for the RC deficits seen in many PD patients and may play an important role in PD pathogenesis.

Fig. 1.

Parkin promotes mitophagy in vivo. (A) Experimental workflow. (B) parkin and Atg7 mutations prolong mitochondrial protein half-life. Box-and-whisker plots of fold change in half-life show median, quartiles, and extreme values. parkin mean and SD = 1.30 ± 0.22; Atg7 = 1.47 ± 0.30. *P < 0.005, mutant vs. control, by nested ANOVA. (C and D) The effects of parkin and Atg7 mutations on half-life correlate significantly for (C) individual mitochondrial proteins but not for (D) proteins from other organellar targets of autophagy (ribosomes, endoplasmic reticulum, and peroxisomes). n = 147 mitochondrial proteins; n = 58 other organellar proteins. All correlations reported as Pearson r. (E) The effects of parkin mutation on mitochondrial protein half-life do not correlate significantly with the effects of SOD2 deficiency (n = 103).

Fig. 2.

Parkin has a selective effect on turnover of RC proteins. (A and B) Plots comparing the influence of parkin and Atg7 on the half-lives of RC proteins (A) and all other mitochondrial proteins (B). Dashed lines indicate equal effect from both mutations; the half-lives of proteins above the dashed line are more greatly influenced by parkin mutation than Atg7 mutation. (C) The percentage of RC and non-RC proteins with half-lives that are more greatly affected by parkin than Atg7 mutation (n = 36 RC proteins; n = 111 non-RC). The RC is significantly enriched in proteins with greater parkin than Atg7 effect on half-life. *P = 0.003 by χ² test. (D) Mutation in parkin has a larger effect on the half-lives of membrane-bound RC subunits than on those of nonmembrane RC subunits (mean fold change = 1.45 ± 0.19 vs. 1.19 ± 0.14). Horizontal lines indicate the median. ^†P = 4.8 × 10⁻⁶ by Student t test.

Fig. 3.

PINK1 null mutants have a selective impairment of RC protein turnover. (A) PINK1 null mutation prolongs the mean half-life of RC proteins but not other mitochondrial proteins. Box-and-whisker plots of fold change in half-life show median, quartiles, and extreme values. Mean fold change: total mito = 1.04 ± 0.16 (n = 147 proteins; P = 0.082 by nested ANOVA), non-RC mito = 0.99 ± 0.13 (n = 102), RC = 1.17 ± 0.16 (n = 45). *P < 0.0005, mutant vs. control, by nested ANOVA. (B) Mutation in PINK1 has a larger effect on membrane-bound than on nonmembrane RC subunits (mean fold change = 1.24 ± 0.13 vs. 1.11 ± 0.16). Horizontal lines indicate the median. ^†P < 0.005 by t test. (C and D) The effects of PINK1 mutation on RC protein half-lives strongly correlate with (C) the effects of mutation in parkin but not (D) the effects of mutation in Atg7 (n = 36 for parkin; n = 34 for Atg7). (E) The effects of PINK1 mutation on the half-lives of non-RC mitochondrial proteins correlate significantly with the effects of parkin mutation (n = 94). The regression line has a negative y intercept, suggesting a uniform shift to faster mitochondrial protein turnover in PINK1.

Fig. 4.

Possible mechanisms of selective RC turnover. (1) Chaperone-mediated extraction and proteasomal degradation. (2) Transport to the lysosome through mitochondria-derived vesicles.