Analysis of the LIR motif during PINK1/Parkin mitophagy

To determine the role of the LIR motif during PINK1/Parkin mitophagy, we first assessed the Atg8 recruitment profile of OPTN and NDP52 following PINK1/Parkin activation. LIR amino acid sequences vary between autophagy receptors, with different Atg8 binding specificities reported for each protein. For example, the LIR motif in OPTN has a high affinity for GABARAP39, but the affinity can switch toward LC3B when OPTN is phosphorylated at S177 by TANK binding kinase (TBK1)40. NDP52 contains a non-canonical LIR motif (also termed CLIR) that is highly selective for LC3C binding23. To assess the Atg8 recruitment profile of OPTN and NDP52 during PINK1/Parkin mitophagy, we utilised penta knockout (KO) cells which lack the five major autophagy receptors (OPTN, NDP52, TAX1BP1, NBR1 and p62)14.

Atg8 localization was assessed using HA-tagged variants of all six mammalian Atg8s (LC3A, LC3B, LC3C, GBRP, GBRPL1 and GBRPL2), to avoid the functional impairment associated with GFP-tagged LC3/GABARAPs14,38. Penta KO cell lines stably expressing untagged Parkin and individual HA-Atg8s were rescued with either GFP-OPTN or GFP-NDP52, before treatment with oligomycin and antimycin A (OA) for 3 h to activate PINK1/Parkin mitophagy. HA-tagged Atg8s were not recruited to mitochondria in the absence of autophagy receptor expression (Fig. 1a), whereas all HA-tagged Atg8s were recruited in penta KOs rescued with either GFP-OPTN or GFP-NDP52 (Fig. 1b, c). Neither OPTN nor NDP52 displayed a preference for a particular Atg8 family member during mitophagy (Fig. 1b, c).

Fig. 1 The LIR motif within autophagy receptors is dispensable for Atg8 recruitment during PINK1/Parkin mitophagy. a–f Representative immunofluorescence images of penta KO HeLa cells stably expressing untagged Parkin and either HA-LC3A, HA-LC3B, HA-LC3C, HA-GBRP, HA-GBRPL1 or HA-GBRPL2, without receptor expression (a), or rescued by co-expression of either GFP-OPTN (b), GFP-NDP52 (c), GFP-p62 (d), GFP-OPTN(F178A) (e) or GFP-NDP52(V136S) (f); immunostained for HSP60, HA & GFP after 3 h OA treatment. Atg8-positive mitochondria are indicated by arrowheads. Scale bars: overviews, 10 µm; insets, 2 µm Full size image

To confirm that Atg8 recruitment was dependent on the LIR motif, we analysed penta KO cells rescued with LIR motif mutant GFP-OPTN(F178A), or GFP-NDP52(V136S). Unexpectedly, both GFP-OPTN(F178A) and GFP-NDP52(V136S) could still recruit all HA-tagged Atg8s (Fig. 1e, f). Co-immunoprecipitation experiments confirmed that the LIR point mutants of OPTN and NDP52 prevented autophagy receptor interaction with Atg8s (Supplementary Fig. 1a, b). OPTN(F178A) binding to HA-LC3B (Supplementary Fig. 1a) and NDP52(V136S) binding to HA-LC3C (Supplementary Fig. 1b) were inhibited. Despite their inability to bind Atg8 family members, LIR-mutant OPTN(F178A) and NDP52 (V136S) both significantly recruited HA-LC3B and HA-LC3C, respectively, following PINK1/Parkin mitophagy activation (Supplementary Fig. 1c, d). In contrast, penta KO cells rescued with GFP-p62 failed to recruit any Atg8s despite the presence of a functional LIR motif within p62 (Fig. 1d). The LIR motif within autophagy receptors is therefore dispensable for both Atg8 recruitment and selective recognition of mitochondria. The ULK1 autophagy initiation complex can be recruited by NDP52 and OPTN, but not p6214. Thus, the recruitment of the ULK1 complex and activation of downstream autophagy machineries is likely to govern Atg8 recruitment32,34.

The LIR motif promotes efficient mitophagy

Our results reveal that the LIR motif is not essential for Atg8 family recruitment nor selectivity, however, several studies have demonstrated the importance of the LIR motif for selective autophagy17,23,24. We therefore assessed the mitophagy activity of LIR-mutant GFP-OPTN(F178A) and GFP-NDP52(V136S). Penta KO cells rescued with GFP-tagged WT and LIR-mutant autophagy receptors, or p62 as a negative control14, were treated with OA for 21 h before immunoblotting for the mitochondrial DNA encoded protein CoxII to assess mitophagy (Fig. 2a, b). LIR-mutant GFP-OPTN(F178A) robustly degraded CoxII similarly to WT GFP-OPTN, whereas GFP-NDP52(V136S) had a significant mitophagy defect relative to WT GFP-NDP52. Despite the mitophagy deficiency observed for LIR-mutant GFP-NDP52(V136S), it could still drive mitophagy to a significant degree when compared to the penta KO control which lacks autophagy receptor expression (Fig. 2a; compare lanes 2 and 7, 2b). The ability of OPTN(F178A) and NDP52(V136S) to drive CoxII degradation indicates that the LIR motif is not essential for mitochondrial clearance. However, the decrease in mitophagy levels observed for NDP52(V136S) pointed toward a defect in mitophagy efficiency. Given that mitophagy efficiency defects may only be apparent at earlier time points24, we utilised mtKeima to measure mitochondrial delivery to lysosomes during the first 3 h of OA incubation (Fig. 2c–h; Supplementary Fig. 1j). Indeed, LIR-mutant GFP-OPTN(F178A) was observed to have a significant defect in the rate of mitophagy (Fig. 2c–e), while GFP-NDP52(V136S) had a much greater mitophagy defect (Fig. 2f–h) consistent with the CoxII degradation data (Fig. 2a, b). Expression of GFP-p62 failed to rescue mitophagy in any of the conducted assays (Fig. 2b; Supplementary Fig. 1f, i). Thus, the LIR motif within OPTN and NDP52 functions to drive efficient mitochondrial clearance during PINK1/Parkin mitophagy.

Fig. 2 The LIR motif within OPTN and NDP52 is essential for efficient mitophagy. a, b Penta KO HeLa cells with or without untagged Parkin expression rescued by stable expression of the indicated GFP-tagged receptors were analysed by immunoblotting after 21 h incubation with OA (a) for quantification of the remaining CoxII levels (b). c–h, penta KO HeLa cells stably expressing mtKeima, untagged Parkin and either GFP-OPTN (c), GFP-OPTN(F178A) (d), GFP-NDP52 (f) or GFP-NDP52(V136S) (g), were imaged by live-cell confocal microscopy ((c, d, f, g); GFP-receptor channels provided in Supplementary Fig. 1g, h), and analysed by Fluorescence Activated Cell Sorting (FACS) to quantify the percentage of 561 nm mtKeima-positive cells (e, h), after time-course incubation with OA. (Representative FACS plots provided in Supplementary Fig. 1j; FACS analysis and representative images for penta KO & GFP-p62 provided in Supplementary Fig. 1e, f, i). Data in (b) are mean ± s.d. from four independent experiments. Data in (e, h) are mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (b one-way ANOVA; e, h two-way ANOVA). ns: not significant. Scale bars: 10 µm Full size image

To determine how the LIR motif might contribute to mitophagy efficiency, we analysed the stages of mitophagy following autophagy receptor recruitment. OPTN and NDP52 play a key role during autophagosome initiation by promoting the recruitment of the ULK1 complex14. We therefore quantified the formation of autophagosome initiation foci containing the ULK1 complex subunit Atg1329. Penta KO cells expressing WT or LIR-mutant autophagy receptors were immunolabelled for endogenous Atg13 after an OA treatment time-course. The analysis revealed that Atg13 foci in penta KO cells expressing GFP-OPTN(F178A) were significantly smaller than those in cells expressing WT GFP-OPTN (Fig. 3a–c), although the overall number of Atg13 foci was not significantly different (Fig. 3b). In contrast, cells expressing GFP-NDP52(V136S) contained both fewer and smaller Atg13 foci relative to cells expressing WT GFP-NDP52 (Fig. 3a, e, f). Recruitment of the initiation machinery to mitochondria by WT and LIR-mutant autophagy receptors was also analysed using subcellular fractionation (Supplementary Fig. 3f,g and i,j). Mitochondrial fractions from penta KO cells expressing OPTN(F178A) or NDP52(V136S) had reduced levels of the ULK1 complex subunit FIP200 relative to WT controls, corroborating the microscopy analyses. Consistent with the defects in Atg13 foci formation (Fig. 3a) and FIP200 recruitment (Supplementary Figure 3f–g and i–j), the recruitment of downstream autophagy effectors including WIPI2b (Supplementary Fig. 2a–c) and Atg16L1 (Supplementary Fig. 2d–f) were also defective in cells expressing LIR-mutant autophagy receptors. Penta KO cells either lacking autophagy receptor expression or expressing GFP-p62 failed to initiate mitophagy as demonstrated by a lack of Atg13 foci (Fig. 3a; Supplementary Fig. 2a, d). Importantly, our analyses of Atg13 foci also revealed that the intensity of GFP-OPTN(F178A) and GFP-NDP52(V136S) at phagophores was significantly reduced relative to WT GFP-OPTN (Fig. 3d) and GFP-NDP52 (Fig. 3g). Taken together, our results reveal that mutation of the LIR motif in OPTN and NDP52 causes an autophagosome formation defect at early stages of mitophagy including phagophore formation. In addition, the reduction of autophagy receptor intensity at phagophore formation sites indicates that mutation of the LIR motif affects events upstream and/or during autophagosome initiation.

Fig. 3 OPTN and NDP52 recruitment is influenced by LIR-mediated interactions with Atg8s. a Representative immunofluorescence images of penta KO HeLa cells stably expressing untagged Parkin rescued by expression of the indicated GFP-tagged receptors; immunostained for Atg13, HSP60 and GFP after treatment with OA for 3 h. (Equivalent images for TAX1BP1 & NBR1 are provided in Supplementary Fig. 5b). b–g Automated image analysis of the average Atg13 foci count per cell (b, e), Atg13 foci volume (c, f) and intensity of foci in the GFP channel (d, g), in penta KO HeLa cells stably expressing untagged Parkin, rescued by stable expression of either GFP-OPTN/GFP-OPTN(F178A) (b–d) or GFP-NDP52/GFP-NDP52(V136S) (e–g), immunostained for Atg13, HSP60 and GFP after time-course incubation with OA. (Data from other time points shown in Supplementary Fig. 2g-j). h, i, j, l Representative immunofluorescence images (h, i) and automated image analysis of GFP-tagged receptor translocation (j, l) in penta KO HeLa cells stably expressing untagged Parkin after rescue expression of either GFP-OPTN/GFP-OPTN(F178A) (h, j) or GFP-NDP52/GFP-NDP52(V136S) (i, l), immunostained for HSP60 and GFP after time-course incubation with OA (times indicated). k, m Automated image analysis of GFP-OPTN (k) or GFP-NDP52 (m) translocation in WT and hexa KO HeLa cells stably expressing untagged Parkin, after time-course incubation with OA and immunostaining for HSP60 & GFP. (Representative images for (k, m) provided in Supplementary Fig. 2q, r). Data in (b–g) and (j–m) are mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). ns: not significant. Scale bars: overviews, 10 µm; insets, 2 µm Full size image

We next asked whether the reduced intensity of LIR-mutant OPTN and NDP52 at autophagosome formation sites was caused by reduced autophagy receptor translocation. GFP-OPTN(F178A) (Fig. 3h, j) and NDP52(V136S) (Fig. 3i, l) translocated significantly slower than their wild-type counterparts. Mitochondrial translocation of NDP52(V136S) was particularly low, consistent with the greater mitophagy defect observed in penta KO cells expressing this mutant (Fig. 2). Defects in mitochondrial translocation of OPTN(F178A) and NDP52(V136S) were also detected via analysis of isolated mitochondrial fractions (Supplementary Fig. 3f–k). To confirm that reduced translocation of LIR-mutant OPTN and NDP52 was due to their inability to bind Atg8s, and to exclude the potential effect of protein alteration from mutated residues, we then assessed WT GFP-OPTN and GFP-NDP52 translocation rates in Atg8 hexa KO cells that lack all six LC3/GABARAPs (Fig. 3k, m; Supplementary Fig. 2q, r). The absence of Atg8 proteins severely impaired the translocation of both WT GFP-OPTN (Fig. 3k; Supplementary Fig. 2q) and GFP-NDP52 (Fig. 3m; Supplementary Fig. 2r), which displayed very similar translocation rates to the LIR mutants expressed in penta KO cells (Fig. 3j, l). Autophagy receptor translocation during PINK1/Parkin mitophagy is thought to be entirely dependent on binding to ubiquitin chains, with both OPTN and NDP52 previously shown to preferentially bind to ubiquitin chains phosphorylated at S65 by PINK114,17. However, analysis of pS65-Ub levels on mitochondria in WT and hexa KO cells confirmed that hexa KO cells did not have reduced levels of pS65-Ub chains (Supplementary Fig. 3a), thus eliminating the possibility that altered pS65-Ub levels had affected autophagy receptor translocation. Collectively, these results demonstrate that OPTN and NDP52 recruitment efficiency during PINK1/Parkin mitophagy is directly influenced by LIR-motif-mediated interactions with LC3s and/or GABARAPs.

LIR-mediated recruitment of autophagy receptors in mitophagy

OPTN and NDP52 recruitment to mitochondria during mitophagy is dependent on ubiquitin binding14,17,40. It is therefore unclear how Atg8s can influence OPTN and NDP52 recruitment. We hypothesised that following the initiation of autophagosome biogenesis, the LIR motif within OPTN and NDP52 may enable ubiquitin-independent recruitment of the autophagy receptors via binding to lipidated Atg8s (Fig. 4a; Supplementary Fig. 4a). Thus, we sought to determine whether the LIR motif was sufficient to mediate the translocation of autophagy receptors independently of ubiquitin binding. First, we analysed the localization of OPTN(D474N) and NDP52(C443K) UBD mutants in penta KO cells following PINK1/Parkin mitophagy activation (Fig. 4b). As expected, both mCherry(mCh)-NDP52(C443K) (Fig. 4c) and mCh-OPTN(D474N) (Fig. 4d) failed to translocate to mitochondria after 3 h OA incubation corroborating previous studies14,41.

Fig. 4 The LIR motif mediates autophagy receptor recruitment after initiation of autophagosome biogenesis. a Schematic of the proposed mechanism by which Atg8s influence receptor translocation, depicting wild-type receptor recruitment via ubiquitin and Atg8 proteins and LIR-mutant receptor recruitment exclusively via ubiquitin (the upstream stages of this model are in depicted in Supplementary Fig. 4a). b, e, h Model schematics depicting the predicted translocation outcomes in cells expressing a UBD mutant receptor alone (b), co-expressing a UBD mutant receptor and a wild-type receptor (e), or co-expressing a UBD/LIR double mutant receptor and a wild-type receptor (h). c, d, f, g, i, j Representative images of penta KO HeLa cells stably expressing untagged Parkin, with co-expression of either mCh-NDP52(C443K) (c), mCh-OPTN(D474N) (d), GFP-OPTN & mCh-NDP52(C443K) (f), GFP-NDP52 & mCh-OPTN(D474N) (g), GFP-OPTN and mCh-NDP52(V136S/C443K) (i), or GFP-NDP52 and mCh-OPTN(F178A/D474N) (j), after 3 h OA incubation and immunostaining for HSP60, GFP and mCherry. k, l Automated image analysis of mCherry translocation in the cell lines shown in (f and i) (k) or in (g and j) (l), after time-course incubation with OA and immunostaining for HSP60, mCherry & GFP. m, n, o Representative images (m) and automated image analysis of mCherry translocation (n) and GFP translocation (o) in the cell lines shown in (g and j), immunostained for HSP60, mCherry and GFP after 3 h incubation with OA in the presence or absence of 1 µM BX795. Data in (k, l, n, o) are mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). ns: not significant. Scale bars: overviews, 10 µm; insets, 2 µm Full size image

Next, we determined whether UBD mutant autophagy receptors can translocate via their LIR motif during phagophore formation by co-expressing a WT autophagy receptor (Fig. 4e). To prevent the effects of homodimerization between UBD mutant and WT autophagy receptors42, cells expressing mCh-OPTN(D474N) were rescued with GFP-NDP52 (Fig. 4g), while cells expressing mCh-NDP52(C433K) were rescued with GFP-OPTN (Fig. 4f). Indeed, the mCh-NDP52(C443K) UBD mutant could translocate to mitochondria when co-expressed with GFP-OPTN (Fig. 4f, k). Likewise, the mCh-OPTN(D474N) UBD mutant translocated to mitochondria in the presence of GFP-NDP52 (Fig. 4g, l). To confirm that the recruitment of UBD mutant autophagy receptors was LIR motif dependent, the experiment was also conducted in cells expressing mCh-OPTN(F178A/D474N) and mCh-NDP52(V136S/C443K) LIR/UBD double mutants (Fig. 4h–j). Consistent with our hypothesis, mCh-NDP52(V136S/C443K) failed to translocate in the presence of GFP-OPTN (Fig. 4i), and mCh-OPTN(F178A/D474N) translocation was significantly defective after 1 h and 2 h OA treatment in the presence of GFP-NDP52 (Fig. 4l). However, after 3 h of OA treatment, mCh-OPTN(F178A/D474N) translocation was observed (Fig. 4j, l). Equivalent translocation of mCh-OPTN(F178A/D474N) did not occur in the absence of GFP-NDP52 (Supplementary Fig. 3b).

NDP52 has been shown to recruit TANK binding kinase (TBK1)43, which is known to bind and phosphorylate OPTN17,24. We therefore asked whether the recruitment of TBK1 by GFP-NDP52 may account for the residual translocation of mCh-OPTN(F178A/D474N). Indeed, incubation with the TBK1 inhibitor BX795 abolished the residual recruitment of mCh-OPTN(F178A/D474N) (Fig. 4m, n), without influencing the recruitment of GFP-NDP52 (Fig. 4o). Previous work has shown that TBK1-mediated phosphorylation of OPTN’s UBD promotes mitochondrial recruitment17, while phosphorylation of the LIR governs Atg8 affinity40. The TBK1-dependent recruitment of mCh-OPTN(F178A/D474N) in Fig. 4m–o, indicates that TBK1 plays an additional role in mediating OPTN recruitment beyond LIR and UBD activity. Taken together, OPTN recruitment during mitophagy additionally involves TBK1 activity along with ubiquitin and Atg8 binding. In contrast, NDP52 relies on ubiquitin and Atg8, but not TBK1 for its recruitment. These results explain why NDP52(V136S) has a greater mitophagy defect than OPTN(F178A) (Fig. 2).

We next explored the functional significance of LIR-mediated autophagy receptor recruitment by asking if the mitophagy defects of LIR-mutant OPTN(F178A) and NDP52(V136S) (observed in Fig. 2), could be rescued via co-expression of a UBD mutant. First, we tested whether the UBD mutants can be recruited to phagophores when co-expressed with a LIR mutant (Fig. 5). As in Fig. 4, mutant OPTN and NDP52 were cross-combined to prevent the effects of homodimerization42. The GFP-OPTN(F178A) LIR mutant was combined with an mCh-NDP52(C443K) UBD mutant (Fig. 5a), and the GFP-NDP52(V136S) LIR mutant was combined with the mCh-OPTN(D474N) UBD mutant (Fig. 5c). Both mCh-NDP52(C443K) (Fig. 5a, b) and mCh-OPTN(D474N) (Fig. 5c, d) were recruited to mitochondria in foci that consistently co-localised with Atg13 labelled phagophores. Recruitment of the UBD mutants to mitochondrial phagophores was dependent on the LIR motif within OPTN and NDP52 (Supplementary Fig. 3c, d). Ubiquitin-independent autophagy receptor translocation via the LIR motif was also observed with p62 (Supplementary Fig. 5c–e), TAX1BP1 (Supplementary Fig. 5f–h), and NBR1 (Supplementary Fig. 5i–k).

Fig. 5 LIR-motif-mediated recruitment of autophagy receptors is required for efficient PINK1/Parkin mitophagy. a, c Representative images of penta KO cells stably expressing untagged Parkin, with co-expression of either GFP-OPTN(F178A) & mCh-NDP52(C443K) (a), or GFP-NDP52(V136S) & mCh-OPTN(D474N) (c), immunostained for Atg13, HSP60, mCherry and GFP after 3 h incubation with OA. (Equivalent representative images for mCh-OPTN(F178A/D474N) provided in Supplementary Fig. 3b). b, d automated image analysis of mCherry translocation in the cell lines shown in (a) (b) and in (c) (d) after time-course incubation with OA and immunostaining for HSP60, mCherry and GFP. e, f Penta KO HeLa cells stably expressing untagged Parkin rescued by stable expression the indicated receptor combinations were analysed by immunoblotting after 8 h incubation with OA (e), for quantification of the remaining CoxII levels (f). g FACS analysis of the percentage of 561 nm mtKeima-positive cells in penta KO HeLa cells stably expressing mtKeima, untagged Parkin and GFP-OPTN(F178A) alone, or with co-expression of either iRFP670-NDP52(C443K) or iRFP670-NDP52(V136S/C443K). (representative FACS plots for mtKeima and iRFP670 expression provided in Supplementary Fig. 1k). h In silico modelling of mitophagy using lipidated Atg8 as the surrogate output variable, under wild-type NDP52 or the LIR-mutant NDP52(V136S). (See Methods for details; Supplementary Tables 1–2 for modelled reactions; Supplementary Fig. 4 for overview of the model). i The proposed model of LIR-mediated autophagy receptor coalescence in which autophagy receptors are initially recruited by S65 phospho-ubiquitin. The post-initiation lipidation of Atg8s to the expanding phagophore subsequently recruits more autophagy receptors via their LIR motif. The additional autophagy receptors accelerate autophagosome biogenesis to generate a positive feedback loop. j Representative images of methanol fixed penta KO cells stably expressing untagged Parkin with co-expression of GFP-OPTN(F178A) & mCh-NDP52(C443K), immunostained for LC3A/B, HSP60, GFP and mCherry after 1 h OA treatment in the presence or absence of 1 µM wortmannin. Data in (b, d, f, g) are mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). ns: not significant. Scale bars: overviews, 10 µm; insets, 2 µm Full size image

Next, we determined whether the recruitment of UBD mutant autophagy receptors was sufficient to rescue the mitophagy defects identified for the LIR mutants OPTN(F178A) and NDP52(V136S) (Fig. 2). The mitophagy defect of GFP-NDP52(V136S) was significantly rescued by co-expression with mCh-OPTN(D474N) but not mCh-OPTN(F178A/D474N), whereas expression of the mCh-OPTN UBD mutants alone did not contribute to CoxII degradation (Fig. 5e, f). In addition, the GFP-NDP52(V136S) mitophagy defect was not rescued by co-expression of mCh-p62(ΔUBD), mCh-TAX1BP1(ΔUBD) or mCh-NBR1(ΔUBD) (Supplementary Fig. 5m, n). The mitophagy efficiency defect of OPTN(F178A) was detected using the mtKeima assay (Fig. 2c–e), therefore this assay was also used for rescue experiments (Supplementary Fig. 1k). The reduced mitophagy efficiency observed for GFP-OPTN(F178A) was significantly rescued by co-expression of iRFP670-NDP52(C443K), but not by the iRFP670-NDP52(V136S/C443K) LIR/UBD double mutant (Fig. 5g). These results demonstrate that LIR-mediated recruitment of OPTN and NDP52 following phagophore formation is critical for efficient PINK1/Parkin mitophagy.

A positive feedback model of selective autophagy

Our results point toward a model in which OPTN and NDP52 are initially recruited to mitochondria via their UBD. After their recruitment, OPTN and NDP52 promote the recruitment and activation of the ULK1 complex14. Subsequent activation of the phosphoinositide 3-kinase complex by ULK1 stimulates local production of PtdIns(3)P at the phagophore nucleation site29, which recruits WIPI2b34. Recruitment of the Atg8 conjugation machinery (Atg12-Atg5-Atg16L1 complex) by WIPI2b defines the site of LC3/GABARAP lipidation33. The Atg8-positive phagophores then recruit additional OPTN and NDP52 via LIR-mediated interactions, which drives further rounds of ULK1 translocation and Atg8 lipidation. This forms an Atg8-dependent positive feedback loop that amplifies the rate of autophagosome biogenesis. The positive feedback loop is lost in the absence of LIR-mediated interactions between autophagy receptors and LC3/GABARAPs (Fig. 5i). This model also provides a mechanistic explanation for the reduced autophagosome size and reduced autophagosome formation efficiency observed during PINK1/Parkin mitophagy in cells lacking Atg8s37,38.

To test the positive feedback model, we generated a computational model of PINK1/Parkin activation and autophagosome formation. The in silico model combines the PINK1/Parkin pS65-Ub-dependent positive feedback loop8,9, and the LIR-dependent positive feedback loop identified in this study. A mathematical kinetic model of autophagy initiation by NDP52 was formulated using a system of 21 ordinary differential equations (ODEs) with starting conditions and rate constants derived from empirical time-course data (Figs 2, 3, 4). The model scheme is shown in Supplementary Fig. 4 and a detailed model description is presented in Methods. The system of ODEs (Supplementary Tables 1–2) was then numerically solved using Mathematica (Wolfram Research) to quantify total lipidated Atg8 (Atg8PE), which was the surrogate model variable chosen to represent mitophagy output of the system. Upon simulated depolarization of mitochondria (reaction 4), the modelled numerical outputs recapitulated the switch-like response of mitophagy induced by WT NDP52 over time (compare Fig. 5h with 2 h). In contrast, the modelled outputs during mitophagy induction by NDP52(V136S) lacked the positive feedback loop and had significantly diminished mitophagy (Fig. 5h). The PINK1/Parkin mitophagy simulations demonstrated a high degree of robustness, as random perturbation of reaction rate constants had a limited effect on the outcomes (Supplementary Fig. 4d). Taken together, the high level of agreement between in silico model simulations and the biological data strongly support the presence of a positive feedback loop mediated by Atg8s and autophagy receptor LIR binding (Fig. 5i). According to the positive feedback model (Fig. 5i), phosphoinositide 3-kinase (PI3K) inhibitors should block LIR-mediated recruitment of selective autophagy receptors by preventing Atg8 lipidation at initiation sites28,29,31. To test the model, the translocation of mCh-NDP52(C443K) (Fig. 5j) and mCh-OPTN(D474N) (Supplementary Fig. 5a) was assessed in the presence of the PI3K inhibitor Wortmannin (Fig. 5j; Supplementary Fig. 5a). Indeed, wortmannin treatment blocked LIR-mediated OPTN and NDP52 recruitment (Fig. 5j; Supplementary Fig. 5a), without affecting upstream ubiquitin-dependent recruitment of LIR-mutant autophagy receptors (Fig. 5i).

In summary, we have discovered a ubiquitin-independent mechanism of autophagy receptor recruitment which amplifies PINK1/Parkin mitophagy signalling by OPTN and NDP52. We argue that cargo selectivity during PINK1/Parkin mitophagy is achieved via de novo autophagosome biogenesis on the surface of mitochondria14, but not through LIR-mediated bridging with Atg8s on autophagic membranes. This is supported by reports showing that cells lacking Atg8s successfully sequester mitochondria within autophagosomes38, and LC3/GABARAP-lipidation-deficient cells contain autophagic membranes surrounding mitochondria44. By clarifying the role of the LIR motif in mitophagy, we have identified an autophagy associated positive feedback loop that drives efficient mitophagy. PINK1/Parkin mitophagy is therefore regulated by two independent but complementary ubiquitin(-like) positive feedback loops. The first is dependent on phospho-ubiquitin generated by PINK1/Parkin8,9. The second is dependent on the family of ubiquitin-like Atg8 proteins that are lipidated by the E3 ligase-like Atg12-Atg5-Atg16L1 complex33,34. Together, the two positive feedback loops enable OPTN and NDP52 to promote rapid clearance of damaged mitochondria, which would help prevent the release of mitochondrial factors that trigger inflammation and cell death45,46.