Fig. 5: ANKZF1 sequence contains six putative LC3-Interacting Regions (LIRs), and LIR-4 (333-336 residues) is indispensable for LC3-interaction.

A Multiple sequence analysis (MSA) of some of the known mitophagy/autophagy adaptor/receptor proteins shows the presence of a conserved LIR (LC3 interacting region) motifs. B ANKZF1 sequence analysis shows the presence of six different probable LIR motifs consisting of similar sequence like known adaptors/receptors, named as LIR1 to LIR6. C MSA of ANKZF1 sequence from different mammals shows the presence of conservation of LIR2, LIR3, LIR4 and LIR6. D Schematic overview to depict the distribution of 6 LIR-like motifs in ANKZF1 full-length protein sequence, three truncation mutants of ANKZF1 (Δ210, Δ330, Δ370) were designed to screen the probable functional LIR motifs crucial for interaction with LC3. E ANKZF1 recruitment and interaction with LC3 during PMD-induced mitochondrial proteotoxic stress were checked after expressing the truncation mutants of ANKZF1. ANKZF1-FL shows punctate foci that co-localize with RFP-LC3 puncta during proteotoxic stress (panel E, upper row). Δ210-ANKZF1, Δ330-ANKZF1 truncation mutants also show similar behaviour and significantly co-localize with LC3 puncta (panel E, second and third rows from the top) like the full-length protein. Δ370-ANKZF1 shows a diffused cytosolic pattern, indicating no interaction with LC3. Line-scan profile of fluorescence intensity of RFP-LC3 puncta and WT-ANKZF1/ Δ210-ANKZF1/Δ330-ANKZF1 puncta shows merged intensity peak, suggesting co-localization. In contrast, Δ370-ANKZF1-GFP puncta and RFP-LC3 puncta peaks do not merge, suggesting the absence of interaction. ANKZF1 peaks are shown in green and LC3 peaks are shown in red. F Pearson’s correlation coefficient of co-localization was calculated and plotted for LC3 puncta and ANKZF1 (WT-ANKZF1, Δ210-ANKZF1, Δ330-ANKZF1, and Δ370-ANKZF1) puncta during Mito-PMD BFP induced stress conditions, Values represent means ± SEM, N = 3, data did not follow a normal distribution, Kruskal-Wallis a non-parametric test with Dunn’s multiple comparison tests was performed to determine the mean differences, ****P < 0.0001. G ANKZF1 LIR mutants, F333A-L336A, W366A-V369A, and W495A-L498A fused with GFP were co-transfected with RFP-LC3 in HeLa cells along with PMD-BFP. W366A-V369A and W495A-L498A mutants of ANKZF1 show interaction and co-localization with RFP-LC3, like the wild-type ANKZF1 (middle and lower panel). In contrast, the F333A-L336A mutant of ANKZF1 does not show any co-localization with RFP-LC3 during PMD-induced stress (top panel). Line-scan profile of fluorescence intensity of mutant ANKZF1-GFP and RFP-LC3 puncta shows merged intensity peak, suggesting co-localization.