SESN2 facilitates mitophagy by helping Parkin translocation through ULK1 mediated Beclin1 phosphorylation

Mitophagy, the selective degradation of mitochondria by autophagy, is crucial for the maintenance of healthy mitochondrial pool in cells. The critical event in mitophagy is the translocation of cytosolic Parkin, a ubiquitin ligase, to the surface of defective mitochondria. This study elucidates a novel role of SESN2/Sestrin2, a stress inducible protein, in mitochondrial translocation of PARK2/Parkin during mitophagy. The data demonstrates that SESN2 downregulation inhibits BECN1/Beclin1 and Parkin interaction, thereby preventing optimum mitochondrial accumulation of Parkin. SESN2 interacts with ULK1 (unc-51 like kinase 1) and assists ULK1 mediated phosphorylation of Beclin1 at serine-14 position required for binding with Parkin prior to mitochondrial translocation. The trigger for SESN2 activation and regulation of Parkin translocation is the generation of mitochondrial superoxide. Scavenging of mitochondrial superoxide lower the levels of SESN2, resulting in retardation of Parkin translocation. Importantly, we observe that SESN2 mediated cytosolic interaction of Parkin and Beclin1 is PINK1 independent but mitochondrial translocation of Parkin is PINK1 dependent. Together, these findings suggest the role of SESN2 as a positive regulator of Parkin mediated mitophagy.

A. HeLa cells stably expressing YF-tagged Parkin were transfected with scrambled siRNA and three different siRNA targeting SESN2 #1, #2, #3 and colocalization of YFP-Parkin with TOMM20 was scored after counting > 100 cells per condition in three independent experiments. CCCP treatment (3 h). Error bars represent the mean ± SEM (n=3). * p < 0.01. ## p < 0.005 B. HeLa cells stably expressing YFP-Parkin were transfected with scrambled siRNA and siRNA targeting SESN2 and treated with 10 µM CCCP for 3 h. Colocalization of YFP tagged Parkin with TOMM20 was scored after counting >100 cells per condition in three independent experiments. Error bars in the graph represents mean ± SEM (n=3). * p < 0.005 C. FLAG-tagged SESN2 was overexpressed in cells and immunoblot analysis was done to measure the percentage of overexpression. H. HeLa cells stably expressing with mito-Keima were transfected with myc-Parkin and treatment with 10µM CCCP and 5µM O/A was given for 12 h. Green (458 nm, excitation) and red (560, excitation) signal was acquired and represented as green/red signal. # p < 0.01; compared to DMSO (si Cont.), * p < 0.05; compared to DMSO (si SESN2), $ P < 0.05; compared to CCCP or O/A treatment (si SESN2), NS, non-significant; compared to DMSO (si Cont.) I. Hela cells were treated with 10 µM CCCP and 5 µM Oligomycin + 5 µM Antimycin A for 2 h in wild-type and SESN2 knockdown cells and total Annexin +ve population (Annexin +ve and Annexin +ve / +Pi+ was measured by flow cytometry. # p < 0.05, compared to DMSO (si Cont.), * p < 0.01, compared to DMSO (si SESN2.) J. Wild type and SESN2 knockdown Hela cells stably expressing YFP-Parkin were treated with 10 µM CCCP and 5 µM Oligomycin + 5 µM Antimycin A for 2 h and SESN2 knockdown cells were sorted for Annexin +ve signal (PE-conjugated). Mitochondrial fractions were isolated and translocation of YFP-Parkin was measured. Red, Annexin +ve , Black , Annexin -ve in SESN2 knockdown panel.

Fig S3
A. HeLa cells stably expressing YFP-Parkin were treated with 10 µM CCCP (2h) and mito-Tempo, percentage of YFP-Parkin co-localizing with mitochondrial marker TOMM20 was assessed. The graph represents mean ± SEM of counts in >100 cells per sample in three independent experiments. B. HeLa cells stably expressing YFP-Parkin were treated with 40 µM Antimycin A (2 h) and mito-Tempo, percentage of YFP-Parkin co-localizing with mitochondrial marker TOMM20 was assessed. The graph represents mean ± SEM of counts in >100 cells per sample (n=3).
C. Similar to fig. S3A, HeLa cells stably expressing YFP-Parkin were incubated with 5mM Paraquat (2 h) and mito-Tempo, co-localization of YFP-Parkin and TOMM20 was scored and represented in graph as mean ± SEM of counts in >100 cells per sample (n =3).
D. HEK293T cells were treated with 10 µM CCCP, 500 nM Rapamycin and 500 nM Everolimus for 3 h. Analysis of MTOR inhibition and SESN2 levels was done using immunoblotting.
E. Results obtained in fig. 5D were confirmed by analysing isolated mitochondrial fraction from HEK293T cells by immunoblotting using antibodies as indicated. TOMM20, mitochondrial fraction and Actin, cytosolic marker.

Fig S4
A. Knockdown of SESN2 and PINK1 was performed in HEK293T cells and analysed by immunoblotting using antibodies as indicated.
B. Graphical representation of intensity of SESN2 and PINK1 protein bands, obtained in Fig. 6C. Error bars represent as mean ± SEM (n=3).
C. Cells were transfected with PINK1 siRNA, plasmids expressing PINK1 kinase dead domain and wild type PINK1. Post-transfection cells were treated with DMSO (vehicle) and CCCP for 3 h and mitochondrial fractions were isolated for immunoblot analysis.
D. YFP-Parkin co-localization with TOMM20 in HeLa cells stably expressing YFP-Parkin was scored after counting >100 cells per condition in three independent experiments. The error bars in the graph represent mean ± SEM.