Miro-1 calcium sensing EF hands are required for Parkin recruitment to mitochondria Džamilja Safiulina, Vinay Choubey, Malle Kuum, Nana Gogichaishvili, Allen Kaasik Department of Pharmacology, University of Tartu, ESTONIA
Mitochondrial outer membrane protein Miro1 interacts with E3 ligase Parkin upon mitochondrial membrane depolarization. Miro1 protein possesses two GTPase domains and two Ca2+-binding EF hands, which upon Ca2+ binding undergoes a conformational change that might affect the affinity for its binding partners involved in mitochondrial trafficking. We therefore studied whether previously described double mutation E208K and E328K in Miro EF hands (Miro1-EF) will affect Parkin interaction with Miro1. Our results demonstrate that Miro1-EF interacts with Parkin (both overexpressed and endogenous) in lesser extent than wild type Miro1. This was evident in control conditions and when the cells were treated with antimycin and oligomycin. Further, we found that Miro1-EF is not ubiquitinated in the same extent as wild type Miro1 in response to antimycin and oligomycin. However, our results show that Miro1-EF mutant is degraded similarly to wild type upon mitochondrial depolarization. We also found that Miro1-EF overexpression decreases Parkin-YFP translocation to mitochondria in response to antimycin and oligomycin. We further followed the kinetics of Parkin-YFP translocation from cytosol to mitochondria and found that it is significantly slower in Miro1-EF group. This result was consistent in different cell types and different experimental damage (antimycin and oligomycin, laser irradiation). We next tested whether the pathological conditions increasing Ca2+ concentration could induce or affect Parkin translocation in neurons. Our results show modest increase in number of Parkin-YFP positive mitochondria in glutamate treated neurons after glutamate washout. Indeed, Miro1-EF mutant suppressed glutamate induced Parkin translocation in approximately half of treated neurons. Thus, we suggest that calcium binding to Miro1 EF-hands is not only regulating mitochondrial movement but might work also as switch for Parkin recruitment to mitochondria also in (patho)physiological conditions.
Funding. This work was supported by grants from the Estonian Research Council (IUT2-5), the European Regional Development Fund (Project No. 2014-2020.4.01.15-0012) and from the European Union's Horizon 2020 research and innovation programme under grant agreement 692202. VC was supported by grant PUT513 from the Estonian Research Council.