Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

10-POS Board 10 NUCLEAR RECRUITMENT OF DRP6 IS MEDIATED BY CARDIOLIPIN INTERACTION AND REGULATED BY POST-TRANSLATIONAL MODIFICATION Himani Dey 1 ; Usha P Kar 1 ; Abdur Rahaman 1 ; 1 National Institute of Science Education and Research, HBNI, School of Biological Sciences, Bhubaneswar, Orissa, India Dynamins and dynamin related proteins are a large GTPases involved in various cellular processes including vesicle scission, fission and fusion of organelles, vacuolar trafficking and viral resistance. Dynamin Related Protein 6 (Drp6) in Tetrahymena localizes on the nuclear envelope (NE) and as cytoplasmic punctae, and is required for nuclear remodelling during macronucleus development. In this study, we report the mechanism of nuclear recruitment of Drp6 and its regulation. Using a lipid overlay assay with various deletion fragments of Drp6, the Lipid Binding Domain was mapped at the position equivalent to the Pleckstrin Homology (PH) domain of dynamin. Using a point mutation (I553M) in the lipid binding domain, we have demonstrated that the mutant fails to associate with nuclear envelope. Various biophysical and biochemical analysis show that the loss of nuclear recruitment is due to a defect in its interaction with cardiolipin and not in its self-assembly, ultra-structure or GTPase activity. This was further confirmed in vivo by depleting cardiolipn in cells expressing GFP-tagged Drp6. Drp6 has differential localisation at different developmental stages. To investigate if post-translational modification is involved in regulation of differential localisation, Drp6 expressed in Tetrahymena was purified and subjected to mass-spectromertric analysis. Four Serine residues (at positions 86,248,701,705) were found to be phosphorylated. Our cellular and biochemical studies show that phosphorylation at Ser248 prolongs its nuclear association, inhibits its GTPase activity and enhances its affinity for cardiolipin enriched membrane without affecting its self-assembly. These results suggest that phosphorylation at Ser248 regulates nuclear recruitment by affecting its GTPase activity and membrane interaction.

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