SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy

Abstract

The stress-responsive mitochondrial sirtuin SIRT4 controls cellular energy metabolism in a NAD+-dependent manner and is implicated in cellular senescence and aging. Here we reveal a novel function of SIRT4 in mitochondrial morphology/quality control and regulation of mitophagy. We report that moderate overexpression of SIRT4, but not its enzymatically inactive mutant H161Y, sensitized cells to mitochondrial stress. CCCP-triggered dissipation of the mitochondrial membrane potential resulted in increased mitochondrial ROS levels and autophagic flux, but surprisingly led to increased mitochondrial mass and decreased Parkin-regulated mitophagy. The anti-respiratory effect of elevated SIRT4 was accompanied by increased levels of the inner-membrane bound long form of the GTPase OPA1 (L-OPA1) that promotes mitochondrial fusion and thereby counteracts fission and mitophagy. Consistent with this, upregulation of endogenous SIRT4 expression in fibroblast models of senescence either by transfection with miR-15b inhibitors or by ionizing radiation increased L-OPA1 levels and mitochondrial fusion in a SIRT4-dependent manner. We further demonstrate that SIRT4 interacts physically with OPA1 in co-immunoprecipitation experiments. Overall, we propose that the SIRT4-OPA1 axis is causally linked to mitochondrial dysfunction and altered mitochondrial dynamics that translates into aging-associated decreased mitophagy based on an unbalanced mitochondrial fusion/fission cycle.

Subcellular localization and mitotic interactome analyses identify SIRT4 as a centrosomally localized and microtubule associated protein

“…S3). Besides its extramitochondrial/centrosomal localization, SIRT4 was observed as described 23,42 in mitochondria using a co-staining against the mitochondrial marker MTC02 (Fig. S4).…”

“…During our studies on the expression and mitochondrial function of SIRT4 40,42,43 we noticed an extra-mitochondrial localization of endogenous SIRT4 at centrosomes and in part at the mitotic spindle in confocal laser scanning and spinning disk microscopy-based analyses of various human cell lines. We employed two independent anti-human SIRT4 antibodies, SAB1407208 (Sigma-Aldrich) raised against full-length human SIRT4 (a.a.1-314) and H-234 (sc-135053, Santa Cruz Biotechnology) raised against a N-terminally truncated version of human SIRT4 (a.a. 81-314).…”

“…S4). Similar to endogenously expressed SIRT4, C-terminal eGFP fusion proteins of SIRT4 and SIRT4(ΔN28), the latter representing an N-terminally (a.a. 1-28) truncated SIRT4 mutant unable to translocate into mitochondria 42,45 , were also detected at interphase centrosomes of HeLa cervix carcinoma and HT1080 fibrosarcoma cells ( Fig. S6 and suppl.…”

“…Protein network analyses revealed several known (e.g. DNA damage response 37 ; mitochondrial respiratory chain components and glutamate metabolism regulators 47 ; regulation of mitochondrial organization 42,43 ) as well as novel functions and components associated with the mitotic SIRT4 interactome (e.g tRNA aminoacylation and mitochondrial translation; cell cycle regulation; microtubule regulation) ( Fig. S11).…”

“…Immunoprecipitation of GFP fusion proteins using the anti-GFP nanobody or standard immunoprecipitation protocols. The single-domain-anti-GFP antibody ("nanobody") method 76 was employed to immunoprecipitate SIRT4-eGFP fusion proteins essentially as described 42 . Coimmunoprecipitation of -tubulin interacting proteins was performed from total cell lysates using -Tubulin specific antibodies (rabbit anti--tubulin, ab52899, Abcam) and Protein A/G Sepharose beads (Santa Cruz Biotechnology).…”