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Selective autophagy of mitochondria, mitophagy, is linked to mitochondrial quality control and as such is critical to a healthy organism. We have used a CRISPR/Cas9 approach to screen human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and upon acute mitochondrial depolarization. We identify two cullin‐RING ligase substrate receptors, VHL and FBXL4, as the most profound negative regulators of basal mitophagy. We show that these converge, albeit via different mechanisms, on control of the mitophagy adaptors BNIP3 and BNIP3L/NIX. FBXL4 restricts NIX and BNIP3 levels via direct interaction and protein destabilization, while VHL acts through suppression of HIF1α‐mediated transcription of BNIP3 and NIX. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study contributes to an understanding of the aetiology of early‐onset mitochondrial encephalomyopathy that is supported by analysis of a disease‐associated mutation. We further show that the compound MLN4924, which globally interferes with cullin‐RING ligase activity, is a strong inducer of mitophagy, thus providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction. Synopsis: Mitophagy is tightly regulated upon acute mitochondrial depolarization, but its control under basal conditions is less understood. Here, a screen identifies two cullin‐RING ubiquitin ligase adaptors, FBXL4 and VHL, as negative regulators of Parkin‐independent mitophagy, downregulating the common target NIX via distinct mechanisms. A CRISPR‐based screen of E3 ubiquitin ligases identifies cullin‐RING ligase substrate adaptors, FBXL4 and VHL, as strong suppressors of Parkin‐independent mitophagy.Both FBXL4 and VHL limit expression levels of mitophagy receptors NIX and BNIP3.VHL exerts a well‐established transcriptional control of BNIP3 (and NIX) via destabilization of HIF1α, whereas FBXL4 directly ubiquitylates and decreases protein stability of NIX.NIX is the main mediator of enhanced mitophagy observed in FBXL4‐deficient cells.The cullin‐RING ligase inhibitor MLN4924 can be used to enhance mitophagy. [ABSTRACT FROM AUTHOR]

The spindle assembly checkpoint (SAC) restrains anaphase until all chromosomes become bi-oriented on the mitotic spindle. The SAC protein Mad2 can fold into two distinct conformers, open (O) and closed (C), and can asymmetrically dimerize. Here, we describe a monoclonal antibody that specifically recognizes the dimerization interface of C-Mad2. This antibody revealed several conformation-specific features of Mad2 in human cells. Notably, we show that Mad2 requires association with Mad1 to adopt the closed conformation and that the activity of the Mad1:C-Mad2 complex undergoes regulation by p31comet-dependent 'capping'. Furthermore, C-Mad2 antibody microinjection caused an abrupt termination of the SAC and accelerated mitotic progression. Remarkably, microinjection of a Mad1-neutralizing antibody triggered a comparable mitotic acceleration. Our study provides direct in vivo evidence for the model that a kinetochore complex of Mad1:C-Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer. [ABSTRACT FROM AUTHOR]