Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

Abstract

Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.

Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context

“…TMEM256 presumably localizes to the inner membrane ( Kustatscher et al., 2019 ), and its complexome profile supports an interaction with the TIM23 core complex ( Figure S6 K). NCBP2-AS2 displays a high molecular mass form similar to PAM16 (TIMM16), DNAJC19 (PAM18/TIMM14), TIMM21, the complex III assembly factor OCIAD1 ( Le Vasseur et al., 2021 ), the mitochondrial disease gene DNAJC30 ( Figure S6 L; Richter-Dennerlein et al., 2014 ; Tebbenkamp et al., 2018 ), the prohibitin protein family ( Figure S6 F), and the high molecular mass form of TIM23 ( Figure S6 M). The TIM23 complex can perform two different functions, distinguished by its association with the presequence translocase-associated motor (PAM) ( Chacinska et al., 2005 , 2010 ; Mick et al., 2012 ).…”

Applying Sodium Carbonate Extraction Mass Spectrometry to Investigate Defects in the Mitochondrial Respiratory Chain

“…Using a number of additional bioinformatics tools for predicting TMDs, including TMPred ( Hofmann, 1993 ) and HMMTOP (Bioinformatics Toolkit, RRID:SCR_010277) ( Tusnády and Simon, 2001 ), as well as TMHMM (RRID:SCR_014935) ( Krogh et al, 2001 ) and Phobius (RRID:SCR_015643) ( Käll et al, 2004 ) without the overlap constraint used by UniProt, we identified an additional 30 proteins that had TMDs identified by at least two algorithms. A number of these proteins have been experimentally determined to be genuine membrane proteins, including complex I subunit NDUFB2 through high resolution structural studies ( Zhu et al, 2016 ) recently identified complex III assembly factor OCIAD1 through classical carbonate extraction studies ( Le Vasseur et al, 2021 ) and pro-survival protein FUNDC2 through mutation and localization analysis ( Ma et al, 2019 ).…”

A Conserved Role for Asrij/OCIAD1 in Progenitor Differentiation and Lineage Specification Through Functional Interaction With the Regulators of Mitochondrial Dynamics

“…While canonical Notch signaling leads to crystal cell differentiation, non-canonical activation of Notch pathway due to stalling in endosomes of Asrij mutants ( Kulkarni et al, 2011 ) may also contribute to the synergistic effects on phenotype. Asrij/OCIAD1 acts as a transmembrane scaffolding protein that regulates assembly and activation of critical signaling components and molecular complexes across organelles ( Sinha et al, 2013 ; Le Vasseur et al, 2021 ). A recent report shows that OCIAD1 is a client of Prohibitin supramolecular complex that acts as a scaffold for the assembly of mitochondrial electron transport chain Complex III in HEK293T and U2OS cells ( Le Vasseur et al, 2021 ).…”

“…Asrij/OCIAD1 acts as a transmembrane scaffolding protein that regulates assembly and activation of critical signaling components and molecular complexes across organelles ( Sinha et al, 2013 ; Le Vasseur et al, 2021 ). A recent report shows that OCIAD1 is a client of Prohibitin supramolecular complex that acts as a scaffold for the assembly of mitochondrial electron transport chain Complex III in HEK293T and U2OS cells ( Le Vasseur et al, 2021 ). Hence, Asrij may potentially act as mediator of inter-organellar communication that may influence blood cell homeostasis.…”