Identification of the Bok Interactome Using Proximity Labeling.

Laura M Szczesniak,Caden G Bonzerato,Richard J H Wojcikiewicz

doi:10.3389/fcell.2021.689951

Laura M Szczesniak, Caden G Bonzerato + Show 1 more

Open Access

https://doi.org/10.3389/fcell.2021.689951

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Abstract

The function of the Bcl-2 family member Bok is currently enigmatic, with various disparate roles reported, including mediation of apoptosis, regulation of mitochondrial morphology, binding to inositol 1,4,5-trisphosphate receptors, and regulation of uridine metabolism. To better define the roles of Bok, we examined its interactome using TurboID-mediated proximity labeling in HeLa cells, in which Bok knock-out leads to mitochondrial fragmentation and Bok overexpression leads to apoptosis. Labeling with TurboID-Bok revealed that Bok was proximal to a wide array of proteins, particularly those involved in mitochondrial fission (e.g., Drp1), endoplasmic reticulum-plasma membrane junctions (e.g., Stim1), and surprisingly among the Bcl-2 family members, just Mcl-1. Comparison with TurboID-Mcl-1 and TurboID-Bak revealed that the three Bcl-2 family member interactomes were largely independent, but with some overlap that likely identifies key interactors. Interestingly, when overexpressed, Mcl-1 and Bok interact physically and functionally, in a manner that depends upon the transmembrane domain of Bok. Overall, this work shows that the Bok interactome is different from those of Mcl-1 and Bak, identifies novel proximities and potential interaction points for Bcl-2 family members, and suggests that Bok may regulate mitochondrial fission via Mcl-1 and Drp1.

Highlights

The Bcl-2 family mediates the intrinsic apoptosis pathway through the coordinated actions of pro- and anti-apoptotic proteins (Kale et al, 2018)
As Bcl-2 related ovarian killer (Bok) deletion causes mitochondrial fragmentation, we focused on mitochondrial proteins
Several studies report that mitochondria-ER contact site (MERC) are essential for numerous signaling processes, including Ca2+ transfer, lipid trafficking/metabolism, and regulation of cell death or survival (Perrone et al, 2020), and interestingly, a recent study suggests that Bok is integral to the stability of MERCs/mitochondrial-associated membrane (MAM) (Carpio et al, 2021)

Summary

Introduction

The Bcl-2 family mediates the intrinsic apoptosis pathway through the coordinated actions of pro- and anti-apoptotic proteins (Kale et al, 2018). The pro-apoptotic proteins include Bax and Bak, which mediate the release of cytochrome c from mitochondria via mitochondrial outer membrane permeabilization (MOMP), an effect opposed by the anti-apoptotic proteins Bcl-2, Mcl, and Bcl-xL. Bok “knock-out” (KO) from mouse embryonic fibroblasts (MEFs) causes mitochondrial fragmentation, which can be rescued by re-introduction of Bok (Schulman et al, 2019). This phenotype is intriguing, given that Bok is predominantly endoplasmic reticulum (ER)-localized (Echeverry et al, 2013) and constitutively bound to inositol 1,4,5trisphosphate receptors (IP3Rs) (Schulman et al, 2013, 2016), which are tetrameric channels that release Ca2+ from ER stores. Bok has been reported to protect IP3Rs from proteolysis (Schulman et al, 2013), mediate ER stress-induced apoptosis (Carpio et al, 2015), and positively regulate uridine metabolism (Srivastava et al, 2019)

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