Defining the Determinants of the Bok-IP3R Interaction and the Bok Interactome

Abstract

Bok is a Bcl-2 protein family member that is often grouped with the pro-apoptotic family members Bax and Bak due to high sequence homology and because exogenously overexpressed Bok induces apoptosis by causing mitochondrial outer membrane permeabilization. However, the cellular roles of Bok remain unclear, as Bok KO cell lines and mice have failed to demonstrate a significant phenotype under normal conditions. Our lab discovered that Bok interacts with inositol 1,4,5-trisphosphate receptors (IP3Rs), tetrameric Ca2+ channels found in the ER membrane of mammalian cells that play an integral role in cell signaling. While other Bcl-2 family members have been reported to weakly interact with IP3Rs, the Bok-IP3R interaction is much more efficient, with essentially all cellular Bok constitutively bound to IP3Rs. We have generated full-length IP3R1 mutants that resolve the Bok-binding region to a small, unstructured loop in the cytosolic region of IP3R1 between _ helices 72 and 73. Additional bioinformatic analysis has revealed that the Bok-IP3R interaction is likely dependent upon helical and dynamic determinants within this loop. Interestingly, Bok KO cell lines demonstrate mitochondrial fragmentation and only minor changes in mitochondrial bioenergetics. We have investigated the role of Bok in mitochondrial dynamics through an in vivo proximity labeling technique known as TurboID. A TurboID-Bok fusion protein expressed in Bok KO HeLas has identified several mitochondrial fission proteins through mass spectroscopy analysis, suggesting that Bok acts at mitochondria-ER contact sites to inhibit fission, and this function may be independent of the Bok-IP3R interaction. The results provided from binding studies and proximity labeling have furthered our knowledge of the Bok-IP3R interaction and of Bok itself to better define, or re-define, the role of Bok within the cell. Understanding how and why these interactions occur will help us further understand fundamental cellular processes in health and human disease.