Abstract 4529: Mapping the protein interactome of mitochondrial intermembrane space proteases identifies a novel function for HTRA2

Mitochondria possess unique proteases that localize to specific sub-compartments of the organelle. However, the functions of these proteases are largely ill-defined. Here, we used proximity-dependent biotinylation (BioID) to map the interactomes of seven proteases located in the intermembrane space of the mitochondria. The mitochondrial intermembrane space proteases HTRA2, OMA1, YME1L1, LACTB, IMMP1L, IMMP2L and PARL were cloned in-frame with the abortive E. coli biotin ligase BirA*, and expressed in 293 T-REx cells. Cell culture media was spiked with biotin for 24 hrs, the cells lysed, and biotinylated proteins were isolated and identified by mass spectrometry. In total, we identified 342 different proteins as high confidence interactors of the seven mitochondrial proteases. Of these, 272 are assigned a GO mitochondrial annotation, and 230 proteins interacted with only 1 or 2 proteases in our dataset. Validation efforts were focused on high temperature requirement peptidase A 2 (HTRA2). HTRA2 is a serine protease that is released into the cytoplasm during apoptosis where it binds Inhibitor of Apoptosis Proteins (IAPs). However, little is known about the function of HTRA2 in the mitochondria. HTRA2 interacted with 60 mitochondrial, 11 nuclear and 4 cytoplasmic proteins, including its known interactor XIAP, and consistent with its known localization to these cellular compartments. HTRA2 interacted with 8 out of 13 components of the MIB complex, a multiprotein assembly that is essential for proper mitochondrial cristae formation. Knockdown of HTRA2 with shRNA in 293T-REx cells disrupted cristae formation and this phenotype was rescued by expression of an shRNA-resistant HTRA2 cDNA. Compared to normal hematopoietic cells, HTRA2 mRNA expression levels are increased in a subgroup of primary AML cells. HTRA2 knockdown in OCI-AML2 leukemia cells led to a similar disruption of mitochondrial cristae. Knockdown of HTRA2 in OCI-AML2 cells led to increased levels of the MIB subunit IMMT, but not two other MIB complex subunits, SAMM50 and CHCHD3. Finally, in cell-free assays, we demonstrate that recombinant HTRA2 can degrade recombinant IMMT, but not SAMM50 or CHCHD3.Thus, we have mapped the interactomes of the proteases of the mitochondrial intermembrane space. Through this effort, we discovered that HTRA2 regulates protein levels of the MIB complex subunit IMMT and that disruption of this process affects mitochondrial cristae formation. Citation Format: Aaron D. Botham, Etienne Coyaud, Sanjit Nirmalanandhan, Marcela Gronda, Rose Hurren, Neil Maclean, Jonathan St. Germain, Sara Mirali, Estelle Laurent, Brian Raught, Aaron Schimmer. Mapping the protein interactome of mitochondrial intermembrane space proteases identifies a novel function for HTRA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4529.