Global Interactome Mapping of Mitochondrial Intermembrane Space Proteases Identifies a Novel Function for HTRA2

Abstract

A number of unique proteases localize to specific sub‐compartments of the mitochondria, but the functions of these enzymes are poorly defined. Here, in vivo proximity‐dependent biotinylation (BioID) is used to map the interactomes of seven proteases localized to the mitochondrial intermembrane space (IMS). In total, 802 high confidence proximity interactions with 342 unique proteins are identified. While all seven proteases co‐localized with the IMS markers OPA1 and CLPB, 230 of the interacting partners are unique to just one or two protease bait proteins, highlighting the ability of BioID to differentiate unique interactomes within the confined space of the IMS. Notably, high‐temperature requirement peptidase 2 (HTRA2) interacts with eight of 13 components of the mitochondrial intermembrane space bridging (MIB) complex, a multiprotein assembly essential for the maintenance of mitochondrial cristae structure. Knockdown of HTRA2 disrupts cristae in HEK 293 and OCI‐AML2 cells, and leads to increased intracellular levels of the MIB subunit IMMT. Using a cell‐free assay it is demonstrated that HTRA2 can degrade recombinant IMMT but not two other core MIB complex subunits, SAMM50 and CHCHD3. The IMS protease interactome thus represents a rich dataset that can be mined to uncover novel IMS protease biology.

Skd3 (human ClpB) is a potent mitochondrial protein disaggregase that is inactivated by 3-methylglutaconic aciduria-linked mutations

“…Using Gene Ontology (GO) analysis for cellular component, we found that proteins in the inner mitochondrial membrane and intermembrane space were enriched in the insoluble fraction in the absence of Skd3 ( Figure 7—figure supplement 2a ; Ashburner et al, 2000 ; Mi et al, 2019 ; The Gene Ontology Consortium, 2019 ). Importantly, Skd3 has been localized to the mitochondrial intermembrane space ( Botham et al, 2019 ; Hung et al, 2014 ; Yoshinaka et al, 2019 ). Using GO analysis for biological process, we found that proteins involved in calcium import into mitochondria, chaperone-mediated protein transport, protein insertion into the mitochondrial inner membrane, mitochondrial electron transport, mitochondrial respiratory-chain complex assembly, and cellular response to hypoxia are more insoluble in Skd3 knockout cells compared to wild-type cells ( Figure 7c and Figure 7—figure supplement 2b ; Ashburner et al, 2000 ; Mi et al, 2019 ; The Gene Ontology Consortium, 2019 ).…”

“…Since Skd3 appears in evolution alongside Hsp78 in choanoflagellates it may have initially arisen to serve a distinct function. We hypothesize that the increasing number and complexity of mitochondrial inner membrane protein assemblies (such as MICU1/MICU2/MCU and respiratory complex I) in metazoa might necessitate the requirement of Skd3 activity in the inner mitochondrial membrane and intermembrane space to maintain proteostasis in these compartments ( Botham et al, 2019 ; Hung et al, 2014 ; Yoshinaka et al, 2019 ).…”

A proximity-dependent biotinylation map of a human cell: an interactive web resource

“…These results are expected, based on the known interactions and functions of AARS2 and PDHA1, and highly reproducible ( Supplementary Table 2 ). As also discussed elsewhere 9,10,20,21 BioID can thus provide both compartment and sub-compartmental resolution, uncovering distinct protein environments within the same cellular compartment.…”

Mitochondrial ClpP serine protease-biological function and emerging target for cancer therapy

“…Upon loss of mitochondrial membrane potential, OMA1 cleaves OPA1, resulting in OPA1 inactivation and decreased mitochondrial fusion 17 . High temperature requirement peptidase 2 (HTRA2) (also called OMI) is another protease in the mitochondrial intermembrane space, which plays a critical role in maintaining mitochondrial cristae structure by interacting and degrading its substrate in the mitochondrial intermembrane space bridging (MIB) complex, inner membrane mitochondrial protein (IMMT) 18 . HTRA2 is also released into the cytoplasm during apoptosis where it binds and inhibits Baculoviral IAP Repeat Containing (BIRC) proteins (also called inhibitor of apoptosis proteins, IAPs), leading to an increase in caspase activity 19,20 .…”