Mapping Interactome Networks of DNAJC11, a Novel Mitochondrial Protein Causing Neuromuscular Pathology in Mice

Abstract

We previously identified DNAJC11, a mitochondrial outer membrane protein of unknown function, as a novel genetic cause in modeled neuromuscular disease. To understand the physiological role of DNAJC11, we employed a proteomic approach for the identification of the DNAJC11 interactome, through the expression of DNAJC11-FLAG in HEK293FT cells and transgenic mice. Our analysis confirmed known DNAJC11-interacting proteins including members of the MICOS complex that organize mitochondrial cristae formation. Moreover, we identified in both biological systems novel mitochondrial interactions including VDACs that exchange metabolites across the outer mitochondrial membrane. In HEK293FT cells, DNAJC11 preferentially interacted with ribosomal subunits and chaperone proteins including Hsp70 members, possibly correlating DNAJC11 with cotranslational folding and import of mitochondrial proteins in metabolically active cells. Instead, the DNAJC11 interactome in the mouse cerebrum was enriched for synaptic proteins, supporting the importance of DNAJC11 in synapse and neuronal integrity. Moreover, we demonstrated that the DUF3395 domain is critically involved in DNAJC11 protein–protein interactions, while the J-domain determines its mitochondrial localization. Collectively, these results provide a functional characterization for DNAJC11 domains, while the identified interactome networks reveal an emerging role of DNAJC11 in mitochondrial biogenesis and response to microenvironment changes and requirements.

Proteomic Identification of the SLC25A46 Interactome in Transgenic Mice Expressing SLC25A46-FLAG

“…The reverse primer 5′-GCG GCC GCΤ ΑΑC TTA TCG TCG TCA TCC TTG TAA TCA ATG TTA TTT TGA AGA AG-3′ (Eurofins Scientific) contained a NotI site, the 24 bp FLAG sequence, and 18 bp of the 3′ SLC25A46 cDNA. The PCR product of 1301 bp was digested with the restriction enzymes HindIII and NotI (ThermoFisher Scientific) and subcloned between the CBX3/hnRNPA2 promoter (5.5 kb) and the 3′ human beta-globin sequence (2.8 kb) of a plasmid digested also with the HindIII and NotI enzymes . The recombinant plasmid transformed the Escherichia coli strain XL1-blue, and selected bacterial clones were sequenced (CeMIA SA) using the primers described above.…”

“…The samples were centrifuged and resuspended in Laemmli buffer (2% SDS, 10% glycerol, 1% 2-mercaptoethanol, 0.002% bromophenol blue, 0.125 mM Tris/HCl, pH 6.8). As a control, 50 μg of intact mitochondria was resuspended in 1% Triton X-100, centrifuged (12,000 g for 10 min at 4 °C), and treated with proteinase K …”

“…The PCR product of 1301 bp was digested with the restriction enzymes HindIII and NotI (ThermoFisher Scientific) and subcloned between the CBX3/hnRNPA2 promoter (5.5 kb) 43 and the 3′ human beta-globin sequence (2.8 kb) 44 of a plasmid digested also with the HindIII and NotI enzymes. 45 The recombinant plasmid transformed the Escherichia coli strain XL1-blue, and selected bacterial clones were sequenced (CeMIA SA) using the primers described above. The SLC25A46-FLAG transgene was excised from the plasmid as a 9656 kb PmeI fragment and was purified using a S.N.A.P.…”

Mitochondrial Function and Reactive Oxygen/Nitrogen Species in Skeletal Muscle

“…Furthermore, the MICOS complex can directly or indirectly interact with other proteins and play a critical role in maintaining mitochondrial ultrastructure. For example, DNAJC11 helps to maintain proper cristae morphology by interacting with the peripheral MICOS complex ( Violitzi et al, 2019 ).…”

MICOS and the mitochondrial inner membrane morphology – when things get out of shape

“…One of these being DNAJC11, which helps to maintain and uphold proper cristae morphology by interacting with the MICOS complex peripherally. However, the exact mechanism of interaction between DNAJC11 and MICOS or the mitochondrial intermembrane space bridging (MIB) complex, of which MICOS is a part and which is discussed further in detail later in this review, is poorly understood [54,[68][69][70]. Studies in mice have shown that a mutation in the DNAJC11 gene leads to abnormal cristae structures [71].…”