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The small heat shock protein (sHsp) Hsp22 from Drosophila melanogaster (DmHsp22) is part of the family of sHsps in this diptera. This sHsp is characterized by its presence in the mitochondrial matrix as well as by its preferential expression during ageing. Although DmHsp22 has been demonstrated to be an efficient in vitro chaperone, its function within mitochondria in vivo remains largely unknown. Thus, determining its protein-interaction network (interactome) in the mitochondrial matrix would help to shed light on its function(s). In the present study we combined immunoaffinity conjugation (IAC) with mass spectroscopy analysis of mitochondria from HeLa cells transfected with DmHsp22 in non-heat shock condition and after heat shock (HS). 60 common DmHsp22-binding mitochondrial partners were detected in two independent IACs. Immunoblotting was used to validate interaction between DmHsp22 and two members of the mitochondrial chaperone machinery; Hsp60 and Hsp70. Among the partners of DmHsp22, several ATP synthase subunits were found. Moreover, we showed that expression of DmHsp22 in transiently transfected HeLa cells increased maximal mitochondrial oxygen consumption capacity and ATP contents, providing a mechanistic link between DmHsp22 and mitochondrial functions.

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Mitochondria are fully integrated in cell signaling. Reversible phosphorylation is involved in adjusting mitochondrial physiology to the cellular needs. Protein kinase A (PKA) phosphorylates several substrates present at the external surface of mitochondria to maintain cellular homeostasis. However, few targets of PKA located inside the organelle are known. The aim of this work was to characterize the impact and the interactome of PKA located inside mitochondria. Our results show that the overexpression of intramitochondrial PKA decreases cellular respiration and increases superoxide levels. Using proximity-dependent biotinylation, followed by LC-MS/MS analysis and in silico phospho-site prediction, we identified 21 mitochondrial proteins potentially targeted by PKA. We confirmed the interaction of PKA with TIM44 using coimmunoprecipitation and observed that TIM44-S80 is a key residue for the interaction between the protein and the kinase. These findings provide insights into the interactome of intramitochondrial PKA and suggest new potential mechanisms in the regulation of mitochondrial functions.