Thermal Profiling Maps Multilayered Organization of the Mitochondrial Proteome

ABSTRACT:

Mitochondria execute essential metabolic, biosynthetic, and signaling functions, yet the regulatory principles governing their proteome remain incompletely understood. Here, we develop a thermal proteome profiling (TPP) workflow applied directly to purified mitochondria, enabling systematic analysis of organelle-specific protein thermal stability. This approach substantially increases mitochondrial proteome and sequence coverage compared with whole-cell TPP, enhances detection of protein–ligand interactions, and provides high-resolution access to proteoform- and protein–protein interaction (PPI)–level organization. Using complex III and I inhibitors, we show that disrupting electron transport chain (ETC) activity broadly destabilizes mitochondrial matrix proteins, revealing a previously unrecognized requirement for ETC function in maintaining the structural integrity of the mitochondrial gene expression machinery and supporting mitochondrial translation. Deep proteoform analysis identifies extensive mitochondrial proteoform diversity arising from phosphorylation, alternative splicing, and proteolytic processing, including previously uncharacterized C-terminal cleavage of SLC25A24. Co-melting network analysis improves recovery of known mitochondrial PPIs and uncovers novel interactions, leading to the discovery of AK4 as a negative regulator of mitochondrial protein synthesis and L2HGDH as a factor required for coenzyme Q biosynthesis. Finally, applying mitochondrial TPP to Mgme1-deficient mice reveals early, abundance-independent remodeling of mtDNA replication and translation factors, including sequestration of Ssbp1 into condensate-like assemblies driven by accumulation of aberrant mtDNA fragments. Together, this work establishes mitochondrial TPP as a powerful and versatile platform for resolving multilayered regulation of the mitochondrial proteome in vitro and in vivo.

INSTRUMENT(S): Liquid Chromatography MS - positive - reverse-phase

PROVIDER: MTBLS7386 | MetaboLights | 2025-12-08

REPOSITORIES: MetaboLights