S006
Complexome profiling – a new tool to analyze assembly
of mitochondrial protein complexes
Ilka Wittig1, Lea Bleier1, Heinrich Heide1, Valentina Strecker1,
Heiko Giese2, Bettina Schwamb3, Mirco Steger1, Jörg Ackermann2,
Stefan Dröse1, Martin Zörnig3, Ina Koch2 and U. Brandt1
1
Molecular Bioenergetics Group, Medical School, Cluster of Excellence
Frankfurt “Macromolecular Complexes” Goethe-University,
Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany
2
Molecular Bioinformatics Group, Institute of Computer Science,
Faculty of Computer Science and Mathematics, Cluster of Excellence
Frankfurt “Macromolecular Complexes”, Goethe-University,
Robert-Mayer-Strasse 11-15, D-60325 Frankfurt am Main, Germany
3
Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus,
Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt am Main, Germany
Many proteins form stable or dynamic macromolecular associations with other
proteins, lipids or nucleic acids. The formation of protein complexes occurs in
a multi-step process that often requires dynamic interactions of subunits with
assembly factors. Defects in the assembly of mitochondrial complexes can lead
to the accumulation of non-functional assembly intermediates and can lead to
mitochondrial disorders. Methods to analyze the dynamics of complex assembly
are scarce. The recently introduced method complexome profiling combines
classical blue-native electrophoresis (BNE) with modern proteomic methods
of quantitative mass spectrometry and hierarchical clustering to identify known
and unknown macromolecular protein complexes. By complexome profiling we
identified and characterized the mitochondrial membrane protein TMEM126B
as an essential assembly factor of complex I that forms together with the other
known assembly factors CIA, ECSIT and ACAD9 a stable mitochondrial complex
I assembly complex (MCIA). We further analyzed the homologous protein
TMEM126A that was reported to be associated with mitochondrial disorders.
The complexome interaction profiles of mitochondria from the human osteosarcoma cell line 143B revealed that TMEM126A does not belong to the core MCIA
complex. Mitochondria from stable shRNA TMEM126A knockdown cells accumulated early assembly intermediates and showed isolated complex I deficiency,
alike to TMEM126B knockdown mitochondria. In addition we detected several
assembly intermediates of complex I in intact mitochondria from fast proliferating
143B cells by complexome profiling. In summary, complexome profiling serves
as a powerful bottom-up approach to identify the composition of unknown protein
complexes, dynamic assembly processes and helps to understand the molecular
mechanisms of mitochondrial diseases.