Scientists from Heidelberg and Freiburg identify the mechanism by which proteins assemble into stable complexes in mitochondria

A tiny processing step has far-reaching consequences for mitochondrial function: The removal of a single amino acid determines whether new proteins can assemble into stable complexes within these cellular powerhouses. This process is carried out by a specific enzyme that precisely trims the mitochondrial proteins at their beginning. If this step is missing, numerous protein complexes lose their stability. This shows a study conducted by a research team led by Prof. Dr. Nora Vögtle of Heidelberg University and Prof. Dr. Pitter Huesgen of the University of Freiburg. The scientists identified a previously unknown mechanism by which human cells stabilize their mitochondrial protein balance.

Human cells consist of various organelles that perform vital tasks as functional systems. These include the mitochondria. These cellular powerhouses are of central importance for energy production but are also involved in numerous metabolic and signaling processes. Most mitochondrial proteins are synthesized outside the organelle and are then transported into the mitochondria. To this end, they carry a kind of molecular address sequence at their beginning – the so-called N-terminus – which is removed by specialized enzymes after import. The proteins can then assemble into stable complexes to assume their mature, functional form.

An enzyme – a biochemical catalyst – called ICP55 is involved in this maturation process. After an initial enzyme has removed the address sequence at the N-terminus, ICP55 cleaves off exactly one additional amino acid. Until now, it was not known which proteins undergo these two steps or what significance this second, tiny processing step has for the function of human mitochondria. The research team led by Prof. Vögtle and Prof. Huesgen therefore systematically investigated which of the imported proteins are processed by ICP55 and what the consequences are when the enzyme is absent.

Using proteomic methods, the researchers identified the mature N-terminus of 446 mitochondrial proteins. In the process, the scientists were able to identify 107 proteins that are clearly targeted for processing by ICP55. To investigate the consequences of defective protein maturation, Prof. Vögtle's research group generated human cells lacking this enzyme. In this context, it was revealed that large protein complexes, in particular, lose their stability. Among those affected were the respiratory chain complexes, which are of fundamental importance for cellular energy production, as well as the HSP60 complex, which helps newly imported or stress-damaged proteins fold correctly.

Using the method of complexome profiling, the researchers also analyzed the overall protein organization of the mitochondria. In the absence of ICP55, numerous proteins were less frequently found in fully assembled large complexes and more frequently in smaller complexes or as individual protein subunits. In addition to the respiratory chain and HSP60, the mitochondrial ribosomes – the mitochondria’s protein factories – and metabolic protein complexes were also affected. Using HSP60 as an example, the scientists were also able to directly demonstrate that a single additional amino acid is indeed sufficient to cause this destabilization. Complexes that had already formed broke down again in the test tube, resulting in an increase in individual HSP60 protein subunits.

“We were surprised to find that removing a single amino acid has such a fundamental impact on the stability of mitochondrial protein complexes,” emphasizes Prof. Vögtle. “This means that the precise trimming of the N-terminus is crucial for proteins to subsequently assemble into stable complexes,” says the scientist, who leads a research group on mitochondrial proteostasis at the Center for Molecular Biology at Heidelberg University. “The exact sequence of processing by ICP55 has been shown to be a fundamental prerequisite for the formation of stable functional units,” explains Prof. Huesgen. The biochemist conducts research in the field of proteomics at the Faculty of Biology at the University of Freiburg.

According to the two scientists, the latest findings provide a potential molecular starting point for understanding how disruptions in precise protein maturation can impair mitochondrial function. They could also contribute to a better understanding of mitochondrial diseases. Researchers from the Biochemistry Center at Heidelberg University, the Jülich Research Center, and the University of Fribourg (Switzerland) were also involved in the work. The research was funded by the German Research Foundation as well as other funding programs. The results of the research appeared in “Nature Structural & Molecular Biology”.