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Membrane proteins and the import business of mitochondria

The powerhouses of cells are surprisingly dependent on external help. More than ninety per cent of all proteins required by the mitochondria are produced outside the outer mitochondrial membrane. How are these proteins transported across the membrane and how do they find their way into the mitochondria? A group of researchers led by Prof. Dr. Chris Meisinger at the University of Freiburg has been investigating the role of large protein complexes in the outer mitochondrial membrane for many years. The researchers have discovered previously unknown protein import and sorting pathways. However, their research has also provided evidence for the mitochondrias' important role in another cellular process: they play a key role in cellular signalling, for example in programmed cell death. It is assumed that membrane protein complexes also play a key role in this process.

Many diseases are the result of mitochondrial defects. Mitochondria are normally the powerhouses of the cells: electrons are transferred from carbon compounds to oxygen in the interior of mitochondria with the aid of a complicated system of protein complexes. This leads to the release of energy. If something goes wrong during this process, combined disorders of the energy-dependent tissues such as the skeletal muscles, the heart and the central nervous system will typically arise. But the mitochondria are also associated with diseases that are not related to the energy metabolism, for example the development of tumours or neurodegenerative diseases such as Alzheimer’s and Parkinson’s. There is growing evidence that mitochondria also play a key role in cellular signalling, for example in apoptosis (programmed cell death). Mitochondria trigger apoptotic processes when they are triggered by external signals to do so. Therefore, the mitochondria are key in maintaining a well functioning tissue complex that is free of abnormal or damaged cells.

A fertile comprehensive approach

Research group led by Prof. Dr. Chris Meisinger (left) at the Institute of Biochemistry and Molecular Biology at the University of Freiburg. © Prof. Dr. Chris Meisinger

"Although mitochondria contain their own DNA, only around one per cent of the proteins required by Saccharomyces mitochondria for their work, can be produced by the mitochondria themselves," said Prof. Dr. Chris Meisinger from the Institute of Biochemistry and Molecular Biology at the University of Freiburg. "Thus, 99% of all required proteins are encoded in the cell nucleus and need to be transported into the mitochondria." This is also the case for the mitochondria in higher organisms, including humans. Huge molecular sluices in the mitochondrial membranes, so-called protein import complexes, play a key role in importing nuclear proteins into the mitochondria. The so-called TOM complex (transporter of the outer membrane) is located in the outer of the two mitochondrial membranes. It recognises arriving proteins that are allocated to the mitochondria and guides them to a channel in the membrane. But what happens then? How do the proteins know whether they need to integrate into the outer membrane, or whether they are destined for the intermembrane space, whether their journey continues across the inner membrane into the so-called mitochondrial matrix?

“In order to gain detailed insights into the transport of proteins into mitochondria, we have been pursuing a comprehensive approach,” said Meisinger going on to add “we are using functional proteomics methods to do this.” Meisinger’s team and the research group led by Dr. Albert Sickmann in Würzburg scanned the mitochondrial proteome a few years ago, i.e. identified all proteins found in mitochondria in the model organism Saccharomyces. Of the 851 proteins identified, the function of one quarter is still unknown. Meisinger and his colleagues then searched for promising candidates in this pool, especially those that were involved in the import of proteins. The majority of proteins found in yeast mitochondria are not required by the organism. When grown in a petri dish with a high enough amount of glucose, the unicellular yeast simply switches to anaerobic energy production, which still works with a minimal set of proteins. The majority of proteins involved in the import of proteins are nevertheless essential, despite the minimal programme. “We have therefore been looking for molecules without which the yeast is unable to survive in a glucose medium,” said Meisinger going on to add “in the hope they had something to do with protein import.”

A view into primitive times

After many laborious biochemical and molecular biological experiments in which the researchers systematically switched off genes suspected of being involved in the import of proteins, they found several components of a large molecular machine located in the outer mitochondrial membrane. This complex, which Meisinger and his team termed ‘sorting and assembly machinery’ (SAM complex) after carrying out additional experiments, is closely connected with the TOM complex. Proteins with a complicated structure that enter the mitochondria by way of the TOM channel and are integrated into the outer mitochondrial membrane are guided to the SAM complex, which integrates them into the membrane and ensures that they are embedded in the right steric conformation. One component of the SAM complex is already found in the bacterial ancestors of the mitochondria. This clarifies the evolutionary importance of the sorting machinery. “We even assume that SAM existed before TOM, because the bacteria did not need to import proteins from the outside,” said Meisinger. “This became necessary when the bacteria were taken inside eukaryotic cells as endosymbionts, thereby giving rise to mitochondria.”

The two membranes of mitochondria and the protein import and sorting machinery acting in and around them. © Prof. Dr. Chris Meisinger
In addition to the SAM complex, Meisinger’s group of researchers, in cooperation with the research group of Prof. Nikolaus Pfanner from the same institute, discovered the molecule Mia40, a factor for the import of proteins into the intermembrane space of mitochondria. Mia40 helps the proteins to fold correctly and assume the conformation that is necessary for their correct function. The membrane-bound TOM and SAM protein import and sorting complexes and sorting helpers such as Mia40 are indispensable for the mitochondria that need to import the majority of required proteins from the outside. Mitochondria need the imported proteins to produce the energy required by the cells. But the machinery located in the outer mitochondrial membrane can do a lot more. “We have scanned the subproteome of the outer membrane and discovered around one hundred proteins,” said Meisinger. “It is interesting to note that more than ten per cent of these proteins are signalling molecules.” For a long time scientists had assumed that mitochondria simply supply the energy that is required by the cells and otherwise have little to do with the rest of the cell. But Meisinger’s findings have shown that the mitochondria are an essential signalling platform in the cells’ interior.

Communication and the inevitable suicide

Other investigations showed that a large percentage of mitochondrial proteins possess phosphorylation sites, areas to which small phosphate groups can be attached. Phosphorylation is typical for molecules that are involved in signalling cascades. “This shows us that there are many signal transduction pathways in mitochondria,” said Meisinger. And these signalling pathways are connected with the rest of the cell via proteins in the outer mitochondrial membrane. The example of signalling molecules that trigger programmed cell death clearly shows this. For example, signalling molecules transmit information from the cell nucleus about severe DNA damage caused upon exposure to excessive UV light. They activate membrane proteins in the outer mitochondrial membrane that transfer the suicide signal into the interior of mitochondria, which react to the signal by releasing cytochrome C into the cytosol. This is the moment when the cell starts to undergo programmed cell death and there is no way back.

“We are currently investigating the proteins in the outer mitochondrial membrane with which the suicide signals interact and what subsequently happens inside the mitochondria on the molecular level,” said Meisinger. The researchers found that SAM and TOM also interact with signalling pathways and will focus on these interactions in future projects. Something that has already become clear is that the mitochondria have long been underestimated and that they might turn out to be of great importance for the pharmaceutical industry. And since the proteins in the outer mitochondrial membrane are key for the function of mitochondria, they will most likely also increasingly come into the focus of medical and pharmaceutical research.

Further information:
Prof. Dr. Chris Meisinger
Institute of Biochemistry and Molecular Biology, and
Excellence Cluster for Biological Signalling Studies (bioss)
Stefan-Meier-Straße 17
D-79104 Freiburg
Tel.: +49 (0)761/203-5287
E-mail: chris.meisinger(at)biochemie.uni-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/membrane-proteins-and-the-import-business-of-mitochondria