Jump to content
Powered by

Like a bridge of molecules

Proteins are the active part of cells. They recognise sequences, transport nutrients and information as well as getting rid of waste. Proteins that go from one side of a membrane through to the other serve as transporters and channels and help molecules across membranes. Dr. Thomas Becker and his colleagues from the Institute for Biochemistry and Molecular Biology at the University of Freiburg are studying these complex processes. They are particularly interested in how transmembrane proteins are integrated into the mitochondrial membranes of yeast cells, the protein complexes involved and whether the lipid composition of the membranes plays a role in this process.

Dr. Thomas Becker – a specialist in transport pathways in biological membranes. © Dr. Thomas Becker, University of Freiburg

The endosymbiotic theory states that plant chloroplasts, mitochondria and possibly other organelles have evolved from free-living prokaryotes that were taken inside another cell, thus giving rise to the first eukaryotes. The two membranes that enclose the mitochondria are the most striking evidence that mitochondria arose from bacteria. The two membranes give the mitochondria five distinct parts: the outer mitochondrial membrane, the space between the outer and inner membranes (intermembrane space), the inner mitochondrial membrane, the infoldings of the inner membrane (cristae) and the space within the inner membrane (matrix).

Both membranes are composed of phospholipid bilayers into which proteins are integrated and they have different properties. While the inner membrane, which contains proteins of the electron transport chain and the ATP synthase enzyme, is similar to the inner bacterial membrane, the phospholipid and protein composition of the outer membrane is more similar to eukaryotic plasma membranes. The outer mitochondrial membrane, which encloses the entire organelle, is closest to the cytosol, and therefore contains proteins that play an important role in cellular signalling and transport.

Protein transport is mediated by TOM and SAM

Microscopic image of two yeast cells in which the mitochondria are labelled with a fluorescent dye. © Dr. Lukasz Opalinski

There are two kinds of transmembrane proteins, those with alpha-helical transmembrane regions and those with beta-barrels. The alpha-helical regions are mostly composed of hydrophobic amino acid residues which are in contact with the inner, also hydrophobic, part of the lipid bilayer. The beta-barrel transmembrane proteins look, as the name suggests, like a barrel, however without a bottom or lid. “These proteins act as membrane pores through which small charged molecules can pass.” said Dr. Thomas Becker from the Institute for Biochemistry and Molecular Biology at the University of Freiburg. Beta-barrel protains - porins, for example - are only present in the outer mitochondrial membrane, the outer membrane of bacteria and the chloroplast. 

But how do beta-barrel transmembrane proteins enter the outer membrane? All of them are synthesised in the cytosolic ribosomes from where they are guided by chaperones to the surface of the mitochondria where they assume their final folded three-dimensional structure. The translocase of the outer membrane (TOM) complex, which consists of at least 19 proteins, is located in the outer membrane and forms a channel through which proteins can move into the intermembrane space of the mitochondrion. Two of the TOM proteins are receptors that recognise the cleavable signalling sequence of the proteins to be imported into the intermembrane space. “After synthesis, the proteins move from the cytosol into the intermembrane space before they are incorporated into the membrane; this might appear strange, but makes sense,” said Becker, describing how proteins are integrated into the outer membrane. “Outer membrane proteins enter the membrane from the intermembrane space. It is here where the necessary protein machinery is located. It is worth pointing out that the same mechanism is found both in prokaryotes, i.e. bacteria, as well as eukaryotic mitochondria.” 

SAM is responsible for folding and membrane insertion

Different pathways are used for transporting alpha-helical and beta-barrel proteins that are destined for the inner or outer mitochondrial membrane. © modified from Becker et al., 2008; Biochim Biophys Acta (1777, 557-563)

In addition to TOM, there is SAM. “SAM is the sorting and assembly machinery of the outer membrane; it is located on the intermembrane space side of the membrane. It operates after the translocation of the proteins and mediates insertion of beta-barrel proteins into the outer mitochondrial membrane,” Becker explained. 

Becker’s team, along with researchers headed up by Prof. Dr. Nikolaus Pfanner, Dr. Nils Wiedemann, Prof. Dr. Carola Hunte and Prof. Dr. Bettina Warscheid, showed in a recent publication that the complexes TOM and SAM are in direct contact with each other and work very closely together. “The two complexes are connected with each other by way of their subunits, just like the planks of a hanging bridge where some of the planks are closer to each other than others,” Becker said. TOM recognises and transfers beta-barrel proteins, while SAM assembles the final protein and mediates its integration into the membrane. Proteins are passed from one complex to the other. “When the protein binds to the complex SAM it is already partially in the membrane,” the biochemist said. Beta-barrel proteins include the central pore-forming components of TOM and SAM as well as a number of proteases and lipases. 

Little is yet known about how alpha-helical proteins are transported and embedded into the outer mitochondrial membrane. Becker assumes that this does not require the central TOM subunit, but instead utilises an insertase which releases the protein laterally into the membrane. However, further research needs to be carried out to substantiate Becker’s assumption.

Membrane mosaic affects insertion of proteins into membranes

In cooperation with a group of researchers led by Prof. Dr. Günther Daum from Graz Technical University, Becker’s team recently discovered another interesting detail in the protein integration process. “Most research is focussed on protein components that are required for importing proteins into the membrane,” Becker said. “We decided to have a closer look at phosphatidylethanolamine (PE), the second most abundant class of phospholipids found in the outer mitochondrial membrane,” Becker said, highlighting their plans for examining the role of PE in the integration process. Becker’s team analysed yeast mutants with lower than normal PE levels and found that while this did not affect the integration of alpha-helical proteins into the membrane, beta-barrel proteins seemed to require these phospholipids for effective integration into the membrane. The researchers found that although the TOM complex was still intact, it bound less effectively to the precursor protein. “This suggests that the TOM complex requires this phospholipid for optimal activity,” Becker added. He continued: “Phosphatidylethanolamine creates some tension in the membrane as the substance somehow promotes the membrane’s dynamics and flexibility.”

More and more protein components are being identified

Protein complexes that consist of several individual proteins mediate the import and integration of proteins into membranes. Becker’s team uses what are known as affinity tags to isolate mitochondrial protein complexes, determine their composition and any previously unknown interaction partners. Dr. Becker is still looking for answers to many questions, including: how are alpha-helical proteins able to enter the membrane and how many functions do mitochondrial chaperones have? “Around ten years ago, we only knew of the TOM and two TIM (translocase of the inner membrane) complexes, none of the others was known. It’s the technological progress which will lead to the increased knowledge and discovery of new protein components.”

Further information:

Dr. Thomas Becker
Institute for Biochemistry and Molecular Biology
University of Freiburg
Stefan-Meier-Str. 17
79110 Freiburg
Tel.: +49 (0)761/ 203-5243
Fax: +49 (0)761/ 203-5261
E-mail: thomas.becker(at)biochemie.uni-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/like-a-bridge-of-molecules