FOR 929: Dynamics of Bacterial Membrane Proteins
Biology
Final Report Abstract
The cell membrane is the essential barrier between the cell and its environment, and mediates a huge array of import – and export processes, as well as the generation of energy. The membrane contains about one fourth of all proteins encoded in a cell, but the organization and dynamics of membrane proteins is largely ill-defined. Recent advances in imaging techniques have made it possible to gain insight into processes such as the assembly of large protein complexes in live cells, the dynamics of protein interactions as well as information of when and where within cells proteins perform their functions. Especially for bacteria, cell biological approaches have dramatically changed the view on the architecture of these single cell organisms. A multitude of proteins assembles at specific places within the cell at certain times in the cell cycle, and also the membrane contains more asymmetrically localized proteins than previously appreciated. The membrane contributes a large surface to the environment, and frequently, only few copies of membrane proteins are synthesized. Moreover, large complexes of proteins exist in the membrane, which have to be assembled from many parts, and which may cooperate in the activities. So called “supercomplexes” have been postulated for proteins of the electron chain of oxidative phosphorylation (OXPHOS), and the recent advances in imaging techniques have made it possible to investigate if such structures indeed exist in living cells, and if so, how stable these are. Major goals of the consortium were the generation and application of novel tools to understand the dynamics of transport processes through bacterial membrane, signal transfer across the membrane, and functional interactions between cytosolic and membrane proteins. Overall, the group has produced 28 publications in the second funding period, several of which were published in high impact journals. In the two funding periods, 6 joint publications have arisen. MreB is an actin-like protein that is essential for the maintenance of rod shape in many bacteria, and is also essential for viability. A major finding of the Graumann group was the formation of 200 to 1700 nm long filaments underneath the cell membrane, which move in a generally perpendicular pattern relative to the long axis of the cells. MreB has intrinsic affinity to the membrane, and mutations in the filament interface affect cell shape, revealing that MreB’s ability to polymerise is an essential feature, similar to actin. FtsY is an essential component of the machinery that leads to the insertion of proteins into the cell membrane, it is the receptor of SRP, which binds to nascent chains of proteins as they are translated. The group of H.-G. Koch has found that FtsY binds to the membrane via two short lipid-binding helices with a preference for negatively charged lipids. On the other hand, FtsY binds to the SecY subunit of the SecYEG translocon, where FtsY occupies the ribosome-binding site of the SecYEG translocon. These findings suggest a hand-over mechanism of nascent membrane proteins from the targeting machinery to the insertion machinery. The SecYEG translocon can only translocate non-folded proteins across the cell membrane, folded proteins (e.g. those that carry a covalently linked cofactor) require the activity of the Tat machinery. Using fluorescence labeling, AG Müller visualized the assembly of TatA, TatB, and TatC in living cells, to demonstrate that this process occurs at distinct sites of the inner membrane, and in strict response to newly synthesized substrate proteins. In mitochondria, the enzymes of the respiratory electron chain are thought to be organized into local supercomplexes. AG Friedrich has succeeded in With labelling all respiratory enzyme complexes in E. coli and in using real-time multicolor singlemolecule fluorescence imaging and super-resolution particle tracking on live cells. Interestingly, enzyme complexes are compartmentalized as assemblages of distinct, dynamic islands indicating an organization that is different to that found in mitochondria. We could demonstrate that the PTS network operates as an integral sensor of sugar influx into the cell and transmits this information linearly to regulate other cellular functions, including chemotaxis system and control of the production of second messenger cAMP. The Phosphotransferase system (PTS) mediates coupling of sugar import with glycolytic flux, and also integrates uptake with conventional chemoreceptor-mediated stimuli within the chemotaxis pathway, to elicit a coordinated behavioral response. AG Sourjik further showed that influx through the PTS system correlates with the metabolic “value” of a sugar, which apparently represents an optimal regulatory strategy evolved by bacteria. AG Einsle has shown that formate transporter FocA has an unusual architecture that enables the protein to switch between passive and active transport modes, dependent on pH conditions, without major structural rearrangements, and that conformational changes of the N-termini mainly act as a barrier for further export of formate. Nitrite channel NirC was also crystallized and shown to be structurally similar to FocA concerning the substrate channel. Nevertheless, relative conductivities for anions vary considerably, FocA prefers formate over nitrite, while NirC shows the opposite behavior. Shewanella oneidensis is able to transfer electrons from intracellular oxidation processes through the periplasm to the MtrAB outer membrane complex, which is able to further transfer the eletcrons to e.g. iron, to reduce Fe3+ to Fe2+. The group of Johannes Gescher has characterized in detail protein/protein interactions involved in periplasmic electron transfer, and has identified a so far unknown outer membrane protein complex encoded on a mobile genomic element, which can complement for a lack of MtrAB, and which is proposed to be involved in the reduction of extracellular dimethyl sulfoxide, extending the range of electron acceptors for Shewanella.
