Membrane Protein Dimerization in Cell-Derived Lipid Membranes Measured by FRET with MC Simulations.

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease.

Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent processive metalloprotease FtsH and cleaves MgtE, the major high-affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn2+ /Zn2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn2+ and Zn2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of "ERAD" in bacteria and exemplifies an ancestral function of rhomboid-superfamily proteins.

Resolving oxidative damage to methionine by an unexpected membrane-associated stereoselective reductase discovered using chiral fluorescent probes.

Nonenzymatic oxidative processes in living organisms are among the inevitable consequences of respiration and environmental conditions. These oxidative processes can lead to the formation of two stereoisomers (R and S) of methionine sulfoxide, and the redox balance between methionine and methionine sulfoxide in proteins has profound implications on their function. Methionine oxidation can be reverted enzymatically by methionine sulfoxide reductases (Msrs). The two enzyme classes known to fulfill this role are MsrA, reducing the (S)-isomer, and MsrB, reducing the (R)-isomer of methionine sulfoxide. They are strictly stereoselective and conserved throughout the tree of life. Under stress conditions such as stationary phase and nutrient starvation, Escherichia coli upregulates the expression of MsrA but a similar effect has not been described for MsrB, raising the conundrum of which pathway enables reduction of the (R)-isomer of methionine sulfoxide in these conditions. Using the recently developed chiral fluorescent probes Sulfox-1, we show that in stationary phase-stressed E. coli, MsrA does have a stereocomplementary activity reducing the (R)-isomer of methionine sulfoxide. However, this activity is not provided by MsrB as expected, but instead by the DMSO reductase complex DmsABC, widely conserved in bacteria. This finding reveals an unexpected diversity in the metabolic enzymes of redox regulation concerning methionine, which should be taken into account in any antibacterial strategies exploiting oxidative stress. DATABASE: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013610.

Sensitive Versatile Fluorogenic Transmembrane Peptide Substrates for Rhomboid Intramembrane Proteases.

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor.

The genome of Streptomyces coelicolor encodes six potential WD-40 genes. Two of them, the wdpB (SCO5953) and the wdpC (SCO4422) genes, were studied to determine their function. Deletion of the wdpB gene resulted in a considerable decrease of aerial hyphae formation, leading to a conditionally bald phenotype, and reduced undecylprodigiosin production. In addition, the aerial hyphae of the ΔwdpB mutant strain were unusually branched and showed the signs of irregular septation and precocious lysis. Disruption of wdpC resulted in the reduction of undecylprodigiosin and delayed actinorhodin production. The ΔwdpC mutant strain showed precocious lysis of hyphae and delayed sporulation without typical curling of aerial hyphae in the early sporulation stage. The whole-genome transcriptome analysis revealed that deletion of wdpB affects the expression of genes involved in aerial hyphae differentiation, sporulation and secondary metabolites production. Deletion of wdpC caused downregulation of several gene clusters encoding secondary metabolites. Both the wdp genes seem to possess transcriptional autoregulatory function. Overexpression and genetic complementation studies confirmed the observed phenotype of both mutants. The results obtained suggest that both genes studied have a pleiotropic effect on physiological and morphological differentiation.