How chaperones, insertases and translocases facilitate insertion and folding of complex cytoplasmic proteins into cellular membranes is not fully understood. Here, we utilize single-molecule force spectroscopy to observe YidC, a transmembrane chaperone/insertase, sculpting the folding trajectory of the polytopic α-helical membrane protein lactose permease (LacY). In the absence of YidC, unfolded LacY inserts individual structural segments into the membrane; however, misfolding dominates the process so that folding cannot be completed. YidC prevents LacY from misfolding by stabilizing the unfolded state from which LacY inserts structural segments stepwise into the membrane until folding is completed. During stepwise insertion, YidC and membrane together stabilize the transient folds. Remarkably, the order of insertion of structural segments is stochastic, thereby indicating that LacY can fold along variable pathways towards the native structure. Since YidC is essential in membrane protein biogenesis and LacY a paradigm for the major facilitator superfamily, our observations have general relevance.

In a variety of bacteria, the phosphotransferase protein IIA Glc plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H + , using a mechanism in which sugar-and H + -binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA Glc (inducer exclusion). Here we report the thermodynamic features of the IIA Glc -LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA Glc binds to LacY with a K d of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA Glc binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA Glc blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA Glc binding. The precise mechanism of the inhibition of LacY by IIA Glc elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA Glc .ITC | PTS | sugar/cation symport | protein-protein interactions | protein conformation C arbohydrate uptake in bacteria is catalyzed by a collection of sugar permeases that belong to different families of transport proteins. In Escherichia and Salmonella, the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions and plays a key role in catabolite repression resulting in preferential utilization of glucose (a constitutive process) that is transported by vectorial phosphorylation catalyzed by the PTS (1-5). The phosphotransferase protein IIA Glc plays a direct role in this regulation of inducible transport systems. The lac operon (6) with lacZ encoding β-galactosidase and lacY encoding lactose permease (LacY) and the mel operon (7, 8) with melA encoding α-galactosidase and melB encoding melibiose permease (MelB) are subject to IIA Glc regulation (9-13). Both LacY and MelB catalyze electrogenic symport of a galactoside with a cation (14-21). Expression of the structural genes requires the participation of both a global transcriptional activator (the cAMP-CAP complex) and a specific inducer (lactose or melibiose, respectively) (3, 22, 23). IIA Glc regulates both cAMP (24) and inducer levels, and this study focuses on regulation of LacY, which influences inducer entry into the cell.With maltose permease, an ABC permease also under PTS regulation, two molecules of IIA Glc bind to the cytoplasmic ATPase subunits and c...

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor–acceptor pairs during solution-based smFRET. We use this “caged FRET” methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules.

Excessive consumption of fructose in the Western diet has been associated with metabolic disorders such as type 2 diabetes and obesity. Altered expression and activity of the fructose uniporter GLUT5, a member of the family of GLUT transporters that facilitate the diffusion of monosaccharides across membranes, has been linked to such disorders. Using Saccharomyces cerivisiae as an expression host, and fluorescence-based methods, we identified mammalian GLUT5 orthologues that are suitable for biochemical and structural studies. Here, we present a crystal structure of GLUT5 from Bos Taurus (bovine) in an inward-facing conformation, refined against data extending tõ 3.1 Å resolution. Like GLUT1, GLUT5 shows the typical Major Facilitator Superfamily (MFS) fold, which consists of two 6-TM bundles, and four additional helices that form a soluble domain on the cytoplasmic side of the membrane. The substrate-binding site is highly similar to that observed in the recent crystal structure of human GLUT1, and to those of other GLUT isoforms based on amino acid sequence. However, there are notable differences. In particular, the substrate-binding site of GLUT5 is larger, because the equivalent of a tryptophan residue lining the cavity that contains the substrate-binding site in GLUT1 is an alanine in GLUT5. Furthermore, we have identified a single point mutation that switches the substrate preference of GLUT5 from D-fructose to D-glucose. Overall, our structural and biochemical data provide novel insights into the structure and substrate specificity of GLUT5, a member of the family of the medically relevant GLUT transporters.