Structure of AMP-PNP-bound vitamin B12 transporter BtuCD-F.

The ATP-binding cassette (ABC) transporter BtuCD mediates the uptake of vitamin B(12) across the inner membrane of Escherichia coli. Previous structures have shown the conformations of apo states, but the transport mechanism has remained unclear. Here we report the 3.5 Å crystal structure of the transporter-binding protein complex BtuCD-BtuF (BtuCD-F) trapped in an ß-γ-imidoadenosine 5'-phosphate (AMP-PNP)-bound intermediate state. Although the ABC domains (BtuD subunits) form the expected closed sandwich dimer, the membrane-spanning BtuC subunits adopt a new conformation, with the central translocation pathway sealed by a previously unrecognized cytoplasmic gate. A fully enclosed cavity is thus formed approximately halfway across the membrane. It is large enough to accommodate a vitamin B(12) molecule, and radioligand trapping showed that liposome-reconstituted BtuCD-F indeed contains bound B(12) in the presence of AMP-PNP. In combination with engineered disulphide crosslinking and functional assays, our data suggest an unexpected peristaltic transport mechanism that is distinct from those observed in other ABC transporters.

Conformational cycle of the vitamin B12 ABC importer in liposomes detected by double electron-electron resonance (DEER).

Double electron-electron resonance is used here to investigate intermediates of the transport cycle of the Escherichia coli vitamin B12 ATP-binding cassette importer BtuCD-F. Previously, we showed the ATP-induced opening of the cytoplasmic gate I in TM5 helices, later confirmed by the AMP-PNP-bound BtuCD-F crystal structure. Here, other key residues are analyzed in TM10 helices (positions 307 and 322) and in the cytoplasmic gate II, i.e. the loop between TM2 and TM3 (positions 82 and 85). Without BtuF, binding of ATP induces detectable changes at positions 307 and 85 in BtuCD in liposomes. Together with BtuF, ATP triggers the closure of the cytoplasmic gate II in liposomes (reported by both positions 82 and 85). This forms a sealed cavity in the translocation channel in agreement with the AMP-PNP·BtuCD-F x-ray structure. When vitamin B12 and AMP-PNP are simultaneously present, the extent of complex formation is reduced, but the short 82-82 interspin distance detected indicates that the substrate does not affect the closed conformation of this gate. The existence of the BtuCD-F complex under these conditions is verified with spectroscopically orthogonal nitroxide and Gd(III)-based labels. The cytoplasmic gate II remains closed also in the vanadate-trapped state, but it reopens in the ADP-bound state of the complex. Therefore, we suggest that the substrate likely trapped in ATP·BtuCD-F can be released after ATP hydrolysis but before the occluded ADP-bound conformation is reached.

High-mass matrix-assisted laser desorption ionization-mass spectrometry of integral membrane proteins and their complexes.

Analyzing purified membrane proteins and membrane protein complexes by mass spectrometry has been notoriously challenging and required highly specialized buffer conditions, sample preparation methods, and apparatus. Here we show that a standard matrix-assisted laser desorption/ionization (MALDI) protocol, if used in combination with a high-mass detector, allows straightforward mass spectrometric measurements of integral membrane proteins and their complexes, directly following purification in detergent solution. Molecular weights can be determined precisely (mass error ≤ 0.1%) such that high-mass MALDI-MS was able to identify the site for N-linked glycosylation of the eukaryotic multidrug ABC transporter Cdr1p without special purification steps, which is impossible by any other current approach. After chemical cross-linking with glutaraldehyde in the presence of detergent micelles, the subunit stoichiometries of a series of integral membrane protein complexes, including the homomeric PglK and the heteromeric BtuCD as well as BtuCDF, were unambiguously resolved. This thus adds a valuable tool for biophysical characterization of integral membrane proteins.

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.

Direct observation of molecular arrays in the organized smooth endoplasmic reticulum.

BACKGROUND: Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization. RESULTS: Here, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets. CONCLUSION: Our results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering.

An emerging consensus for the structure of EmrE.

The archetypical member of the small multidrug-resistance family is EmrE, a multidrug transporter that extrudes toxic polyaromatic cations from the cell coupled to the inward movement of protons down a concentration gradient. The architecture of EmrE was first defined from the analysis of two-dimensional crystals by cryoelectron microscopy (cryo-EM), which showed that EmrE was an unusual asymmetric dimer formed from a bundle of eight alpha-helices. The most favoured interpretation of the structure was that the monomers were oriented in opposite orientations in the membrane in an antiparallel orientation. A model was subsequently built based upon the cryo-EM data and evolutionary constraints and this model was consistent with mutagenic data indicating which amino-acid residues were important for substrate binding and transport. Two X-ray structures that differed significantly from the cryo-EM structure were subsequently retracted owing to a data-analysis error. However, the revised X-ray structure with substrate bound is extremely similar to the model built from the cryo-EM structure (r.m.s.d. of 1.4 A), suggesting that the proposed antiparallel orientation of the monomers is indeed correct; this represents a new structural paradigm in membrane-protein structures. The vast majority of mutagenic and biochemical data corroborate this structure, although cross-linking studies and recent EPR data apparently support a model of EmrE that contains parallel dimers.

GFP-LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy.

Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP-LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP-LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP-Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin-positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP-LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP-LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP-LC3, a fusion protein widely used as a marker for autophagic vesicles and pre-autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes.

The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells.

The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism.

Structure of AMP-PNP-bound BtuCD and mechanism of ATP-powered vitamin B12 transport by BtuCD-F.

The reaction mechanism of BtuCD-F-catalyzed vitamin B12 transport into Escherichia coli is currently unclear. Here we present the structure of the last missing state in the form of AMP-PNP-bound BtuCD, trapped by a disulfide cross-link. Our structural and biochemical data allow a consistent mechanism to be formulated, thus rationalizing the roles of substrate, ATP and substrate-binding protein.

Asymmetric states of vitamin B12 transporter BtuCD are not discriminated by its cognate substrate binding protein BtuF.

BtuCD is an ABC transporter catalyzing the uptake of vitamin B12 across the Escherichia coli inner membrane. A previously reported X-ray structure of BtuCD in complex with the periplasmic vitamin B12-binding protein BtuF revealed asymmetry of the transmembrane BtuC subunits. The functional relevance of this asymmetry has remained uncertain. Here we report the X-ray structure of a catalytically impaired BtuCD mutant in complex with BtuF, where the BtuC subunits adopt a distinct asymmetric conformation. The structure suggests that BtuF does not discriminate between, or impose, asymmetric conformations of BtuCD. It also explains the conformational disorder observed in BtuCDF crystals.

Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals.

The 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The pharmacology of TSPO has been extensively studied, but little is known about its biochemistry, oligomeric state, and structure. We have expressed, purified, and characterized a homologous protein, TspO from Rhodobacter sphaeroides, and reconstituted it as helical crystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determined a three-dimensional structure of TspO at 10 A resolution. The structure suggests that monomeric TspO comprises five transmembrane alpha helices that form a homodimer, which is consistent with the dimeric state observed in detergent solution. Furthermore, the arrangement of transmembrane domains of individual TspO subunits indicates a possibility of two substrate translocation pathways per dimer. The structure provides the first insight into the molecular architecture of TSPO/PBR protein family that will serve as a framework for future studies.

Electron crystallography reveals plasticity within the drug binding site of the small multidrug transporter EmrE.

EmrE is a Small Multidrug Resistance transporter (SMR) family member that mediates counter transport of protons and hydrophobic cationic drugs such as tetraphenylphosphonium (TPP+), ethidium, propidium and dequalinium. It is thought that the selectivity of the drug binding site in EmrE is defined by two negatively charged glutamate residues within a hydrophobic pocket formed from six of the alpha-helices, three from each monomer of the asymmetric EmrE homodimer. It is not apparent how such a binding pocket accommodates drugs of various sizes and shapes or whether the conformational changes that occur upon drug binding are identical for drugs of diverse chemical nature. Here, using electron cryomicroscopy of EmrE two-dimensional crystals we have determined projection structures of EmrE bound to three structurally different planar drugs, ethidium, propidium and dequalinium. Using image analysis and rigorous comparisons between these density maps and the density maps of the ligand-free and TPP+-bound forms of EmrE, we identify regions within the transporter that adapt differentially depending on the type of ligand bound. We show that all three planar drugs bind at the same pocket within the protein as TPP+. Furthermore, our analysis indicates that, while retaining the overall fold of the protein, binding of the planar drugs is accompanied by small rearrangements of the transmembrane domains that are different to those that occur when TPP+ binds. The regions in the EmrE dimer that are remodelled surround the drug binding site and include transmembrane domains from both monomers.

Peptide-based interactions with calnexin target misassembled membrane proteins into endoplasmic reticulum-derived multilamellar bodies.

Oligomeric assembly of neurotransmitter transporters is a prerequisite for their export from the endoplasmic reticulum (ER) and their subsequent delivery to the neuronal synapse. We previously identified mutations, e.g., in the gamma-aminobutyric acid (GABA) transporter-1 (GAT1), which disrupted assembly and caused retention of the transporter in the ER. Using one representative mutant, GAT1-E101D, we showed here that ER retention was due to association of the transporter with the ER chaperone calnexin: interaction with calnexin led to accumulation of GAT1 in concentric bodies corresponding to previously described multilamellar ER-derived structures. The transmembrane domain of calnexin was necessary and sufficient to direct the protein into these concentric bodies. Both yellow fluorescent protein-tagged versions of wild-type GAT1 and of the GAT1-E101D mutant remained in disperse (i.e., non-aggregated) form in these concentric bodies, because fluorescence recovered rapidly (t(1/2) approximately 500 ms) upon photobleaching. Fluorescence energy resonance transfer microscopy was employed to visualize a tight interaction of GAT1-E101D with calnexin. Recognition by calnexin occurred largely in a glycan-independent manner and, at least in part, at the level of the transmembrane domain. Our findings are consistent with a model in which the transmembrane segment of calnexin participates in chaperoning the inter- and intramolecular arrangement of hydrophobic segment in oligomeric proteins.

Concentrative export from the endoplasmic reticulum of the gamma-aminobutyric acid transporter 1 requires binding to SEC24D.

Re-uptake of gamma-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 ((566)RL(567)), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D ((733)DD(734)) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of (733)DD(734) by (733)VN(734)), and mutation of (566)RL(567) to (566)AS(567) (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components.

The conserved glutamate (Glu136) in transmembrane domain 2 of the serotonin transporter is required for the conformational switch in the transport cycle.

The alternate access model provides the theoretical framework for understanding how transporters translocate hydrophilic substrates across the lipid bilayer. The model postulates at least two conformations of a transporter, an outward and an inward facing conformation, which seal the translocation pathway to the interior and exterior of the cell, respectively. It is not clear how the conformational switch is triggered in neurotransmitter/sodium symporters, but Na+ is likely to play an essential role. Here, we focused on Glu136 of the serotonin transporter (SERT); this residue is conserved in transmembrane domain 2 of neurotransmitter/sodium symporters and related proteins. Three substitutions were introduced, resulting in SERT-E136D, SERT-E136Q, and SERT-E136A, which were all correctly inserted into the plasma membrane. SERT-E136Q and SERT-E136A failed to support substrate influx into cells, whereas SERT-E136D did so at a reduced rate. Binding experiments with the inhibitor 2beta-[3H]carbomethoxy-3beta-(4-iodophenyl)tropane (beta-[3H]CIT) supported the conjecture that the mutant transporters preferentially adopted the inward facing conformation: beta-[3H]CIT interacted with SERT in a manner consistent with binding to the outward facing state. Accordingly, the Na+-induced acceleration of beta-[3H]CIT association was most pronounced in wild-type SERT, followed by SERT-E136D > SERT-E136Q > SERT-E136A. Similarly, SERT-E136Q supported substrate efflux in a manner indistinguishable from wild-type SERT, whereas SERT-E136A was inactive. Thus, in the absence of Glu136, the conformational equilibrium of SERT is shifted progressively (SERT-E136D > SERT-E136Q > SERT-E136A) to the inward facing conformation.

The ubiquitin-specific protease Usp4 regulates the cell surface level of the A2A receptor.

Many membrane proteins incur a folding problem during biosynthesis; only a fraction thereof is exported from the endoplasmic reticulum (ER), because quality control is stringent. This is also true for G protein-coupled receptors. Here, we identify the deubiquitinating enzyme Usp4 as an interaction partner of the A2a adenosine receptor, a Gs-coupled receptor. Usp4 binds to the carboxyl terminus of the A2A receptor and allows for its accumulation as deubiquinated protein. This relaxes ER quality control and enhances cell surface expression of functionally active receptor. The effect of Usp4 on the A2A receptor was specific because 1) it was not seen in C-terminally truncated versions of the receptor; 2) it was not mimicked by Usp14, another member of the ubiquitin-specific protease family; and 3) it was not seen with the metabotropic glutamate receptor-5, another G protein-coupled receptor with a high propensity for intracellular retention. These observations show that deubiquinating enzymes can regulate quality control in the ER.

Oligomerization of the {gamma}-aminobutyric acid transporter-1 is driven by an interplay of polar and hydrophobic interactions in transmembrane helix II.

The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers. Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization. In artificial transmembrane segments, interhelical interactions are stabilized by polar residues. We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the gamma-aminobutyric acid transporter-1 (GAT1). We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and beta-lactamase fragment complementation. Of all generated mutants, only Y86A- (but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly. The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Tyr(86) and Glu(101); Tyr(86) participates in hydrophobic stacking. Glu(101) is in the a-position of the leucine heptad repeat (where positions 1-7 are denoted a-g, and each leucine is in the central d-position). Thus, Glu(101) is in the position predicted for the hydrogen bond donor (i.e. sandwiched between Leu(97) and Leu(104), which are one helical turn above and below Glu(101)). These key residues, namely Tyr(86) and Glu(101), are conserved in related transporters from archaeae to humans; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments.

Two discontinuous segments in the carboxyl terminus are required for membrane targeting of the rat gamma-aminobutyric acid transporter-1 (GAT1).

Like all members of the Na(+)/Cl(-)-dependent neurotransmitter transporter family, the rat gamma-aminobutyric acid transporter-1 (GAT1) is sorted and targeted to specialized domains of the cell surface. Here we identify two discontinuous signals in the carboxyl terminus of GAT1 that cooperate to drive surface expression. This conclusion is based on the following observations. Upon deletion of the last 37 amino acids, the resulting GAT1-Delta37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-Delta27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-Delta27 reached 50% of the wild type level. Additional extensions up to the position -3 (GAT1-Delta3) did not further enhance surface expression. Thus the last three amino acids (AYI) comprise a second distal signal. The sequence AYI is reminiscent of a type II PDZ-binding motif; accordingly substituting Glu for Ile abrogated the effect of this motif. Neither the AYI motif nor the last 10 residues rescued the protein from intracellular retention when grafted onto GAT1-Delta37 and GAT1-Delta32; the AYI motif was dispensable for targeting of GAT1 to the growth cone of differentiating PC12 cells. We therefore conclude that the two segments act in a hierarchical manner such that the proximal motif ((569)VMI(571)) supports endoplasmic reticulum export of the protein and the distal AYI motif places GAT1 under the control of the exocyst.