The ABCs of membrane transporters in health and disease (SLC series): introduction

The field of transport biology has steadily grown over the past decade and is now recognized as playing an important role in manifestation and treatment of disease. The SLC (solute carrier) gene series has grown to now include 52 families and 395 transporter genes in the human genome. A list of these genes can be found at the HUGO Gene Nomenclature Committee (HGNC) website (see www.genenames.org/genefamilies/SLC). This special issue features mini-reviews for each of these SLC families written by the experts in each field. The existing online resource for solute carriers, the Bioparadigms SLC Tables (www.bioparadigms.org), has been updated and significantly extended with additional information and cross-links to other relevant databases, and the nomenclature used in this database has been validated and approved by the HGNC. In addition, the Bioparadigms SLC Tables functionality has been improved to allow easier access by the scientific community. This introduction includes: an overview of all known SLC and "non-SLC" transporter genes; a list of transporters of water soluble vitamins; a summary of recent progress in the structure determination of transporters (including GLUT1/SLC2A1); roles of transporters in human diseases and roles in drug approval and pharmaceutical perspectives.

Mechanism for active membrane fusion triggering by morbillivirus attachment protein.

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.

AGROBACTERIUM VIRB10 CONTRIBUTIONS TO TYPE IV SUBSTRATE SECRETION, T-PILUS BIOGENESIS, AND OUTER MEMBRANE PORE FORMATION

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) identify residues/domains contributing to VirB10 stability, oligomerization, and function, and (iii) monitor structural changes accompanying energy activation or substrate translocation. These studies were aided by recent structural resolution of a periplasmic domain of a VirB10 homolog and a ‘core’ complex composed of homologs of VirB10 and two outer membrane associated subunits, VirB7 and VirB9. By use of the substituted cysteine accessibility method (SCAM), I confirmed the bitopic topology of VirB10. Through phenotypic studies of Ala-Cys insertion mutations, I identified “uncoupling” mutations in the TM and β-barrel domains that blocked T-pilus assembly but permitted substrate transfer. I showed that cysteine replacements in the C-terminal periplasmic domain yielded a variety of phenotypes in relation to protein accumulation, oligomerization, substrate transfer, and T-pilus formation. By SCAM, I also gained further evidence that VirB10 adopts different structural states during machine biogenesis. Finally, I showed that VirB10 supports substrate transfer even when its TM domain is extensively mutagenized or substituted with heterologous TM domains. By contrast, specific residues most probably involved in oligomerization of the TM domain are required for biogenesis of the T-pilus.

Active water in protein-protein communication within the membrane: the case of SRII-HtrII signal relay.

We detect internal water molecules in a membrane-embedded receptor-transducer complex and demonstrate water structure changes during formation of the signaling state. Time-resolved FTIR spectroscopy reveals stimulus-induced repositioning of one or more structurally active water molecules to a significantly more hydrophobic environment in the signaling state of the sensory rhodopsin II (SRII)-transducer (HtrII) complex. These waters, distinct from bound water molecules within the SRII receptor, appear to be in the middle of the transmembrane interface region near the Tyr199(SRII)-Asn74(HtrII) hydrogen bond. We conclude that water potentially plays an important role in the SRII --> HtrII signal transfer mechanism in the membrane's hydrophobic core.

Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive inside rule.

Transmembrane domain orientation within some membrane proteins is dependent on membrane lipid composition. Initial orientation occurs within the translocon, but final orientation is determined after membrane insertion by interactions within the protein and between lipid headgroups and protein extramembrane domains. Positively and negatively charged amino acids in extramembrane domains represent cytoplasmic retention and membrane translocation forces, respectively, which are determinants of protein orientation. Lipids with no net charge dampen the translocation potential of negative residues working in opposition to cytoplasmic retention of positive residues, thus allowing the functional presence of negative residues in cytoplasmic domains without affecting protein topology.

Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Cardiolipin membrane domains in prokaryotes and eukaryotes.

Cardiolipin (CL) plays a key role in dynamic organization of bacterial and mitochondrial membranes. CL forms membrane domains in bacterial cells, and these domains appear to participate in binding and functional regulation of multi-protein complexes involved in diverse cellular functions including cell division, energy metabolism, and membrane transport. Visualization of CL domains in bacterial cells by the fluorescent dye 10-N-nonyl acridine orange is critically reviewed. Possible mechanisms proposed for CL dynamic localization in bacterial cells are discussed. In the mitochondrial membrane CL is involved in organization of multi-subunit oxidative phosphorylation complexes and in their association into higher order supercomplexes. Evidence suggesting a possible role for CL in concert with ATP synthase oligomers in establishing mitochondrial cristae morphology is presented. Hypotheses on CL-dependent dynamic re-organization of the respiratory chain in response to changes in metabolic states and CL dynamic re-localization in mitochondria during the apoptotic response are briefly addressed.

Redistribution of plasma membrane phosphatidylserine during erythroid development

Phosphatidylserine (PS) is not only one of the structural components of the plasma membrane, it also plays an important role in blood coagulation, and cell-cell interactions during aging and apoptosis.^ Here we studied some alterations that occur in membrane phosphatidylserine asymmetry during erythroid differentiation-associated apoptosis and erythrocyte aging and characterized some aspects in the regulation of PS asymmetry.^ Erythroleukemia cells, frequently used to study erythroid development, undergo apoptosis when induced to differentiate along the erythroid lineage. In the case of K562 cells induced to differentiate with hemin, this event is characterized by DNA fragmentation that correlates with downregulation of the survival protein BCL-xL and ultimately the result is cell death. We showed here that reorientation of PS from the inner-to-outer plasma membrane leaflet and inhibition of the aminophospholipid translocase are also events observed upon hemin treatment. We observed that constitutive expression of BCL-2 did not inhibit the alterations caused by hemin in membrane lipid asymmetry and only slightly prevented hemin-induced DNA fragmentation. On the other hand, BCL-2 effectively inhibited actinomycin D and staurosporine-induced DNA fragmentation and the appearance of PS at the outer leaflet of these cells. z.VAD.fmk, a widely used caspases inhibitor, blocked DNA fragmentation induced by both hemin and actinomycin D but only inhibited PS externalization in cells treated with actinomycin D.^ These results showed that PS externalization occurs during differentiation-related apoptosis. Unlike the pharmacologically-induced event, however, hemin-induced PS redistribution seems to be regulated by a mechanism independent of BCL-2 and caspases.^ Membrane PS is externalized not only during apoptosis but also during red blood cell senescence. To study this event, we artificially induced cellular aging by in vitro storage or vesiculation in the presence of the amphipathic lipid dilauroylphosphatidylcholine. These cells were monitored for age-dependent changes in cell density by Percoll gradient centrifugation and assessed for alterations in membrane lipid asymmetry and their ability to be cleared in vivo. These experiments demonstrated a progressive increase in red cell density upon vesiculation and in vitro aging. The clearance rate of cells obtained after vesiculation, was biphasic in nature, showing a very rapid component together with a second component consistent with the clearance rates of control populations. Quantitation of PS in the outer leaflet of red cells revealed that membrane redistribution of PS occurred upon in vitro storage and vesiculation. Inhibition of the aminophospholipid translocase with the sulfhydryl-oxidant reagent pyridyldithioethylamine resulted in higher PS externalization and enhanced clearance of vesiculated RBC.^ These observations not only suggest that vesiculation may be the mechanism responsible for some of the characteristic changes in cell density and PS asymmetry that occur upon cell aging, but also confirm the role of PS in the recognition and clearance of senescent cells. ^

Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Rapid effects of 1alpha,25-dihydroxyvitamin D3 on osteoblasts: Studies of membrane transducers that affect calcium signals

1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] exerts pleiotropic effects on osteoblasts via both long-term nuclear receptor-mediated and rapid membrane-initiated pathways during bone remodeling and mineral homeostasis. This study explored the membrane transducers that mediate rapid effects of 1,25(OH)2D3 on osteoblasts, including sphingomyelinase (SMase) and L-type voltage sensitive calcium channels (VSCCs). ^ It was previously demonstrated that 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through VSCCs in ROS 17/2.8 osteoblasts, however the molecular identity of 1,25(OH)2D 3-regulated VSCC has not been known. In this study, on the basis of in vitro tests of three unique ribozymes specifically cleaving a1C mRNA, I transfected ROS 17/2.8 cells with vectors coding recombinant ribozyme modified with U1 snRNA structure, and successfully selected stable clonal cells in which the expression of a1C was strikingly reduced. Ca2+ influx studies in these cells compared to control transfectants showed selective attenuation of depolarization- and 1,25(OH)2D3-regulated Ca2+ responses. These results allow us to conclude that the cardiac ( a1C ) subtype of the L-type VSCC is the major membrane transducer of Ca 2+ influx in osteoblasts. ^ I also demonstrated that 1,25(OH)2D3 induces a rapid hydrolysis of membrane sphingomyelin (SM) in ROS 17/2.8 cells, with the concomitant generation of ceramide, detectable at 15 minute, and maximal at 1 hour after addition. Sphingosine, sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC), downstream products of SM hydrolysis, but not ceramide, elicit Ca 2+ release from intracellular stores. Considering ceramide, sphingosine, and SPP as second messengers modulating intracellular kinases or phosphatases, these findings implicate sphingolipid-signaling pathways in transducing rapid effects of 1,25(OH)2D3 on osteoblasts. In structure/function analyses of sphingolipid signaling, it was observed that psychosine elicits Ca2+ release from intracellular stores. This challenges the dogma that sphingosine phosphorylation permits mobilization of Ca2+ , because psychosine is a sphingosine analog galactosylated at 1-carbon, preventing phosphorylation at that site. Psychosine is the pathological metabolite found in patients with Krabbe's disease, suggesting that psychosine disrupts the physiological sphingolipid signaling by chronic release of Ca2+ from intracellular stores. ^ Slower SM turnover than Ca2+ influx through VSCCs in response to 1,25(OH)2D3 demonstrates ceramide does not mediate the 1,25(OH)2D3-induced Ca2+ signaling, a conclusion endorsed further by the failure of ceramide to induce Ca 2+ signaling. ^

Ceramide in Plasma Membrane Repair

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca2+ and an efflux of cytoplasmic proteins. In order to ensure cellular survival, lesions have to be identified, plugged and removed from the membrane. The Ca2+-driven fusion of lysosomes with the plasma membrane leads to hydrolysis of sphingomyelin by acid sphingomyelinase and a formation of ceramide platforms in the outer leaflet of the lipid bilayer. We propose that the negative curvature, promoted by tighter packing of lipids in the outer layer, leads to an inward vesiculation of the damaged area for its endocytotic uptake and internal degradation. In contrast, the activation of neutral sphingomyelinase triggers the production of ceramide within the inner leaflet of the lipid bilayer, thereby promoting an outward curvature, which enables the cell to shed the membrane-containing toxin pore into the extracellular space. In this process, ceramide is supported by members of the annexin protein family which act as Ca2+ sensors and as membrane fusion agents.