A Call for Systematic Research on Solute Carriers

Solute carrier (SLC) membrane transport proteins control essential physiological functions, including nutrient uptake, ion transport, and waste removal. SLCs interact with several important drugs, and a quarter of the more than 400 SLC genes are associated with human diseases. Yet, compared to other gene families of similar stature, SLCs are relatively understudied. The time is right for a systematic attack on SLC structure, specificity, and function, taking into account kinship and expression, as well as the dependencies that arise from the common metabolic space.

NaSi-1 and Sat-1: structure, function and transcriptional regulation of two genes encoding renal proximal tubular sulfate transporters

Sulfate (SO42-) is an important anion regulating many metabolic and cellular processes. Maintenance Of SO42- homeostasis occurs in the renal proximal tubule via membrane transport proteins. Two SO42- transporters that have been characterized and implicated in regulating serum SO42- levels are: NaSi- 1, a Na+-SO4 (2-) cotransporter located at the brush border membrane and Sat-1, a SO4 (2-) -anion exchanger located on the basolateral membranes of proximal tubular cells. Unlike Sat-1, for which very few studies have looked at regulation of its expression, NaSi- 1 has been shown to be regulated by various hormones and dietary conditions in vivo. To study this further, NaSj- I (SLC13A1) and Sat- I (SLC26A1) gene structures were determined and recent studies have characterized their respective gene promoters. This review presents the current understanding of the transcriptional regulation of NaSj- I and Sat- 1, and describes possible pathogenetic implications which arise as a consequence of altered SO(4)(2-)homeostasis. (c) 2005 Elsevier Ltd. All rights reserved.

Experiment: Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid-base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H(+)-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH(e) and pH(i)) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO(2) conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO(2). Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH(e) whenever seawater pH changes. However, measurements of pH(i) demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na(+) and HCO(3)(-), suggesting a bicarbonate buffer mechanism involving secondary active Na(+)-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH(i) enables calcification to proceed despite decreased pH(e). However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.

Haem uptake is essential for egg production in the haematophagous blood fluke of humans, Schistosoma mansoni

Schistosomes ingest host erythrocytes, liberating large quantities of haem. Despite its toxicity, haem is an essential factor for numerous biological reactions, and may be an important iron source for these helminths. We used a fluorescence haem analogue, palladium mesoporphyrin, to investigate pathways of haem acquisition, and showed that palladium mesoporphyrin accumulates in the vitellaria (eggshell precursor glands) and ovary of female Schistosoma mansoni. Furthermore, incubation of adult females in 10-100 μm cyclosporin A (IC50 = 2.3 μm) inhibits the uptake of palladium mesoporphyrin to these tissues, with tenfold reductions in fluorescence intensity of the ovary. In vitro exposure to cyclosporin A resulted in significant perturbation of egg production, reducing egg output from 34 eggs per female to 5.7 eggs per female over the incubation period, and retardation of egg development. We characterized a S. mansoni homologue of the haem-responsive genes of Caenorhabditis elegans. The gene (Smhrg-1) encodes a protein with a molecular weight of approximately 17 kDa. SmHRG-1 was able to rescue growth in haem transport-deficient HEM1Δ yeast. Transcriptional suppression of Smhrg-1 in adult S. mansoni worms resulted in significant delay in egg maturation, with 47% of eggs from transcriptionally suppressed worms being identified as immature compared with only 27% of eggs laid by control worms treated with firefly luciferase. Our findings indicate the presence of transmembrane haem transporters in schistosomes, with a high abundance of these molecules being present in tissues involved in oogenesis.

Identification de nouveaux partenaires protéiques des récepteurs couplés aux protéines G contrôlant leur transport du reticulum endoplasmique à la membrane plasmique

Les récepteurs couplés aux protéines G (RCPGs) forment la plus grande et la plus diversifiée des familles de protéines localisées à la surface cellulaire et responsables de la transmission de signaux à l’intérieur des cellules. D’intenses recherches effectuées au cours des trente dernières années ont mené à l’identification de dizaines de protéines interagissant avec les RCPGs et contrôlant la signalisation, la désensibilisation, l’internalisation et la dégradation de ces importantes cibles pharmacologiques. Contrairement aux processus régulant l’activité des récepteurs à partir de la membrane plasmique, les mécanismes moléculaires contrôlant la biosynthèse des RCPGs dans le reticulum endoplasmique (RE) et leur transport jusqu’à la surface cellulaire sont très peu caractérisés. Une meilleure compréhension de ces processus nécessite l’identification de la machinerie protéique responsable de la maturation des RCPGs. Un crible protéomique basé sur le transfert d’énergie de résonance de bioluminescence (BRET), qui permet la mesure d’interactions protéiques dans les cellules vivantes, a mené à l’identification de plusieurs nouvelles protéines localisées dans la voie de sécrétion et interagissant potentiellement avec les RCPGs. Ces protéines étant localisées dans les compartiments cellulaires (reticulum endoplasmique et appareil de Golgi) responsables de la synthèse, du repliement adéquat et du transport à la membrane plasmique des récepteurs, il est très probable qu’elles soient impliquées dans le contrôle de l’expression des RCPGs à la surface cellulaire. La caractérisation de l’homologue humain de cornichon 4 (CNIH4), un nouvel intéracteur des RCPGs identifié dans le crible, a démontré que cette protéine localisée dans les compartiments précoces de la voie de sécrétion (RE et ERGIC) interagit de façon sélective avec les RCPGs. De plus, la suppression de l’expression endogène de cette protéine préalablement non-caractérisée, diminue le transport à la membrane plasmique d’un récepteur, indiquant que CNIH4 influence positivement l’export des RCPGs du RE. Ceci est supporté par l’observation que la surexpression de CNIH4 à de faibles niveaux favorise la maturation d’un récepteur mutant normalement retenu dans le RE. Nous avons également pu démontrer que CNIH4 est associée à la protéine Sec23, une des composantes de l’enveloppe des vésicules COPII qui sont responsables du transport des protéines du RE vers le Golgi, suggérant que CNIH4 pourrait favoriser le recrutement des récepteurs dans ces vésicules. La surexpression de CNIH4 à de très hauts niveaux provoque également la rétention intracellulaire des récepteurs. Cet effet dominant négatif pourrait être causé par la titration d’un autre facteur d’export des RCPGs. Une deuxième étude a permis de révéler que la protéine transmembranaire 9 (TMEM9), un nouvel intéracteur des RCPGs également identifié dans le crible, interagit sélectivement avec les récepteurs et avec CNIH4. La surexpression de cette protéine aux fonctions précédemment inconnues, rétablit le transport normal d’un récepteur en présence de CNIH4 surexprimée. De plus, la co-expression de TMEM9 potentialise la capacité de CNIH4 à augmenter la maturation d’un récepteur mutant normalement retenu dans le RE, suggérant que ces deux protéines forment un complexe régulant la maturation des RCPGs. Au cours de cette thèse, de nouvelles protéines interagissant avec les RCPGs et contrôlant leur expression à la membrane plasmique ont donc été identifiées, permettant une meilleure compréhension des mécanismes régulant le transport des récepteurs du RE à la surface cellulaire.

Vibrational spectroscopic and electrochemical investigations of multi-centered heme proteins in biomimetic membrane architectures

Membrane proteins play a major role in every living cell. They are the key factors in the cell’s metabolism and in other functions, for example in cell-cell interaction, signal transduction, and transport of ions and nutrients. Cytochrome c oxidase (CcO), as one of the membrane proteins of the respiratory chain, plays a significant role in the energy transformation of higher organisms. CcO is a multi centered heme protein, utilizing redox energy to actively transport protons across the mitochondrial membrane. One aim of this dissertation is to investigate single steps in the mechanism of the ion transfer process coupled to electron transfer, which are not fully understood. The protein-tethered bilayer lipid membrane is a general approach to immobilize membrane proteins in an oriented fashion on a planar electrode embedded in a biomimetic membrane. This system enables the combination of electrochemical techniques with surface enhanced resonance Raman (SERRS), surface enhanced reflection absorption infrared (SEIRAS), and surface plasmon spectroscopy to study protein mediated electron and ion transport processes. The orientation of the enzymes within the surface confined architecture can be controlled by specific site-mutations, i.e. the insertion of a poly-histidine tag to different subunits of the enzyme. CcO can, thus, be oriented uniformly with its natural electron pathway entry pointing either towards or away from the electrode surface. The first orientation allows an ultra-fast direct electron transfer(ET) into the protein, not provided by conventional systems, which can be leveraged to study intrinsic charge transfer processes. The second orientation permits to study the interaction with its natural electron donor cytochrome c. Electrochemical and SERR measurements show conclusively that the redox site structure and the activity of the surface confined enzyme are preserved. Therefore, this biomimetic system offers a unique platform to study the kinetics of the ET processes in order to clarify mechanistic properties of the enzyme. Highly sensitive and ultra fast electrochemical techniques allow the separation of ET steps between all four redox centres including the determination of ET rates. Furthermore, proton transfer coupled to ET could be directly measured and discriminated from other ion transfer processes, revealing novel mechanistic information of the proton transfer mechanism of cytochrome c oxidase. In order to study the kinetics of the ET inside the protein, including the catalytic center, time resolved SEIRAS and SERRS measurements were performed to gain more insight into the structural and coordination changes of the heme environment. The electrical behaviour of tethered membrane systems and membrane intrinsic proteins as well as related charge transfer processes were simulated by solving the respective sets of differential equations, utilizing a software package called SPICE. This helps to understand charge transfer processes across membranes and to develop models that can help to elucidate mechanisms of complex enzymatic processes.

Atomic force microscopy for the study of membrane proteins

Fundamental biological processes such as cell-cell communication, signal transduction, molecular transport and energy conversion are performed by membrane proteins. These important proteins are studied best in their native environment, the lipid bilayer. The atomic force microscope (AFM) is the instrument of choice to determine the native surface structure, supramolecular organization, conformational changes and dynamics of membrane-embedded proteins under near-physiological conditions. In addition, membrane proteins are imaged at subnanometer resolution and at the single molecule level with the AFM. This review highlights the major advances and results achieved on reconstituted membrane proteins and native membranes as well as the recent developments of the AFM for imaging.

Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity.

The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.

Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN

The four mammalian golgins, p230/golgin-245, golgin-97, GCC88 and GCC185 are targeted to trans-Golgi network ITGN) membranes by their C-terminal GRIP domain in a G-protein-dependent process. The Arf-like GTPase, Arl1, has been shown to mediate TGN recruitment of p230/golgin245 and golgin-97 by interaction with their GRIP domains; however, it is not known whether all the TGN golgins bind to Arl1 and whether they are all recruited to the same or different TGN domains. Here we demonstrate differences in membrane binding properties and TGN domain recruitment of the mammalian GRIP domain proteins. Overexpression of full-length GCC185 resulted in the appearance of small punctate structures dispersed in the cytoplasm of transfected cells that were identified as membrane tubular structures by immunoelectron microscopy. The cytoplasmic GCC185-labelled structures were enriched for membrane binding determinants of GCC185 GRIP, whereas the three other mammalian GRIP family members did not colocalize with the GCC185-labelled structures. These GCC185-labelled structures included the TGN resident protein alpha2,6 sialyltransferase and excluded the recycling TGN protein, TGN46. The Golgi stack was unaffected by overexpression of GCC185. Overexpression of both full-length GCC185 and GCC88 showed distinct and nonoverlapping structures. We also show that the GRIP domains of GCC185 and GCC88 differ in membrane binding properties from each other and, in contrast to p230/golgin245 and golgin-97, do not interact with Arl1 in vivo. Collectively these results show that GCC88, GCC185 and p230/golgin245 are recruited to functionally distinct domains of the TGN and are likely to be important for the maintenance of TGN subdomain structure, a critical feature for mediating protein sorting and membrane transport.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis.

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.

Amino acid interaction preferences in helical membrane proteins

Membrane proteins are involved in a number of important biological functions. Yet, they are poorly understood from the structure and folding point of view. The external environment being drastically different from that of globular proteins, the intra-protein interactions in membrane proteins are also expected to be different. Hence, statistical potentials representing the features of inter-residue interactions based exclusively on the structures of membrane proteins are much needed. Currently, a reasonable number of structures are available, making it possible to undertake such an analysis on membrane proteins. In this study we have examined the inter-residue interaction propensities of amino acids in the membrane spanning regions of the alpha-helical membrane (HM) proteins. Recently we have shown that valuable information can be obtained on globular proteins by the evaluation of the pair-wise interactions of amino acids by classifying them into different structural environments, based on factors such as the secondary structure or the number of contacts that a residue can make. Here we have explored the possible ways of classifying the intra-protein environment of HM proteins and have developed scoring functions based on different classification schemes. On evaluation of different schemes, we find that the scheme which classifies amino acids to different intra-contact environment is the most promising one. Based on this classification scheme, we also redefine the hydrophobicity scale of amino acids in HM proteins.

Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-beta Conformation

Identifying the structures of membrane bound proteins is critical to understanding their function in healthy and diseased states. We introduce a surface enhanced Raman spectroscopy technique which can determine the conformation of membrane-bound proteins, at low micromolar concentrations, and also in the presence of a substantial membrane-free fraction. Unlike conventional surface enhanced Raman spectroscopy, our approach does not require immobilization of molecules, as it uses spontaneous binding of proteins to lipid bilayer-encapsulated Ag nanoparticles. We apply this technique to probe membrane-attached oligomers of Amyloid-beta(40) (A beta(40)), whose conformation is keenly sought in the context of Alzheimer's disease. Isotope-shifts in the Raman spectra help us obtain secondary structure information at the level of individual residues. Our results show the presence of a beta-turn, flanked by two beta-sheet regions. We use solid-state NMR data to confirm the presence of the beta-sheets in these regions. In the membrane-attached oligomer, we find a strongly contrasting and near-orthogonal orientation of the backbone H-bonds compared to what is found in the mature, less-toxic A beta fibrils. Significantly, this allows a ``porin'' like beta-barrel structure, providing a structural basis for proposed mechanisms of A beta oligomer toxicity.

Coordination of platinum therapeutic agents to met-rich motifs of human copper transport protein1.

Platinum therapeutic agents are widely used in the treatment of several forms of cancer. Various mechanisms for the transport of the drugs have been proposed including passive diffusion across the cellular membrane and active transport via proteins. The copper transport protein Ctr1 is responsible for high affinity copper uptake but has also been implicated in the transport of cisplatin into cells. Human hCtr1 contains two methionine-rich Mets motifs on its extracellular N-terminus that are potential platinum-binding sites: the first one encompasses residues 7-14 with amino acid sequence Met-Gly-Met-Ser-Tyr-Met-Asp-Ser and the second one spans residues 39-46 with sequence Met-Met-Met-Met-Pro-Met-Thr-Phe. In these studies, we use liquid chromatography and mass spectrometry to compare the binding interactions between cisplatin, carboplatin and oxaliplatin with synthetic peptides corresponding to hCtr1 Mets motifs. The interactions of cisplatin and carboplatin with Met-rich motifs that contain three or more methionines result in removal of the carrier ligands of both platinum complexes. In contrast, oxaliplatin retains its cyclohexyldiamine ligand upon platinum coordination to the peptide.

Nuclear trafficking and functions of endocytic proteins implicated in oncogenesis

A subset of proteins predominantly associated with early endosomes or implicated in clathrin-mediated endocytosis can shuttle between the cytoplasm and the nucleus. Although the endocytic functions of these proteins have been extensively studied, much less effort has been expended in exploring their nuclear roles. Membrane trafficking proteins can affect signalling and proliferation and this can be achieved either at a nuclear or endocytic level. Furthermore, some proteins, such as Huntingtin interacting protein 1, are known as cancer biomarkers. This review will highlight the limits of our understanding of their nuclear functions and the relevance of this to signalling and oncogenesis.