C-terminal Cytoplasmic Tail Research Articles

The Crystal Structure of the Plexin-Semaphorin-Integrin Domain/Hybrid Domain/I-EGF1 Segment from the Human Integrin β2 Subunit at 1.8-Å Resolution

Integrins are modular (alphabeta) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane. Interdomain motions play a key role in signal transduction by propagating structural changes through the molecule, thus controlling the activation state and adhesive properties of the integrin. We expressed a soluble fragment of the human integrin beta2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-A resolution. The structure reveals an elongated molecule with a rigid architecture stabilized by nine disulfide bridges. The PSI domain is located centrally and participates in the formation of extended interfaces with the hybrid domain and I-EGF1 domains, respectively. The hybrid domain/PSI interface involves the burial of an Arg residue, and contacts between PSI and I-EGF1 are mainly mediated by well conserved Arg and Trp residues. Conservation of key interacting residues across the various integrin beta subunits sequences suggests that our structure represents a good model for the entire integrin family. Superposition with the integrin beta3 receptor in its bent conformation suggests that an articulation point is present at the linkage between its I-EGF1 and I-EGF2 modules and underlines the importance of this region for the control of integrin-mediated cell adhesion.

Identification of CD36 molecular features required for its in vitro angiostatic activity

Thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP-1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor-A (VEGF-A). To demonstrate that the TSP-1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36-deficient endothelial cells. Both TSP-1 and the agonist anti-CD36 mAb SMO, which mimics TSP-1 activity, reduced the VEGF-A165-induced migration and sprouting of CD36-ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C-terminal cytoplasmic tail by site-directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP-1. In contrast, point mutation of C466 did not alter TSP-1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP-1 down-modulates the VEGF receptor-2 (VEGFR-2) and p38 mitogen-associated protein kinase phosphorylation induced by VEGF-A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C-terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP-1 and extend our knowledge of regulation of VEGFR-2-mediated biological activities on ECs.

Comparative genomics on Vangl1 and Vangl2 genes

WNT signals are transduced to the beta-catenin pathway or the planar cell polarity (PCP) pathway. WNT - beta-catenin pathway is implicated in carcinogenesis, while WNT-PCP pathway is implicated in cell motility and metastasis. Drosophila Van Gogh (Vang), Frizzled (Fz), Starry night (Stan), Prickle (Pk) and Diego (Dgo) are PCP signaling molecules. Vangl1 (Strabismus 2) and Vangl2 (Strabismus 1 or Ltap) are mammalian homologs of Drosophila Vang interacting with PRICKLE1, PRICKLE2, ANKRD6, DVL1, DVL2, DVL3, KAI1 and MAGI3. Here we identified and characterized rat Vangl1 and Vangl2 genes by using bioinformatics. Rat Vangl1 gene, consisting of eight exons, was located within AC098913.7 and AC108524.6 genome sequences. Rat Vangl2 gene, consisting of eight exons, was located within AC118856.3 and AC115243.5 genome sequences. Exon-intron structure of mammalian Vangl1 and Vangl2 orthologs was well conserved. E47 and double ELK1-binding sites were conserved among promoters of mammalian Vangl1 orthologs. PAX4, NFkappaB, HNF4, SOX9, RFX1, and POU2F1 (OCT1)-binding sites were conserved among promoters of mammalian Vangl2 orthologs. Rat Vangl1 (526 aa) and Vangl2 (521 aa) were four-transmembrane proteins with 71.5% total-amino-acid identity. Ser cluster motif (SxxSxxSxxSxxSxxS) in the N-terminal cytoplasmic region and PDZ-binding motif in the C-terminal cytoplasmic tail were evolutionarily conserved among vertebrate Vangl1 and Vangl2 orthologs. This is the first report on rat Vangl1 and Vangl2 genes as well as on comparative genomics for Vangl1 and Vangl2 orthologs.

The Δe13 Isoform of the Calcitonin Receptor Forms a Six-Transmembrane Domain Receptor with Dominant-Negative Effects on Receptor Surface Expression and Signaling

The CTRdelta e13 splice variant of the rabbit calcitonin receptor, which lacks the 14 amino acids of the seventh transmembrane domain (TMD) that are encoded by exon 13, is poorly expressed on the cell surface, fails to mobilize intracellular calcium or activate Erk, and inhibits the cell surface expression of the full-length C1a isoform. Nuclear magnetic resonance- and fluorescence-activated cell sorter-based experiments showed that the residual seventh TMD of CTRdelta e13 fails to partition into the lipid bilayer, resulting in an extracellular C terminus. Truncating the receptor after residue 397 to delete the cytoplasmic tail resulted in reduced cell surface expression and an inability to mobilize intracellular calcium or activate Erk, but the truncated receptor did not inhibit C1a cell surface expression. In contrast, when the receptor was truncated after residue 374 to eliminate the entire seventh TMD domain and the C-terminal domain, the resulting receptor reduced the cell surface expression of C1a in a manner similar to that of CTRdelta e13. Thus, normal cell surface expression, mobilization of intracellular calcium, and Erk activation requires the cytoplasmic C-terminal tail of the CTR, whereas the absence of the seventh TMD in the transmembrane helical bundle causes the dominant-negative effect on the surface expression of C1a.

Secretory carrier membrane proteins interact and regulate trafficking of the organellar (Na+,K+)/H+ exchanger NHE7

The mammalian (Na(+),K(+))/H(+) exchanger NHE7 resides chiefly in the trans-Golgi network (TGN) and post-Golgi vesicles where it is thought to contribute to organellar pH homeostasis. However, the mechanisms that underlie the targeting and regulation of NHE7 are unknown. To gain insight into these processes, yeast two-hybrid methodology was used to screen a human brain cDNA library for proteins that interact with the cytoplasmic C-terminus of NHE7. One binding partner we identified was SCAMP2, a member of the secretory carrier membrane protein (SCAMP) gene family. Direct association of these two proteins was further supported by co-immunolocalization and co-immunoprecipitation analyses using transfected cells, by their co-sedimentation in membrane fractions resolved on sucrose density gradients, and by in vitro protein binding assays. Other members of the SCAMP family, such as SCAMP1 and SCAMP5, also associated with NHE7. The majority of the NHE7-SCAMP complexes accumulated at the TGN, but a minor fraction also resided in recycling vesicles. Biochemical analyses indicated that the C-terminal cytoplasmic tail of NHE7 bound preferentially to a highly conserved cytoplasmic loop between the second and the third transmembrane segments (TM2-TM3 loop) of SCAMP2. A deletion mutant of SCAMP2 lacking this region (SCAMP2/Delta184-208) bound weakly to NHE7, but caused a significant fraction of NHE7 and wild-type SCAMP2 to redistribute to a pool of scattered recycling vesicles without noticeably affecting the location of other resident TGN (syntaxin 6) or Golgi cisternae (GM130) proteins. Conversely, a GFP-tagged TM2-TM3 construct of SCAMP2 interacted with NHE7, but also led to the redistribution of NHE7 to dispersed vesicular structures. We propose a model wherein SCAMPs participate in the shuttling of NHE7 between recycling vesicles and the TGN.

The delta e13 isoform of the calcitonin receptor forms a six transmembrane domain receptor with dominant negative effects on the signaling of the calcitonin receptor

Like many other class B G protein-coupled receptors, there are several splice variants of the calcitonin receptor. A splice variant of the rabbit CTR (delta e13 CTR) lacks 14 amino acids of the C-terminus of the 7th transmembrane domain that are encoded by exon 13. This isoform is poorly expressed on the cell surface and fails to mobilize intracellular calcium or activate Erk. Moreover, it exerts a dominant-negative effect on CTR signaling by inhibiting the cell surface expression of the C1a isoform. Here, the structural and topological features responsible for these features of the delta e13 isoform are characterized. Using NMR-based experiments, we found that the remainder of the 7th transmembrane domain fails to insert into the lipid bilayer and the C-terminus of delta e13 is therefore extracellular. A truncation mutant lacking the cytoplasmic tail mimicked delta e13 CTR's reduced cell surface expression and its inability to mobilize intracellular calcium or activate Erk, but not its inhibition of C1a cell surface expression. In contrast, a CTR construct lacking the entire 7th transmembrane domain and C-terminal domain reduced the cell surface expression of C1a, similar to the effect of delta e13. We conclude that the cytoplasmic C-terminal tail of the calcitonin receptor is necessary for cell surface expression, mobilization of intracellular calcium, and Erk activation, while the failure of the 7th transmembrane domain to remain in the lipid bilayer is responsible for the dominant-negative effect of the delta e13 CTR isoform.

The Mep2p ammonium permease controls nitrogen starvation‐induced filamentous growth in Candida albicans

Nitrogen starvation is one of the signals that induce Candida albicans, the major fungal pathogen of humans, to switch from yeast to filamentous growth. In response to nitrogen starvation, C. albicans expresses the MEP1 and MEP2 genes, which encode two ammonium permeases that enable growth when limiting concentrations of ammonium are the only available nitrogen source. In addition to its role as an ammonium transporter, Mep2p, but not Mep1p, also has a central function in the induction of filamentous growth on a solid surface under limiting nitrogen conditions. When ammonium is absent or present at low concentrations, Mep2p activates both the Cph1p-dependent mitogen-activated protein (MAP) kinase pathway and the cAMP-dependent signalling pathway in a Ras1p-dependent fashion via its C-terminal cytoplasmic tail, which is essential for signalling but dispensable for ammonium transport. In contrast, under ammonium-replete conditions that require transporter-mediated uptake Mep2p is engaged in ammonium transport and signalling is blocked such that C. albicans continues to grow in the budding yeast form. Mep2p is a less efficient ammonium transporter than Mep1p and is expressed at much higher levels, a distinguishing feature that is important for its signalling function. At sufficiently high concentrations, ammonium represses filamentous growth even when the signalling pathways are artificially activated. Therefore, C. albicans has established a regulatory circuit in which a preferred nitrogen source, ammonium, also serves as an inhibitor of morphogenesis that is taken up into the cell by the same transporter that mediates the induction of filamentous growth in response to nitrogen starvation.

The Ammonium Transporter RhBG: REQUIREMENT OF A TYROSINE-BASED SIGNAL AND ANKYRIN-G FOR BASOLATERAL TARGETING AND MEMBRANE ANCHORAGE IN POLARIZED KIDNEY EPITHELIAL CELLS

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.

Ligand-dependent complex formation between the Angiotensin II receptor subtype AT2 and Na +/H + exchanger NHE6 in mammalian cells

Involvement of Angiotensin II (Ang II) in the regulation of sodium levels by modulating the Na +/H + exchangers is demonstrated in many tissues. Screening of a mouse 17-day fetus cDNA library with the Angiotensin II receptor AT2 as the bait in yeast two-hybrid assay led us to identify an AT2-interacting mouse fetus peptide that shared 98% amino acid identity with the corresponding region of the human NHE6. NCBI Blast search showed that the clone 6430520C02 (GenBank™ Accession # AK032326) of the mouse genome project carried the complete sequence of this new mouse NHE6 isoform. The human and mouse NHE6 peptides share 97% overall homology. Further analysis showed that the region spanning the third intracellular loop and C-terminal cytoplasmic tail of the AT2 directly interacted with a 182 amino acid region that spans the predicted 5th intracellular loop and the initial part of the C-terminus of the mouse NHE6 in yeast two-hybrid assay. This 182-amino acid region that interacted with the AT2 also shares 98% homology with the corresponding region of rat NHE6 and therefore is highly conserved across species. We detected widespread expression of this NHE6 isoform in several rat tissues including 10-day fetus, 17-day fetus, and 30-day post-natal tissues of heart, brain, kidney and muscle. Moreover, the AT2 co-immunoiprecipitated with a hemagglutinin tagged NHE6 when expressed in human cell line MCF-7, and activated by AngII. This ligand-dependent complex formation between the AT2 and NHE6 suggests that the hormone Ang II may act as a regulator of NHE6, and Ang II-mediated direct protein–protein interaction between AT2 and NHE6 could be a mechanism for modulating the functions of the ubiquitously expressed NHE6 in different tissues.

Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein.

We performed a structure-function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop-loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

Extensive phosphorylation of Smoothened in Hedgehog pathway activation

The transmembrane protein Smoothened (Smo) is activated in response to the extracellular protein signal, Hedgehog (Hh), and transmits this state of pathway activity into the cell. Previous studies in Drosophila have correlated pathway activation with Smo accumulation and increased phosphorylation. Using immunopurification and mass spectrometry, we identify here 26 serine/threonine residues within the Smo C-terminal cytoplasmic tail that are phosphorylated in Hh-stimulated cells. By systematically substituting alanine or glutamic acid to block or simulate phosphorylation, we provide evidence for a functional role of collective phosphorylation of a subset of phosphoresidues in pathway activation. This role is indicated by the ability of altered Smo proteins to produce changes in transcription of Hh-responsive genes in vivo and in cultured cells. These altered Smo proteins also affect biochemical indicators of pathway activity, such as Smo accumulation and phosphorylation of other pathway components. The prevalence and arrangement of phosphoresidues within the Smo cytoplasmic tail at recognition sites for cAMP-dependent protein kinase and casein kinase 1 suggest a role for these kinases in Smo phosphorylation, and such a role is supported by the effects of manipulating kinase activities in cultured cells. Our studies confirm and extend previous studies showing a positive effect for cAMP-dependent protein kinase and uncover a positive role for casein kinase 1alpha in Hh pathway activation.

Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction.

Hyperpolarization-activated cation currents (I(h)) are carried by channels encoded by a family of four genes (HCN1-4) that are differentially expressed within the brain in specific cellular and subcellular compartments. HCN1 shows a high level of expression in apical dendrites of cortical pyramidal neurons and in presynaptic terminals of cerebellar basket cells, structures with a high density of I(h). Expression of I(h) is also regulated by neuronal activity. To isolate proteins that may control HCN channel expression or function, we performed yeast two-hybrid screens using the C-terminal cytoplasmic tails of the HCN proteins as bait. We identified a brain-specific protein, which has been previously termed TRIP8b (for TPR-containing Rab8b interacting protein) and PEX5Rp (for Pex5p-related protein), that specifically interacts with all four HCN channels through a conserved sequence in their C-terminal tails. In situ hybridization and immunohistochemistry show that TRIP8b and HCN1 are colocalized, particularly within dendritic arbors of hippocampal CA1 and neocortical layer V pyramidal neurons. The dendritic expression of TRIP8b in layer V pyramidal neurons is disrupted after deletion of HCN1 through homologous recombination, demonstrating a key in vivo interaction between HCN1 and TRIP8b. TRIP8b dramatically alters the trafficking of HCN channels heterologously expressed in Xenopus oocytes and human embryonic kidney 293 cells, causing a specific decrease in surface expression of HCN protein and I(h) density, with a pronounced intracellular accumulation of HCN protein that is colocalized in discrete cytoplasmic clusters with TRIP8b. Finally, TRIP8b expression in cultured pyramidal neurons markedly decreases native I(h) density. These data suggest a possible role for TRIP8b in regulating HCN channel density in the plasma membrane.

Recessive mutations in PTHR1 cause contrasting skeletal dysplasias in Eiken and Blomstrand syndromes

Eiken syndrome is a rare autosomal recessive skeletal dysplasia. We identified a truncation mutation in the C-terminal cytoplasmic tail of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) type 1 receptor (PTHR1) gene as the cause of this syndrome. Eiken syndrome differs from Jansen and Blomstrand chondrodysplasia and from enchondromatosis, which are all syndromes caused by PTHR1 mutations. Notably, the skeletal features are opposite to those in Blomstrand chondrodysplasia, which is caused by inactivating recessive mutations in PTHR1. To our knowledge, this is the first description of opposite manifestations resulting from distinct recessive mutations in the same gene.

Roles of the intracellular regions of angiotensin II receptor AT2 in mediating reduction of intracellular cGMP levels

We have shown previously that the angiotensin II (Ang II) receptor AT2 reduces the intracellular levels of cGMP in Xenopus oocytes when activated by ligand binding, and the C-terminal cytoplasmic tail of the AT2 acts as a negative regulator of this function. Here we report the effects of mutations in the 2nd and 3rd intracellular loops of AT2 on AT2-mediated cGMP reduction. Mutating the highly conserved DRY motif (D141G-R142G-Y143A) of the 2nd ICL implicated in activating G α subunit of trimeric G-proteins did not affect AT2-mediated cGMP reduction. Moreover, anti-G iα antibody or phosphodiesterase inhibitor IBMX did not inhibit AT2-mediated cGMP reduction, suggesting that G iα activation and subsequent phosphodiesterase activation are not involved in this function. In contrast, mutations T250R-R251N and L255F-K256R located in the C-terminus of the 3rd ICL of AT2 retained ligand-binding properties of the wild-type AT2, and its ability to interact with the ErbB3 in yeast two-hybrid assay, but abolished AT2-mediated cGMP reduction. Similarities in the roles of ICLs of AT2 in AT2-mediated cGMP reduction in oocytes, and AT2-mediated SHP1 activation in COS-7 cells, (need of 3rd ICL for both functions and lack of involvement of DRY motif), suggest that the cascade of events in these two signaling mechanisms could be similar, and that an oocyte-specific SHP1-like protein may be involved in AT2-mediated cGMP reduction in these cells.

The C-terminal Tail of Presenilin Regulates Omi/HtrA2 Protease Activity

Presenilin mutations are responsible for most cases of autosomal dominant inherited forms of early onset Alzheimer disease. Presenilins play an important role in amyloid beta-precursor processing, NOTCH receptor signaling, and apoptosis. However, the molecular mechanisms by which presenilins regulate apoptosis are not fully understood. Here, we report that presenilin-1 (PS1) regulates the proteolytic activity of the serine protease Omi/HtrA2 through direct interaction with its regulatory PDZ domain. We show that a peptide corresponding to the cytoplasmic C-terminal tail of PS1 dramatically increases the proteolytic activity of Omi/HtrA2 toward the inhibitor of apoptosis proteins and beta-casein and induces cell death in an Omi/HtrA2-dependent manner. Consistent with these results, ectopic expression of full-length PS1, but not PS1 lacking the C-terminal PDZ binding motif, potentiated Omi/HtrA2-induced cell death. Our results suggest that the C terminus of PS1 is an activation peptide ligand for the PDZ domain of Omi/HtrA2 and may regulate the protease activity of Omi/HtrA2 after its release from the mitochondria during apoptosis. This mechanism of Omi/HtrA2 activation is similar to the mechanism of activation of the related bacterial DegS protease by the outer-membrane porins.

The 15-amino acid motif of the C terminus of the beta2-adrenergic receptor is sufficient to confer insulin-stimulated counterregulation to the beta1-adrenergic receptor.

Insulin counterregulates catecholamine action in part by inducing the sequestration of beta2-adrenergic receptors. Although similar to agonist-induced sequestration, insulin-induced internalization of beta2-adrenergic receptors operates through a distinct and better-understood cellular pathway. The effects of insulin treatment on the function and trafficking of both beta1- and beta2-adrenergic receptors were tested. The beta2-adrenergic receptors were counterregulated and internalized in response to insulin. The beta1-adrenergic receptors, in sharp contrast, are shown to be resistant to the ability of insulin to counterregulate function and induce receptor internalization. Using chimeric receptors composed of beta1-/beta2-adrenergic receptors in tandem with mutagenesis, we explored the role of the C-terminal cytoplasmic tail of the beta2-adrenergic receptors for insulin-induced counterregulation. Substitution of the C-terminal cytoplasmic tail of the beta2-adrenergic receptor on the beta1-adrenergic receptor enabled the chimeric G protein-coupled receptor to be functionally and spatially regulated by insulin. Truncation of the beta2-adrenergic receptor C-terminal cytoplasmic tail to a 15-amino acid motif harboring a potential Src homology 2-binding domain at Y350 and an Akt phosphorylation site at S345,346 was sufficient to enable receptor regulation by insulin.

Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein.

We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of approximately 500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.

F exclusion of bacteriophage T7 occurs at the cell membrane

The F plasmid PifA protein, known to be the cause of F exclusion of bacteriophage T7, is shown to be a membrane-associated protein. No transmembrane domains of PifA were located. In contrast, T7 gp1.2 and gp10, the two phage proteins that trigger phage exclusion, are both soluble cytoplasmic proteins. The Escherichia coli FxsA protein, which, at higher concentrations than found in wild-type cells, protects T7 from exclusion, is shown to interact with PifA. FxsA is a polytopic membrane protein with four transmembrane segments and a long cytoplasmic C-terminal tail. This tail is not important in alleviating F exclusion and can be deleted; in contrast, the fourth transmembrane segment of FxsA is critical in allowing wild-type T7 to grow in the presence of F PifA. These data suggest that the primary event that triggers the exclusion process occurs at the cytoplasmic membrane and that FxsA sequesters PifA so that membrane damage is minimized.

Renal epithelial traffic jams and one-way streets.

The kidney contains the body’s second largest number of different cell types, modulating the great diversity of different functions that allow complex homeostasis to occur. A feature that unites all these cell types in the postglomerular nephron is that they are highly polarized; one surface being

A C-terminal Region Dictates the Apical Plasma Membrane Targeting of the Human Sodium-dependent Vitamin C Transporter-1 in Polarized Epithelia

The human sodium-dependent vitamin C transporter (hSVCT1) mediates sodium-dependent cellular uptake of the essential micronutrient l-ascorbic acid (vitamin C). However, the molecular determinants that control the cell surface expression, subcellular distribution, and dynamics of hSVCT1 remain undefined. To identify molecular determinants involved in hSVCT1 targeting in polarized epithelia, we used live cell imaging approaches to resolve the targeting and trafficking dynamics of hSVCT1 truncation mutants in renal and intestinal cells. Confocal imaging demonstrated that hSVCT1 was expressed at the apical cell surface and video rate measurements revealed hSVCT1 also resided in a heterogeneous population of intracellular organelles with discrete dynamic properties. By progressive truncation of the cytoplasmic C-terminal tail of hSVCT1, we delimited an essential role for an embedded ten amino acid sequence PICPVFKGFS (amino acids 563-572) in defining the physiological targeting of hSVCT1. Intriguingly, this sequence bears significant homology to recently identified apical targeting motifs in two other sodium-dependent transporters, and we suggest this conservation is reflected topologically through the adoption of a beta-turn confirmation in the cytoplasmic C-tail of each transporter. Our results provide the first direct resolution of functional hSVCT1 expression at the apical cell surface of polarized epithelia and define an apical targeting signal of relevance to transporters of diverse substrate specificity.