Conserved residues F316 and G476 in the concentrative nucleoside transporter 1 (hCNT1) affect guanosine sensitivity and membrane expression, respectively

2005 ◽  
Vol 288 (1) ◽  
pp. C39-C45 ◽  
Author(s):  
Yurong Lai ◽  
Eun-Woo Lee ◽  
Carl C. Ton ◽  
Shashi Vijay ◽  
Huixia Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Membrane Expression ◽  
Concentrative Nucleoside Transporter ◽  
Green Fluorescent

The functional significance of two highly conserved amino acid residues, F316 [putative transmembrane domain (TM)7] and G476 (putative TM11), in the concentrative nucleoside transporter hCNT1 (SLC28A1) was examined by performing site-directed mutagenesis. Conservative mutations at these positions (F316A, F316Y, G476A, and G476L) were generated and expressed in Madin-Darby canine kidney (MDCK) cells as fusion polypeptides with green fluorescent protein (GFP). Unlike wild-type hCNT1, G476A-GFP and G476L-GFP were not expressed in the plasma membrane in undifferentiated or differentiated MDCK cells and had no functional activity. Like wild-type hCNT1, F316A-GFP and F316Y-GFP were expressed in the plasma membrane of undifferentiated MDCK cells and in the apical membrane of differentiated MDCK cells. Remarkably, transport of [3H]uridine by F316Y-GFP or F316A-GFP was highly sensitive to inhibition by guanosine. Furthermore, genotyping of exon 11 of hCNT1 (TM7) in a panel of 260 anonymous human DNA samples revealed a novel F316H variant (TT>CA; 1/260). When expressed in MDCK cells, [3H]uridine transport by F316H was also found to be sensitive to inhibition by guanosine (IC50 = 148 μM). The effect of the F316H mutation resembles the N4 type nucleoside transporter phenotype previously reported to be present in human kidneys. We suggest that the N4 transport system is a naturally occurring variant of hCNT1, perhaps at the F316 position. Collectively, our data show that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1. In addition, we have discovered that hCNT1 displays natural variation at position F316 and that the variant F316H confers on the transporter an unusual sensitivity to inhibition by guanosine.

scholarly journals The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane

2012 ◽  
Vol 302 (12) ◽  
pp. C1713-C1730 ◽  
Author(s):  
Hong C. Li ◽  
Volodymyr Kucher ◽  
Emily Y. Li ◽  
Laura Conforti ◽  
Kamyar A. Zahedi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sodium Bicarbonate ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Functional Activity ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Sodium Bicarbonate Cotransporter ◽  
Green Fluorescent

The NH2 terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH2-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH2 terminus of NBCe1A are important in its accurate targeting to the plasma membrane.

Subcellular redistribution of the renal betaine transporter during hypertonic stress

2003 ◽  
Vol 285 (5) ◽  
pp. C1091-C1100 ◽  
Author(s):  
Stephen A. Kempson ◽  
Vaibhave Parikh ◽  
Lixuan Xi ◽  
Shaoyou Chu ◽  
Marshall H. Montrose
Keyword(s):  
Plasma Membrane ◽  
Posttranscriptional Regulation ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Live Cells ◽  
Hypertonic Medium ◽  
Hypertonic Stress ◽  
Antibody Staining ◽  
Renal Inner Medulla ◽  
Green Fluorescent

The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the NH2 terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent Km for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2-3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins.

Activity of the renal Na+-K+-2Cl− cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl− transport

2006 ◽  
Vol 290 (5) ◽  
pp. F1094-F1102 ◽  
Author(s):  
Anahí Paredes ◽  
Consuelo Plata ◽  
Manuel Rivera ◽  
Erika Moreno ◽  
Norma Vázquez ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Fluorescent Protein ◽  
Functional Expression ◽  
Site Directed Mutagenesis ◽  
Intrinsic Activity ◽  
Xenopus Laevis Oocytes ◽  
Type I ◽  
Wild Type ◽  
Glycosylation Sites ◽  
Green Fluorescent

The renal-specific Na+-K+-2Cl− cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na+-Cl− cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by 86Rb+ influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced ∼20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl− affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

scholarly journals Analysis of Localization of Mutated Tissue-Nonspecific Alkaline Phosphatase Proteins Associated with Neonatal Hypophosphatasia Using Green Fluorescent Protein Chimeras1

1998 ◽  
Vol 83 (11) ◽  
pp. 3936-3942
Author(s):  
Guiming Cai ◽  
Toshimi Michigami ◽  
Takehisa Yamamoto ◽  
Natsuo Yasui ◽  
Kenichi Satomura ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Enzymatic Activity ◽  
Fluorescent Protein ◽  
Clinical Severity ◽  
Wild Type ◽  
Neonatal Patient ◽  
Tissue Nonspecific Alkaline Phosphatase ◽  
Green Fluorescent

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity are usually correlated in hypophosphatasia; patients with perinatal hypophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no respiratory problems and survived. He was compound heterozygous for the conversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucleotide T at 1735 (delT1735), causing the frame shift with the result of the addition of 80 amino acids at the C-terminal of the protein. Because the C-terminal portion of TNSALP is known to be important for TNSALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein chimeras. The expression vectors containing the complementary DNA of fusion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation, were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wild-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that the delT1735 mutant lost the activity and that the F310L mutant exhibited an enzymatic activity level that was 72% of the normal level. The F310L mutation was also detected in another neonatal patient with relatively mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458–4461, 1996), suggesting that residual ALP activity of the F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical severity in hypophosphatasia.

scholarly journals Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 Protein into the Chitin Ring and Septum Is Required for Proper Morphogenesis in Yeast

2009 ◽  
Vol 20 (22) ◽  
pp. 4856-4870 ◽  
Author(s):  
Eleonora Rolli ◽  
Enrico Ragni ◽  
Julia Calderon ◽  
Silvia Porello ◽  
Umberto Fascio ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Separation ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Cell Envelope ◽  
Phenotypic Analysis ◽  
Neck Size ◽  
Bud Neck ◽  
Green Fluorescent

Gas1p is a glucan-elongase that plays a crucial role in yeast morphogenesis. It is predominantly anchored to the plasma membrane through a glycosylphosphatidylinositol, but a fraction was also found covalently bound to the cell wall. We have used fusions with the green fluorescent protein or red fluorescent protein (RFP) to determine its localization. Gas1p was present in microdomains of the plasma membrane, at the mother-bud neck and in the bud scars. By exploiting the instability of RFP-Gas1p, we identified mobile and immobile pools of Gas1p. Moreover, in chs3Δ cells the chitin ring and the cross-linked Gas1p were missing, but this unveiled an additional unexpected localization of Gas1p along the septum line in cells at cytokinesis. Localization of Gas1p was also perturbed in a chs2Δ mutant where a remedial septum is produced. Phenotypic analysis of cells expressing a fusion of Gas1p to a transmembrane domain unmasked new roles of the cell wall-bound Gas1p in the maintenance of the bud neck size and in cell separation. We present evidence that Crh1p and Crh2p are required for tethering Gas1p to the chitin ring and bud scar. These results reveal a new mechanism of protein immobilization at specific sites of the cell envelope.

scholarly journals Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

1999 ◽  
Vol 10 (6) ◽  
pp. 2033-2050 ◽  
Author(s):  
Karen Jordan ◽  
Joell L. Solan ◽  
Michel Dominguez ◽  
Michael Sia ◽  
Art Hand ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Dye Transfer ◽  
Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress

2014 ◽  
Vol 307 (1) ◽  
pp. F107-F115 ◽  
Author(s):  
Eva S. Schweikhard ◽  
Stephen A. Kempson ◽  
Christine Ziegler ◽  
Birgitta C. Burckhardt
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Gaba Uptake ◽  
Gaba Transporter ◽  
Western Blots ◽  
Hypertonic Stress ◽  
293 Cells ◽  
Gaba Transporter 1 ◽  
Green Fluorescent

Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 μM dioctanoyl- sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl- sn-glycerol restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [3H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation by PMA similar to EGFP-wt-BGT1. In contrast, T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane.

scholarly journals Specific Retrieval of the Exocytic SNARE Snc1p from Early Yeast Endosomes

2000 ◽  
Vol 11 (1) ◽  
pp. 23-38 ◽  
Author(s):  
Michael J. Lewis ◽  
Benjamin J. Nichols ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman ◽  
Hugh R. B. Pelham
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Immunoelectron Microscopy ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Early Endosomes ◽  
Internal Structures ◽  
Green Fluorescent

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome–Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.

scholarly journals TheSchizosaccharomyces pombe spo3+Gene Is Required for Assembly of the Forespore Membrane and Genetically Interacts withpsy1+-encoding Syntaxin-like Protein

2001 ◽  
Vol 12 (12) ◽  
pp. 3955-3972 ◽  
Author(s):  
Taro Nakamura ◽  
Michiko Nakamura-Kubo ◽  
Aiko Hirata ◽  
Chikashi Shimoda
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Membrane Vesicles ◽  
Null Mutant ◽  
Wild Type ◽  
Normal Morphology ◽  
Novel Proteins ◽  
Green Fluorescent

Formation of the forespore membrane, which becomes the plasma membrane of spores, is an intriguing step in the sporulation of the fission yeast Schizosaccharomyces pombe. Here we report two novel proteins that localize to the forespore membrane.spo3+encodes a potential membrane protein, which was expressed only during sporulation. Green fluorescent protein (GFP) fusion revealed that Spo3 localized to the forespore membrane. The spo3 disruptant was viable and executed meiotic nuclear divisions as efficiently as the wild type but did not form spores. One of the spo3 alleles,spo3-KC51, was dose-dependently suppressed bypsy1+, which encodes a protein similar to mammalian syntaxin-1A, a component of the plasma membrane docking/fusion complex. psy1+was essential for vegetative growth, and its transcription was enhanced during sporulation. As expected, Psy1 localized to the plasma membrane during vegetative growth. Interestingly, Psy1 on the plasma membrane disappeared immediately after first meiotic division and relocalized to the forespore membrane as the second division initiated. In thespo3 null mutant, the forespore membrane was initiated but failed to develop a normal morphology. Electron microscopy revealed that membrane vesicles were accumulated in the cytoplasm of immaturespo3Δ asci. These results suggest that Spo3 is a key component of the forespore membrane and is essential for its assembly acting in collaboration with the syntaxin-like protein.

scholarly journals Mutations in sixth transmembrane domain of AQP2 inhibit its translocation induced by vasopression

2000 ◽  
Vol 278 (3) ◽  
pp. F395-F405 ◽  
Author(s):  
Yumi Yamashita ◽  
Keiji Hirai ◽  
Yoshifumi Katayama ◽  
Kiyohide Fushimi ◽  
Sei Sasaki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Collecting Duct ◽  
Water Channel ◽  
Wild Type ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Golgi Network

Vasopression-induced phosphorylation of serine 256 of the aquaporin-2 (AQP2) water channel triggers translocation of the protein from cystolic reservoir vesicles to the apical membrane of collecting duct principal cells. Dileucine motifs are located in the sixth transmembrane domain (6TM) of AQP2 and are known as the signal sequence for internalization, sorting from the trans-Golgi network to endosomes/lysosomes, and basolateral sorting. In this study, involvement of 6TM in vasopressin-induced translocation of the protein was investigated. A series of mutations in 6TM of AQP2 was introduced to rat cDNA and expressed in LLC-PK1 cells. Immunofluorescence microscopy indicated that the mutant AQP2 proteins were retained in the cytoplasm after vasopressin stimulation, which actually promoted the plasma membrane expression of wild-type protein. Immunoelectron microscopy showed that the mutant AQP2 proteins reached the endosomes but did not reach the plasma membrane. These results demonstrate that 6TM has essential domains for vasopressin-induced translocation from endosomes to the plasma membrane.

Sign in / Sign up

Export Citation Format

Share Document