Vesicular localization of the rat ATP-binding cassette half-transporter rAbcb6

2008 ◽  
Vol 294 (2) ◽  
pp. C579-C590 ◽  
Author(s):  
Youssef Abdul Jalil ◽  
Vera Ritz ◽  
Ana Jakimenko ◽  
Christoph Schmitz-Salue ◽  
Heike Siebert ◽  
...  
Keyword(s):  
Transition Metal ◽  
Fluorescent Protein ◽  
Metal Tolerance ◽  
Heavy Metal Tolerance ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Subcellular Fractionation ◽  
Atp Binding ◽  
Lovo Cells ◽  
Atp Binding Cassette

The clarification of subcellular localization represents an important basis toward characterization of ATP-binding cassette (ABC) transporters and resolution of their roles in cellular physiology. Rat Abcb6 (rAbcb6) is a membrane-situated half-transporter belonging to the ABC protein superfamily. To investigate rAbcb6 subcellular distribution, the human colon adenocarcinoma line LoVo, which we found to be devoid of endogenous human ABCB6 mRNA, was employed for heterologous expression of rAbcb6 bearing a COOH-terminal epitope tag (rAbcb6-V5). Following subcellular fractionation, rAbcb6-V5 was observed as an N-glycosylated protein in fractions enriched with lysosomal/endosomal membrane proteins. Indirect immunofluorescence analyses of rAbcb6-V5 using antibodies against a rAbcb6-specific peptide or against the V5-tag revealed a punctate pattern that was colocalized with lysosome-associated membrane protein 1 (LAMP1), a marker of lysosomes/late endosomes. Substantial colocalization of tagged rAbcb6 with lysosomal/late endosomal marker was confirmed with living, unfixed LoVo cells coexpressing rAbcb6 fused to enhanced green fluorescent protein. Vesicular distribution in LoVo cells was consistent with localization of endogenous rAbcb6 expressed in rat primary hepatocyte cultures or in liver sections, as revealed by overlap of rat Lamp1 with rAbcb6 in double immunofluorescence analyses. Since several Abcb6-related half-transporters confer heavy metal tolerance, we investigated whether rAbcb6 expression in LoVo cells might affect sensitivity toward transition metal toxicity. Applying MTT viability assays, we found that expression of either rAbcb6-V5 or untagged rAbcb6 conferred tolerance toward copper, but not to cobalt or zinc. In summary, these results demonstrate that rAbcb6 is a glycosylated protein targeted to intracellular vesicular membranes and suggest involvement of rAbcb6 in transition metal homeostasis.

scholarly journals Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

1992 ◽  
Vol 11 (10) ◽  
pp. 3491-3499 ◽  
Author(s):  
D.F. Ortiz ◽  
L. Kreppel ◽  
D.M. Speiser ◽  
G. Scheel ◽  
G. McDonald ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Fission Yeast ◽  
Metal Tolerance ◽  
Heavy Metal Tolerance ◽  
Vacuolar Membrane ◽  
Atp Binding ◽  
Membrane Transporter ◽  
Atp Binding Cassette

scholarly journals Lack of integrin α-chain endoproteolytic cleavage in furin-deficient human colon adenocarcinoma cells LoVo

1996 ◽  
Vol 317 (3) ◽  
pp. 803-809 ◽  
Author(s):  
Maxime LEHMANN ◽  
Véronique RIGOT ◽  
Nabil G. SEIDAH ◽  
Jacques MARVALDI ◽  
Jean-Claude LISSITZKY
Keyword(s):  
Present Report ◽  
Colon Adenocarcinoma ◽  
Complete Removal ◽  
Human Colon ◽  
Competent Cell ◽  
Integrin Subunit ◽  
Furin Cleavage ◽  
Ht29 Cells ◽  
Endoproteolytic Cleavage ◽  
Lovo Cells

In the present report the biosynthesis of the integrin α-chains endowed with constitutive endoproteolytic cleavage was evaluated in LoVo cells where furin, a subtilisin-like convertase involved in post-translational endoproteolytic processing, is not functional. It was found that cell-surface α3, α6 and αv subunits were not processed endoproteolytically into heavy and light chains as they were in HT29-D4 cells, a furin-competent cell line. Complete removal of N-linked oligosaccharides and pulse–chase experiments confirmed that the cleavage of the α6 integrin subunit occurring 45 min after translation in HT29 cells did not take place in LoVo cells. Apart from cleavage deficiency, α6 subunit glycosylation, association with β4 subunits and targeting to the plasma membrane seemed comparable in LoVo and HT29 cells. The pro-α6 and the pro-α3 subunits immunopurified from LoVo cells were highly sensitive to endoproteolysis by recombinant furin. Furin cleavage was calcium dependent and resulted in the conversion of the 140 kDa pro-α6 into a 120 kDa heavy chain. These results suggest strongly that furin is involved in the endoproteolytic processing of cleavable integrin α subunits.

scholarly journals Normal Formation of a Subset of Intestinal Granules in Caenorhabditis elegans Requires ATP-binding Cassette Transporters HAF-4 and HAF-9, Which Are Highly Homologous to Human Lysosomal Peptide Transporter TAP-Like

2009 ◽  
Vol 20 (12) ◽  
pp. 2979-2990 ◽  
Author(s):  
Hiromi Kawai ◽  
Takahiro Tanji ◽  
Hirohisa Shiraishi ◽  
Mitsuo Yamada ◽  
Ryoko Iijima ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Physiological Role ◽  
Peptide Transporter ◽  
Atp Binding ◽  
Loss Of Function ◽  
Specific Expression ◽  
Atp Binding Cassette

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.

Co-operative function and mutual stabilization of the half ATP-binding cassette transporters HAF-4 and HAF-9 in Caenorhabditis elegans

2013 ◽  
Vol 452 (3) ◽  
pp. 467-475 ◽  
Author(s):  
Takahiro Tanji ◽  
Kenji Nishikori ◽  
Hirohisa Shiraishi ◽  
Masatomo Maeda ◽  
Ayako Ohashi-Kobayashi
Keyword(s):  
Caenorhabditis Elegans ◽  
Antigen Processing ◽  
Fluorescent Protein ◽  
Peptide Transporter ◽  
Deletion Mutants ◽  
Atp Binding ◽  
Physical Interaction ◽  
Atp Binding Cassette Transporters ◽  
Green Fluorescent ◽  
Atp Binding Cassette

Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4–HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.

scholarly journals Correlation of Induction of ATP Binding Cassette Transporter A5 (ABCA5) and ABCB1 mRNAs with Differentiation State of Human Colon Tumor

2007 ◽  
Vol 30 (6) ◽  
pp. 1144-1146 ◽  
Author(s):  
Sumio Ohtsuki ◽  
Mayu Kamoi ◽  
Yuki Watanabe ◽  
Hiroya Suzuki ◽  
Satoko Hori ◽  
...  
Keyword(s):  
Human Colon ◽  
Atp Binding ◽  
Colon Tumor ◽  
Atp Binding Cassette Transporter ◽  
Atp Binding Cassette

scholarly journals Differentiation-dependent autophagy controls the fate of newly synthesized N-linked glycoproteins in the colon adenocarcinoma HT-29 cell line

1995 ◽  
Vol 309 (2) ◽  
pp. 521-527 ◽  
Author(s):  
J J Houri ◽  
E Ogier-Denis ◽  
D De Stefanis ◽  
C Bauvy ◽  
F M Baccino ◽  
...  
Keyword(s):  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Subcellular Fractionation ◽  
Human Colon Cancer ◽  
Lysosomal Compartment ◽  
Lysosomal Fraction ◽  
Undifferentiated Cells ◽  
High Mannose ◽  
Exocytic Pathway ◽  
Ht 29

Our previous results have demonstrated that, in undifferentiated human colon cancer HT-29 cells, a pool of glycoproteins bearing high-mannose oligosaccharides rapidly escapes the exocytic pathway to be degraded in the lysosomal compartment [Trugnan, Ogier-Denis, Sapin, Darmoul, Bauvy, Aubery and Codogno (1991) J. Biol. Chem. 266, 20849-20855]. We report here on the mechanism that governs this degradative pathway. Using pulse-chase experiments in combination with subcellular fractionation, we have observed that the sequestration of high-mannose glycoproteins in lysosomes was impaired by drugs which interfere with the autophagic-lysosomal pathway. The accumulation of high-mannose glycoproteins in the lysosomal fraction was shown to be part of the general autophagic pathway constitutively expressed in undifferentiated cells, as independently measured by the sequestration of the cytosolic enzyme lactate dehydrogenase and electroloaded raffinose. Furthermore, when HT-29 cells were cultured under differentiation-permissive conditions, the decreased accumulation of high-mannose glycoproteins in the lysosomal compartment was correlated with the decrease in autophagy.

Targeting Immunotherapy using the Avidin–Biotin System for a Human Colon Adenocarcinoma In Vitro

1997 ◽  
Vol 25 (1) ◽  
pp. 14-23 ◽  
Author(s):  
M Nakaki ◽  
H Takikawa ◽  
M Yamanaka
Keyword(s):  
Cell Line ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Cancer Drug ◽  
Present System ◽  
Clinical Tool ◽  
Anti Cancer ◽  
Lovo Cells ◽  
Human Colon Adenocarcinoma

A new method of targeting immunotherapy using the avidin–biotin system in vitro was investigated. Both an anti-carcinoembryonic antigen monoclonal antibody (anti-CEA MAb) and an anti-cancer drug, neocarzinostatin (NCS), were biotinylated. A human colon adenocarcinoma cell line (LoVo) was immunized with biotinylated anti-CEA MAb; avidin was added, and the cell line was incubated with various concentrations of biotinylated NCS for either 72 h or 7 min. In the incubation for 72 h, the IC50 was similar (≈0.45 μg/ml) for biotinylated NCS for LoVo cells immunized with biotinylated anti-CEA MAb and those without immunization. In the incubation for 7 min, the IC50 (concentration producing 50% cytotoxicity) of biotinylated NCS for LoVo cells immunized with biotinylated anti-CEA MAb (0.35 μg/ml) was five times less than that of non-immunized LoVo cells (1.8 μg/ml). Thus the present system has the potential to reduce the dosage of anti-cancer drugs needed, and this strategy seems likely to be a valuable clinical tool in targeting immunotherapy.

Regulation of ornithine decarboxylase activity in LoVo cells

1990 ◽  
Vol 258 (6) ◽  
pp. G934-G941
Author(s):  
S. A. McCormack ◽  
L. L. Tague ◽  
E. J. Gragoe ◽  
L. R. Johnson
Keyword(s):  
Ornithine Decarboxylase ◽  
Choline Chloride ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Decarboxylase Activity ◽  
Lovo Cells ◽  
Fetal Bovine ◽  
Na Uptake ◽  
Stimulation Of

The role of Na+ and Na(+)-H+ exchange in the stimulation of ornithine decarboxylase (ODC) activity has been investigated in a human colon adenocarcinoma cell line, LoVo. Asparagine (Asn; 10 mM) or 10% fetal bovine serum (FBS) increased ODC activity from undetectable levels to greater than 500 pmol CO2.mg protein-1.h-1 in 4 h. This increase could be reduced 50% by concentrations of Na(+)-H+ exchange inhibitors that did not reduce protein synthesis. (approximately 0.2 mM for amiloride and 0.05 mM for hexamethyleneamiloride). Asn was able to double the uptake of 22Na+, whether an ionic (choline chloride) or nonionic (D-mannitol) substance was substituted for Na+, and the substitution of these compounds as well as N-methyl-glucamine for Na+ largely prevented the stimulation of ODC by Asn. Another factor influencing ODC activity was extracellular pH (pHo). When pHo was lowered, intracellular pH (pHi) also fell, and ODC activity was reduced. When pHo was raised, pHi also rose, and ODC activity increased. The well-known correlation between increased pHi and Na+ uptake with the stimulation of growth may be due to their influence on ODC activity.

scholarly journals Subcellular localization of rat Abca5, a rat ATP-binding-cassette transporter expressed in Leydig cells, and characterization of its splice variant apparently encoding a half-transporter

2005 ◽  
Vol 393 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Frauke Petry ◽  
Vera Ritz ◽  
Cornelia Meineke ◽  
Peter Middel ◽  
Thomas Kietzmann ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Splice Variant ◽  
Leydig Cells ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Expression Vectors ◽  
Atp Binding ◽  
Hek 293 ◽  
293 Cells ◽  
Atp Binding Cassette

Several transporters belonging to the ABCA subfamily of ABC (ATP-binding cassette) proteins are involved in lipid trafficking. Human ABCA5 and its rat orthologue, rAbca5, represent recently identified subfamily members whose substrate spectrum remains to be defined. The elucidation of (sub)cellular rAbca5 distribution would be expected to provide a basis for optimization of functional analyses. In the present study, we applied in situ hybridization to examine rAbca5 mRNA distribution within sections of rat testis, a tissue expressing high levels of rAbca5 mRNA. We found rAbca5 mRNA to be predominantly expressed in interstitial Leydig cells, which are major sites of testosterone synthesis. To investigate rAbca5 subcellular localization, we constructed expression vectors yielding rAbca5 fused either to EGFP (enhanced green fluorescent protein) or to a peptide bearing the viral V5 epitope. During rAbca5 cDNA cloning, we discovered a splice variant sequence (rAbca5 V20+16), predicted to give rise to a truncated, half-size transporter, which was highly homologous with a human splice variant described by us previously. Quantitative RT (reverse transcription)–PCR demonstrated that the rAbca5 splice variant was expressed in numerous tissues (including testis, brain and lungs), its cDNA amounting to 2.6–11.2% of total rAbca5 cDNA. Transfection of individual rAbca5-EGFP, rAbca5 splice variant-EGFP or transporter-V5 expression plasmids along with organelle marker plasmids into HEK-293 cells (human embryonic kidney 293 cells) revealed that both rAbca5 and splice variant fusion proteins co-localized with marker protein for the Golgi apparatus. Expression of rAbca5 mRNA in Leydig cells, intracellular localization of rAbca5–EGFP/rAbca5–V5 and involvement of rAbca5-related proteins in lipid transport suggest that rAbca5 may participate in intracellular sterol/steroid trafficking.

Immunolocalization and funtional role of scavenger receptor BI (SR-BI) and ATP-binding cassette A1 (ABC1)

2001 ◽  
Vol 120 (5) ◽  
pp. A678-A678
Author(s):  
I SUC ◽  
M BENDAYAN ◽  
E DELVIN ◽  
L BRISSETTE ◽  
C GAROFALO ◽  
...  
Keyword(s):  
Scavenger Receptor ◽  
Atp Binding ◽  
Scavenger Receptor Bi ◽  
Atp Binding Cassette
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