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.
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Research Article|
December 12 2005
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
Frauke Petry;
Frauke Petry
*Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
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Vera Ritz;
Vera Ritz
*Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
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Cornelia Meineke;
Cornelia Meineke
*Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
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Peter Middel;
Peter Middel
†Department of Pathology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
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Thomas Kietzmann;
Thomas Kietzmann
‡Department of Biochemistry, Faculty of Chemistry, University of Kaiserslautern, Erwin-Schroedinger-Str., Gebaeude 54, D-67663 Kaiserslautern, Germany
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Christoph Schmitz-Salue;
Christoph Schmitz-Salue
*Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
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Karen I. Hirsch-Ernst
Karen I. Hirsch-Ernst
1
*Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
1To whom correspondence should be addressed (email khirsche@med.uni-goettingen.de).
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Publisher: Portland Press Ltd
Received:
May 16 2005
Revision Received:
September 12 2005
Accepted:
September 14 2005
Accepted Manuscript online:
September 14 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 393 (1): 79–87.
Article history
Received:
May 16 2005
Revision Received:
September 12 2005
Accepted:
September 14 2005
Accepted Manuscript online:
September 14 2005
Citation
Frauke Petry, Vera Ritz, Cornelia Meineke, Peter Middel, Thomas Kietzmann, Christoph Schmitz-Salue, Karen I. Hirsch-Ernst; 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. Biochem J 1 January 2006; 393 (1): 79–87. doi: https://doi.org/10.1042/BJ20050808
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