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
    Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
  • Vera Ritz
    Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
  • Cornelia Meineke
    Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
  • Peter Middel
    Department of Pathology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
  • Thomas Kietzmann
    Department of Biochemistry, Faculty of Chemistry, University of Kaiserslautern, Erwin-Schroedinger-Str., Gebaeude 54, D-67663 Kaiserslautern, Germany
  • Christoph Schmitz-Salue
    Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany
  • Karen I. Hirsch-Ernst
    Institute of Pharmacology and Toxicology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany

抄録

<jats:p>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.</jats:p>

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