J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M700300-JLR200 on August 6, 2007

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Journal of Lipid Research, Vol. 48, 2354-2364, November 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Topology of the yeast fatty acid transport protein Fat1p: mechanistic implications for functional domains on the cytosolic surface of the plasma membrane

Thomas Obermeyer*,{dagger}, Peter Fraisl*, Concetta C. DiRusso*,{dagger} and Paul N. Black1,*,{dagger}

* Center for Metabolic Disease, Ordway Research Institute, Albany Medical College, Albany, NY 12208
{dagger} Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208

Published, JLR Papers in Press, August 6, 2007.

1 To whom correspondence should be addressed. e-mail: pblack{at}ordwayresearch.org

The fatty acid transport protein (FATP) Fat1p in the yeast Saccharomyces cerevisiae functions in concert with acyl-coenzyme A synthetase (ACSL; either Faa1p or Faa4p) in vectorial acylation, which couples the transport of exogenous fatty acids with activation to CoA thioesters. To further define the role of Fat1p in the transport of exogenous fatty acids, the topological orientation of two highly conserved motifs [ATP/AMP and FATP/very long chain acyl CoA synthetase (VLACS)], the carboxyl 124 amino acid residues, which bind the ACSL Faa1p, and the amino and carboxyl termini within the plasma membrane were defined. T7 or hemagglutinin epitope tags were engineered at both amino and carboxyl termini, as well as at multiple nonconserved, predicted random coil segments within the protein. Six different epitope-tagged chimeras of Fat1p were generated and expressed in yeast; the sidedness of the tags was tested using indirect immunofluorescence and protease protection by Western blotting. Plasma membrane localization of the tagged proteins was assessed by immunofluorescence. Fat1p appears to have at least two transmembrane domains resulting in a Nin–Cin topology. We propose that Fat1p has a third region, which binds to the membrane and separates the highly conserved residues comprising the two halves of the ATP/AMP motif. The Nin–Cin topology results in the placement of the ATP/AMP and FATP/VLACS domains of Fat1p on the inner face of the plasma membrane. The carboxyl-terminal region of Fat1p, which interacts with ACSL, is likewise positioned on the inner face of the plasma membrane. This topological orientation is consistent with the mechanistic roles of both Fat1p and Faa1p or Faa4p in the coupled transport/activation of exogenous fatty acids by vectorial acylation.

Supplementary key words fatty acid transport protein • topology • functional domains

Abbreviations: VLACS, very long chain acyl CoA synthetase


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Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.