Submitted on June 29, 2007
Accepted on August 6, 2007
Topology of the yeast fatty acid transport protein Fat1p: Mechanistic implications for functional domains on the cytosolic surface of the plasma membrane
Thomas Obermeyer, Peter Fraisl, Concetta C. DiRusso, and Paul N. Black
Center for Metabolic Disease, Ordway Research Institute, Albany, NY 12208
Corresponding Author: pblack{at}ordwayresearch.org
The fatty acid transport protein Fat1p in the yeast Saccharomyces cerevisiae functions in concert with acyl CoA synthetase (ACSL; either Faa1p or Faa4p) in vectorial acylation, which couples the transport of exogenous fatty acid to 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/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 (HA) epitope tags were engineered at both amino- and carboxyl- termini, as well as at multiple non-conserved, 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 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-C¬in 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 in 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.
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