The ACB1 gene encoding the acyl-CoA-binding protein (ACBP) was disrupted in Saccharomyces cerevisiae. The disruption did not affect the growth rate on glucose but reduced the growth rate on ethanol slightly. Although the growth rate of the acb1-disrupted cells was unaffected or only slightly affected, the acb1-disrupted strain was unable to compete with wild type cells when grown in mixed culture. The acyl-CoA level in the disrupted cells was increased from 1.5- to 2.5-fold during exponential growth. The increase in the acyl-CoA level was caused solely by an increase in de novo synthesized stearoyl-CoA. Experiments with purified yeast fatty acid synthetase show that it will synthesize long chain acyl-CoAs in the absence of acyl-CoA-binding protein. The addition of ACBP to the incubation medium resulted in a dramatic decrease in the chain length of the synthesized acyl-CoA esters. Despite the fact that the stearoyl-CoA concentration was increased 7-fold and the Δ9-desaturase mRNA level was increased 3-fold, the synthesis of oleic acid was unchanged in the acb1-disrupted strain. The results strongly indicate that ACBP in yeast is involved in the transport of newly synthesized acyl-CoA esters from the fatty acid synthetase to acyl-CoA-consuming processes.

In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions between these proteins. Multicopy extragenic suppressors were selected in strains carrying deletions inFAA1 and FAA4 or FAA1 andFAT1. Each strain is unable to grow under synthetic lethal conditions when exogenous long-chain fatty acids are required, and neither strain accumulates the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ indicating a fatty acid transport defect. By using these phenotypes as selective screens, plasmids were identified encoding FAA1, FAT1, andFAA4 in the faa1Δ faa4Δ strain and encoding FAA1 and FAT1 in thefaa1Δ fat1Δ strain. MulticopyFAA4 could not suppress the growth defect in thefaa1Δ fat1Δ strain indicating some essential functions of Fat1p cannot be performed by Faa4p. Chromosomally encoded FAA1 and FAT1 are not able to suppress the growth deficiencies of the fat1Δfaa1Δ and faa1Δ faa4Δstrains, respectively, indicating Faa1p and Fat1p play distinct roles in the fatty acid import process. When expressed from a 2μ plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity, which indicates vectorial esterification and metabolic trapping are the driving forces behind import. Evidence of a physical interaction between Fat1p and FACS was provided using three independent biochemical approaches. First, a C-terminal peptide of Fat1p deficient in fatty acid transport exerted a dominant negative effect against long-chain acyl-CoA synthetase activity. Second, protein fusions employing Faa1p as bait and portions of Fat1p as trap were active when tested using the yeast two-hybrid system. Third, co-expressed, differentially tagged Fat1p and Faa1p or Faa4p were co-immunoprecipitated. Collectively, these data support the hypothesis that fatty acid import by vectorial acylation in yeast requires a multiprotein complex, which consists of Fat1p and Faa1p or Faa4p.