Disruption of the gene encoding the acyl-CoA-binding protein (ACB1) perturbs acyl-CoA metabolism in Saccharomyces cerevisiae.

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 Delta9-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.

Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family.

Bovine acyl-coenzyme A binding protein is a four-helix bundle protein belonging to a group of homologous eukaryote proteins that binds medium and long-chain acyl-coenzyme A esters with a very high affinity. The three-dimensional structure of both the free and the ligated protein together with the folding kinetics have been described in detail for the bovine protein and with four new sequences reported here, a total of 16 closely related sequences ranging from yeasts and plants to human are known. The kinetics of folding and unfolding in different concentrations of guanidine hydrochloride together with equilibrium unfolding have been measured for bovine, rat and yeast acyl-coenzyme A binding protein. The bovine and rat sequences are closely related whereas the yeast is more distantly related to these. In addition to the three natural variants, kinetics of a bovine mutant protein, Tyr31 --> Asn, have been studied. Both the folding and unfolding rates in water of the yeast protein are 15 times faster than those of bovine. The folding rates in water of the two mammalian forms, rat and bovine, are similar, though still significantly different. A faster unfolding rate both for rat and the bovine mutant protein results from a lower stability of the native states of these. These hydrophobic regions, mini cores, have been identified in the three-dimensional structure of the bovine protein and found to be formed primarily by residues that have been conserved throughout the entire eukaryote evolution from yeasts to both plants and mammals as seen in the sample of 16 sequences. The conserved residues are found to stabilize helix-helix interactions and serve specific functional purposes for ligand binding. The fast one-step folding mechanism of ACBP has been shown to be a feature that seems to be maintained throughout evolution despite numerous differences in sequence and even dramatic differences in folding kinetics and protein stability. The protein study raises the question to what extent does the conserved hydrophobic residues provide a scaffold for an efficient one-step folding mechanism.