Use of Escherichia coli strains containing fad mutations plus a triple plasmid expression system to study the import of myristate, its activation by Saccharomyces cerevisiae acyl-CoA synthetase, and its utilization by S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.
A system is described for studying protein N-myristoylation, a eukaryotic protein modification, in Escherichia coli strains containing components of eukaryotic metabolic pathways that regulate metabolism of myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) substrates. Three recombinant plasmids were used to simultaneously direct synthesis of Saccharomyces cerevisiae Nmt1p, a substrate protein (S. cerevisiae ADP-ribosylation factor 1, Arf1p), and one of the acyl-CoA synthetases produced by S. cerevisiae (Faa1p) in isogenic strains of bacteria with wild type or mutant alleles of genes comprising the regulon for fatty acid degradation (FadR, FadE, FadL and FadD). Incorporation of exogenous tritiated myristate into Arf1p and bacterial phospholipid biosynthetic pathways was analyzed. Removal of FadL, a 448-residue protein necessary for efficient transport of fatty acids across the outer membrane, had no detectable effect on Nmt1p-dependent N-myristoylation of Arf1p. This finding is consistent with the notion that permeation of C14:0 across the bacterial inner membrane can occur by simple diffusion. Studies of strains that contain a mutation in FadE which inhibits beta-oxidation of exogenous fatty acids, confirm that Nmt1p retains its specificity for myristoyl-CoA over palmitoyl-CoA in E. coli. A mutation that inactivates FadD, a 580-residue protein which is the only acyl-CoA synthetase produced by this bacterium, completely blocks incorporation of exogenous myristate into Arf1p. This failure to be incorporated indicates that myristoyl-acyl carrier protein, generated by inner membrane acyl-acyl carrier protein synthetase, is not a substrate for Nmt1p. S. cerevisiae Faa1p can partially complement this mutant fadD allele. It can fully "restore" N-myristoylation of Arf1p. Faa1p can also rescue growth at 37 degrees C of fadD- strains on minimal media supplemented with C12:0, although this rescue becomes less efficient as the chain length of the supplemental fatty acid increases. In addition, S. cerevisiae Faa1p is better able to direct myristoyl-CoA to the bacteria's phospholipid biosynthetic pathways than FadD, while FadD is more efficient at directing myristoyl-CoA to the genetically engineered protein N-myristoylation pathway. Since cellular acyl-CoA synthetase activity in S. cerevisiae has been distributed to at least two functionally differentiated proteins, this system should be useful for comparing their structure-activity relationships as well as their interactions with Nmt1p in an organelle-free environment.
Knoll
L J
LJ
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Gordon
J I
JI
D000217:Acyltransferases / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001692:Biological Transport; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D050612:Fatty Acid Transport Proteins; D017353:Gene Deletion; D005798:Genes, Bacterial; D009154:Mutation; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D010957:Plasmids; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D050612:Fatty Acid Transport Proteins; D009227:Myristic Acids; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C065691:fadL protein, E coli; D019814:Myristic Acid; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase