Myristic acid auxotrophy caused by mutation of S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.
The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C. Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.
Duronio
R J
RJ
Department of Molecular Biology, Washington University School of Medicine, St. Louis, Missouri 63110.
Rudnick
D A
DA
Johnson
R L
RL
Johnson
D R
DR
Gordon
J I
JI
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000378:metabolism; D004271:DNA, Fungal; D003652:Decanoic Acids / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000096:biosynthesis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009227:Myristic Acids / Q000378:metabolism*; D010641:Phenotype; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic; D013696:Temperature; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D004271:DNA, Fungal; D003652:Decanoic Acids; D005227:Fatty Acids; D009227:Myristic Acids; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase