He L-carnitine-dependent strains described within this study offer exciting platforms for studying the function of the carnitine shuttle in wholesome and diseased human cells. Several eukaryotes use a citrate-oxaloacetate shuttle, consisting of mitochondrial citrate synthase, a mitochondrial citrate transporter, and cytosolic ATP-dependent citrate lyase, for export ofacetyl units from their mitochondria (480). Conversion of mitochondrial acetyl-CoA to acetate, followed by its export and cytosolic ATP-dependent activation to acetyl-CoA, occurs in Trypanosoma brucei (51). The latter mechanism also supports slow development of pyruvate decarboxylase-negative S. cerevisiae mutants, which cannot make use of the PDH bypass for cytosolic acetyl-CoA synthesis (52). The ATP requirement of those naturally occurring acetyl-CoA shuttles is constant with our hypothesis that in vivo concentrations of acetyl-CoA in cytosol and mitochondria of wild-type yeast cells do not enable outward translocation of acetyl units via the energy-independent carnitine shuttle. Quantification of trade-offs among ATP efficiency and in vivo kinetics of cyto-TABLE 4 Particular carnitine acetyltransferase activities in cell extracts of S. cerevisiae strainsaStrain IMX585 IMX868 IMX923 IMX925 IME140 IME320 IME321 IME233 CEN.PK113-7D CEN.PK215-4A IMX745 IMS0482 IMX852 IMX913 IMX932 Brief descriptionb Reference strain CARN sga1 ::pADH1-YAT2 sga1 ::pADH1-YAT2C173G Empty multicopy plasmid Multicopy plasmid pADH1-YAT2 Multicopy plasmid pADH1-YAT2C173G Multicopy plasmid pTDH3-CAT2 CAT2 YAT1 YAT2 cat2 yat1 YAT2 CARN CARN evolution line 1 CARN,pADH1-YAT2 MCT1T641GRTG2G503T CARN,pADH1-YAT2C173G MCT1T641G RTG2G503T CARN,yat2 MCT1T641G RTG2G503T Carbon supply within the medium Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Ethanol Ethanol Glucose Glucose Glucose Glucose Glucose Carnitine acetyltransferase activity ( mol mg protein 1 min 1)c BD 2.1421473-07-5 structure 69 BD BD BD BD BD four.24 1.75 BD three.19 2.39 two.92 3.11 two.82 0.0.52 0.0.14 0.05 0.73 0.71 0.a Strains had been grown in shake flasks containing synthetic medium with either 20 g liter 1 glucose or two (vol/vol) ethanol as the carbon source and harvested in mid-exponential phase.36234-66-9 uses b The composition on the CARN gene set is described in Supplies and Solutions.PMID:36014399 c Carnitine acetyltransferase activities in cell extracts have been obtained from duplicate development experiments and are shown as implies standard deviations. The detection limit of the enzyme assay was 0.01 mol mg protein 1 min 1. BD, under detection.May/June 2016 Volume 7 Problem 3 e00520-mbio.asm.orgVan Rossum et al.solic acetyl-CoA provision by way of distinct pathways demands analysis of mitochondrial and cytosolic acetyl-CoA pools in wild-type and engineered strains. Such studies will, nevertheless, must await development of techniques for correct measurement of acetyl-CoA concentrations in distinct cellular compartments. YAT2, the third gene in which a point mutation stimulated carnitine-dependent growth of acs1 acs2 strains, was reported to encode a carnitine acetyltransferase (15). Yat2 shows substantial sequence identity using the two other yeast carnitine acetyltransferases (28 and 22 amino acid sequence identity with Yat1 and Cat2, respectively [53]). On the other hand, Yat2 is substantially longer than Yat1 and Cat2, by 236 and 253 amino acids, respectively, and its 169-amino-acid C-terminal sequence is conserved only in some closely connected orthologs within the Saccharomycetaceae (54). The mutation in YA.