Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal ␤-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (⌬pox2 ⌬pox3 ⌬pox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.The yeast Yarrowia lipolytica can grow on alkanes or fatty acids as the sole carbon source (for a review, see reference 5). Alkanes must be converted to fatty acids before they can serve as substrates for different metabolic pathways. In mammalian cells, the degradation of fatty acids occurs through ␤-oxidation in mitochondria and peroxisomes. In yeasts, the enzymes in this pathway are present only in peroxisomes. Fatty acid degradation is a multiple-step process requiring four different enzymatic activities. The first step is catalyzed by an acyl-coenzyme A (CoA) oxidase (Aoxp). Saccharomyces cerevisiae contains only one Aox (28), but Y. lipolytica contains five Aox isoenzymes with different substrate specificities and different activity levels ( Fig. 1) (27,28). Aox3p is specific for short-chain acyl-CoAs (17), Aox2p preferentially oxidizes long-chain acylCoAs (18), and the Aox4p and Aox5p activities do not appear to be sensitive to the length of the aliphatic chain of the acyl-CoA (29). Previous studies revealed that the mutants MTLY36 (⌬pox2 ⌬pox3 ⌬pox5) and MTLY37 (⌬pox2 ⌬pox3 ⌬pox4 ⌬pox5) either had a growth defect or did not grow at all, respectively, when cultivated on oleic acid minimal medium (28).Fatty acids can also be utilized as storage molecules when they are incorporated into triacylglycerols. All eukaryotic organisms and some gram-positive bacteria store triacylglycerols in intracellular compartments that are variously termed lipid particles, lipid droplets (LDs), lipid bodies (LBs), oil bodies, oleosomes, or spherosomes (in plants). The structure of these lipid-rich compartments is similar in all cell types and is rather simple: LBs consist of a hydrophobic core formed from neutral lipids, mainly triacylglycerols and/or steryl esters...

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