TY - JOUR
T1 - Genetic evaluation of physiological functions of thiolase isozymes in the n-alkane-assimilating yeast Candida tropicalis
AU - Kanayama, Naoki
AU - Ueda, Mitsuyoshi
AU - Atomi, Haruyuki
AU - Tanaka, Atsuo
PY - 1998/2
Y1 - 1998/2
N2 - The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT- T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-tlaΔ/t1bΔ null mutation abolished the activity of acetoacetyI-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3- ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C10 to C13). All thiolase activities in this yeast disappeared with the ct- tlaΔ/tlbΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-tlbΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C4 substrate butyrate.
AB - The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT- T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-tlaΔ/t1bΔ null mutation abolished the activity of acetoacetyI-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3- ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C10 to C13). All thiolase activities in this yeast disappeared with the ct- tlaΔ/tlbΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-tlbΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C4 substrate butyrate.
UR - http://www.scopus.com/inward/record.url?scp=0031910095&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0031910095&partnerID=8YFLogxK
U2 - 10.1128/jb.180.3.690-698.1998
DO - 10.1128/jb.180.3.690-698.1998
M3 - Article
C2 - 9457876
AN - SCOPUS:0031910095
VL - 180
SP - 690
EP - 698
JO - Journal of Bacteriology
JF - Journal of Bacteriology
SN - 0021-9193
IS - 3
ER -