Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice.
Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases that are found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, much less is known about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase by heterologous expression in the yeast double knockout mutant Deltaypc1Deltaydc1 that lacks the yeast ceramidases YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis-Menten kinetics, with optimum activity between pH 5.7 and 6.0. OsCDase activity was enhanced in the presence of Ca(2+), Mg(2+), Mn(2+) and Zn(2+), but inhibited in the presence of Fe(2+). OsCDase appears to use ceramide instead of phytoceramide as a substrate. Subcellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants.
Pata
Mickael O
MO
School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland.
Wu
Bill X
BX
Bielawski
Jacek
J
Xiong
Tou Cheu
TC
Hannun
Yusuf A
YA
Ng
Carl K-Y
CK
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D006056:Golgi Apparatus / Q000378:metabolism; D012275:Oryza / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013107:Sphingolipids / Q000378:metabolism; D013379:Substrate Specificity
D002518:Ceramides; D018076:DNA, Complementary; D010940:Plant Proteins; D018749:RNA, Plant; D013107:Sphingolipids; D000581:Amidohydrolases; D055572:Ceramidases