Cloning and characterization of genea required for synthesis of hydroxylated inositolphoephoceramides in S. cerevisiae

The CS02 and CSG2 genes are required for growth of s. cerevisiae cells in 10 mM Ca²⁺ and for eyntheaia of mannosylated sphingolipida. Suppressors of the Ca²⁺-sensitive growth phenotype of cagl and csg2 have identified genes required for the hydroxylation of inositolphosphoceramide (IPC) to make the IPC-C and IPC-D species. The SCS7 gene was cloned on the basis of its ability to reverse the suppression of the cag2 Caï+-senaitive phenotype by aca7 mutations. The SCS7 gene encodea a protein containing a cytochrome bj-like domain and a domain which is a member of a super-family of desaturaaea/hydroxylases containing oxo-bridged diron clusters in which the ironbinding motif consists of His-containing sequences. The CCC2 gene which encodes a CuI+-ATPase was cloned as a dose dependent suppressor of csgl; it also suppresses cag2. The amount of IPC-D synthesis ia dependent on the expression level of Ccc2p and the amount of Cu2in the growth media suggesting the presence of a Cu1+-dependent hydroxylating enzyme. The role of Ccc2p is therefore proposed to be transport of Cu3to the intracellular compartment where the hydroxylase resides. Future experiments will be aimed at identifying the Cu2-dopendent hydroxylase which itself would be predicted to be a dose-dependent suppressor of the csg mutants.