PHOSPHATIDYLSERINE DECARBOXYLASE-2 OF SACCHAROMYCES-CEREVISIAE - CLONING AND MAPPING OF THE GENE, HETEROLOGOUS EXPRESSION, AND CREATION OF THE NULL ALLELE

The yeast Saccharomyces cerevisiae expresses two phosphatidylserine de carboxylase (PSD) activities which are responsible for conversion of p hosphatidylserine to phosphatidylethanolamine, and either enzyme alone is sufficient for normal cellular growth, However, strains containing a PSD1 null allele and a mutation leading to loss of PSD2 activity (p sd1-Delta 1::TRP1 psd2) are auxotrophic for ethanolamine, This nutriti onal requirement was utilized to isolate the gene encoding the PSD2 en zyme by complementation. The PSD2 gene encodes a protein of 1138 amino acids with a predicted molecular mass of 130 kDa. The deduced amino a cid sequence shows significant identity (34%) to a PSD-like sequence f rom Clostridium pasteurianum and the yeast PSD1 (19%) at the carboxyl end of the protein, Of particular interest is the presence of a sequen ce, GGST, which may be involved in post-translational processing and p ros thetic group formation similar to other PSD enzymes, The PSD2 amin o acid sequence also shows significant homology to the C-2 regions of protein kinase C and synaptotagmin. Physical mapping experiments demon strate that the PSD2 is located on chromosome 7. The PSD2 gene was het erologously expressed by infection of Sf-9 insect cells with recombina nt baculovirus, resulting in a 10-fold increase in PSD activity. The n ull allele of PSD2 was introduced into yeast strains by one-step gene deletion/disruption with a HIS3 marker gene, Strains expressing wild t ype PSD1 and the psd2-Delta 1::HIS3 allele show a small decrease in ov erall PSD activity, but no noticeable effect upon [H-3] serine incorpo ration into aminophospholipids. Strains containing both the psd1-Delta 1::TRP1 and psd2-Delta 1::HIS3 null alleles, however, express no dete ctable PSD activity, are ethanolamine auxotrophs and show a severe def icit in the conversion of [H-3] serine-labeled phosphatidylserine to p hosphatidylethanolamine. These data indicate that the gene isolated is the structural gene for PSD2 and that the PSD1 and PSD2 enzymes accou nt for all yeast PSD activity.