Trotter P J, Pedretti J, Yates R, Voelker D R
Lord and Taylor Laboratory for Lung Biochemistry, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
J Biol Chem. 1995 Mar 17;270(11):6071-80. doi: 10.1074/jbc.270.11.6071.
The yeast Saccharomyces cerevisiae expresses two phosphatidylserine decarboxylase (PSD) activities which are responsible for conversion of phosphatidylserine 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 (psd1-delta 1::TRP1 psd2) are auxotrophic for ethanolamine. This nutritional requirement was utilized to isolate the gene encoding the PSD2 enzyme by complementation. The PSD2 gene encodes a protein of 1138 amino acids with a predicted molecular mass of 130 kDa. The deduced amino acid sequence shows significant identity (34%) to a PSD-like sequence from Clostridium pasteurianum and the yeast PSD1 (19%) at the carboxyl end of the protein. Of particular interest is the presence of a sequence, GGST, which may be involved in post-translational processing and prosthetic group formation similar to other PSD enzymes. The PSD2 amino acid sequence also shows significant homology to the C2 regions of protein kinase C and synaptotagmin. Physical mapping experiments demonstrate that the PSD2 is located on chromosome 7. The PSD2 gene was heterologously expressed by infection of Sf-9 insect cells with recombinant baculovirus, resulting in a 10-fold increase in PSD activity. The null allele of PSD2 was introduced into yeast strains by one-step gene deletion/disruption with a HIS3 marker gene. Strains expressing wild type PSD1 and the psd2-delta 1::HIS3 allele show a small decrease in overall PSD activity, but no noticeable effect upon [3H]serine incorporation into aminophospholipids. Strains containing both the psd1-delta 1::TRP1 and psd2-delta 1::HIS3 null alleles, however, express no detectable PSD activity, are ethanolamine auxotrophs and show a severe deficit in the conversion of [3H]serine-labeled phosphatidylserine to phosphatidylethanolamine. These data indicate that the gene isolated is the structural gene for PSD2 and that the PSD1 and PSD2 enzymes account for all yeast PSD activity.
酿酒酵母表达两种磷脂酰丝氨酸脱羧酶（PSD）活性，它们负责将磷脂酰丝氨酸转化为磷脂酰乙醇胺，且任何一种酶单独存在就足以支持细胞正常生长。然而，含有PSD1无效等位基因和导致PSD2活性丧失的突变（psd1-Δ1::TRP1 psd2）的菌株对乙醇胺是营养缺陷型。利用这种营养需求通过互补作用分离出编码PSD2酶的基因。PSD2基因编码一个由1138个氨基酸组成的蛋白质，预测分子量为130 kDa。推导的氨基酸序列在蛋白质的羧基末端与巴氏梭菌的一个类PSD序列有显著的一致性（34%），与酵母PSD1也有19%的一致性。特别有趣的是存在一个序列GGST，它可能与翻译后加工以及辅基形成有关，类似于其他PSD酶。PSD2氨基酸序列与蛋白激酶C和突触结合蛋白的C2区域也有显著的同源性。物理图谱实验表明PSD2位于7号染色体上。通过用重组杆状病毒感染Sf-9昆虫细胞，使PSD2基因在异源系统中表达，导致PSD活性增加了10倍。通过用HIS3标记基因进行一步基因缺失/破坏，将PSD2的无效等位基因导入酵母菌株。表达野生型PSD1和psd2-Δ1::HIS3等位基因的菌株总体PSD活性略有下降，但对[3H]丝氨酸掺入氨基磷脂没有明显影响。然而，同时含有psd1-Δ1::TRP1和psd2-Δ1::HIS3无效等位基因的菌株没有可检测到的PSD活性，是乙醇胺营养缺陷型，并且在将[3H]丝氨酸标记的磷脂酰丝氨酸转化为磷脂酰乙醇胺方面表现出严重缺陷。这些数据表明分离出的基因是PSD2的结构基因，并且PSD1和PSD2酶构成了酵母所有的PSD活性。
