Membrane topology of yeast alkaline ceramidase YPC1

被引:5
作者
Ramachandra, Nagaraju [1 ]
Conzelmann, Andreas [1 ]
机构
[1] Univ Fribourg, Dept Biol, CH-1700 Fribourg, Switzerland
基金
瑞士国家科学基金会;
关键词
ceramidase; CREST superfamily; cysteine residue accessibility; topology; yeast phyto-ceramidase 1 (YPC1); INOSITOL PHOSPHORYLCERAMIDE SYNTHASE; SACCHAROMYCES-CEREVISIAE; GPI ANCHORS; LAG1; CLONING; SPHINGOLIPIDS; SPHINGOSINE; SPECIFICITY; HOMOLOGS; PROTEINS;
D O I
10.1042/BJ20130085
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Ypc1p (yeast phyto-ceramidase 1) and Ydc1p (yeast dihydroceramidase 1) are alkaline ceramide hydrolases that reside in the ER (endoplasmic reticulum). Ypc1p can catalyse the reverse reaction, i.e. the condensation of non-esterified fatty acids with phytosphingosine or dihydrosphingosine and overexpression of YPC1 or YDC1 can provide enough ceramide synthesis to rescue the viability of cells lacking the normal acyl-CoA-dependent ceramide synthases. To better understand the coexistence of acyl-CoA-dependent ceramide synthases and ceramidases in the ER we investigated the membrane topology of Ypc1p by probing the cysteine residue accessibility of natural and substituted cysteines with membrane non-permeating mass-tagged probes. The N- and C-terminal ends of Ypc1p are oriented towards the lumen and cytosol respectively. Two of the five natural cysteines, Cys(27) and Cys(219), are essential for enzymatic activity and form a disulfide bridge. The data allow the inference that all of the amino acids of Ypc1p that are conserved in the Pfam PF05875 ceramidase motif and the CREST {alkaline ceramidase, PAQR [progestin and adipoQ (adiponectin) receptor] receptor, Per! (protein processing in the ER 1), SID-1 (sister disjunction 1) and TMEM8 (transmembrane protein 8)} superfamily are located in or near the ER lumen. Microsomal assays using a lysine residue-specific reagent show that the reverse ceramidase activity can only be blocked when the reagent has access to Ypc1p from the lumenal side. Overall the data suggest that the active site of Ypc1p resides at the lumenal side of the ER membrane.
引用
收藏
页码:585 / 594
页数:10
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