Membrane topology of yeast alkaline ceramidase YPC1

Abstract

Condensed title: Topology of yeast alkaline ceramidase YPC1Key words: YPC1, ceramidase, topology, cysteine accessibility, CREST superfamily Abbreviations: DHS, dihydrosphingosine; DTR, dual topology reporter; DTT, dithiothreitol; FOA, 5'-fluoroorotic acid; IPC, inositolphosphorylceramide; LCB, long chain base; MIPC, mannosyl-IPC; M(IP) 2 C, inositolphosphoryl-MIPC; NEM, N-ethylmaleimide; PEG-mal, PEG-5000-maleimide; PHS, phytosphingosine; RT, room temperature; SCAM TM , substituted cysteine accessibility method for TM determination; TM, transmembrane helix; TNBS, trinitrobenzene sulfonic acid; UBI-mal, ubiquitin-maleimide; VLCFA, very long chain fatty acid; wt, wild type. SynopsisYpc1p and Ydc1p are alkaline ceramide hydrolases, which reside in the ER. Ypc1p can catalyze the reverse reaction, i.e. the condensation of free fatty acids with phytosphingosine or dihydrosphingosine and overexpression of YPC1 or YDC1 can provide enough ceramide synthesis as 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 cysteine 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 5 natural cysteines, Cys27 and Cys219, are essential for enzymatic activity and form a disulfide bridge. The data allow inferring that all amino acids of Ypc1p that are conserved in the pfam PF05875 ceramidase motif and the CREST superfamily are located in or near the ER lumen. Microsomal assays using a lysine-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. IntroductionThe mature sphingolipids of Saccharomyces cerevisiae, namely the inositolphosphorylceramides (IPCs), mannosyl-IPCs (MIPCs) and inositolphosphoryl-MIPCs (M(IP) 2 Cs) are major components of the yeast plasma membrane. Moreover, as in higher eukaryotes, the metabolic intermediates of sphingolipid metabolism have been advocated as signaling molecules in many cell biological phenomena and responses (for review see [1]). Ceramide is a central metabolic intermediate (Fig. 1), which can result from breakdown of IPCs by Isc1p [2] or can be synthesized from acyl-CoA and long chain bases (LCBs) by ceramide synthases consisting of Lag1p or Lac1p in complex with (Fig. 1). Yeast Lag1p and Lac1p are functionally redundant but concomitant deletion of LAG1 and LAC1 causes a significant growth defect in the genetic background of W303 cells and is lethal in the YPK9 background [3,4,8]. Insertion of glycosylation sites and factor Xa protease sites into Lag1p and Lac1p, the catalytically active subunits of the yeast ceramide synthase, generated a topological model compris...

Characterization of yeast mutants lacking alkaline ceramidasesYPC1andYDC1

“…On the other hand, Ypc1p may reside in membrane domains enriched in C24:0 and C26:0. Ypc1p has its active site on the luminal side of the ER (Ramachandra & Conzelmann, ), and the concentrations of various fatty acids in the luminal leaflet of the ER are presently not known. We could not detect IPCs with a C16:0 as fatty acid after growing 2∆.YPC1 cells in medium containing C16:0 (not shown).…”

“…Thus, even when expressed from their endogenous promoters, they may contribute to ceramide biosynthesis if Lac1p and Lac1p are not operational. Moreover, the reverse ceramidase activity of Ypc1p and Ydc1p in microsomal detergent extracts from wild-type (WT) cells can easily be detected and is quite substantial, as expression of GAL1 promoter driven YPC1, placed on a 2 l or a centromeric vector, increases the activity only 25 and 2.3-fold over WT levels, respectively (Mao et al, 2000a;Ramachandra & Conzelmann, 2013; therein Supporting Information, Fig. S2d).…”

Cdc1 removes the ethanolamine phosphate of the first mannose of GPI anchors and thereby facilitates the integration of GPI proteins into the yeast cell wall

“…Materials were from sources described recently ( Ramachandra and Conzelmann, 2013 ). [ 3 H] myo -inositol and [ 14 C]serine were from ARC Radiochemicals (St. Louis, MO); rhodamine phalloidin and Prolong Gold antifade reagent were from Invitrogen.…”

Aging-related elevation of sphingoid bases shortens yeast chronological life span by compromising mitochondrial function

“…To exclude the possibility that Ypc1p protein per se may affect the expression of other LCB metabolic enzymes, a catalytically inactive mutant of this enzyme was constructed as a negative control by site-directed mutagenesis as described in the experimental procedure. We mutated Ypc1p by replacing Cys 27 with Phe to generate the Cys 27 > Phe 27 mutant, named Ypc1p-C27F because it was reported that Cys 27 is essential for Ypc1p's reverse activity [24]. We generated a yeast stain that overexpresses Ypc1p or Ypc1p-C27F under the control of a Gal1 promoter.…”

“…To exclude the possibility that Ypc1p per se may induce biological effects independently of its lipid mediator, a catalytically inactive mutant of this enzyme was constructed as a negative control. Ypc1p was mutated by replacing Cys 27 with Phe to generate the Cys 27 > Phe 27 mutant [24], named Ypc1p-C27F, by a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technology, CT) following the manufacturer's manual. The ORF of Ypc1p-C27F was cloned into a pYES2 vector, and the resulting expression construct, pYES2::YPC1 C27F , was transformed into yeast cells as described previously.…”