Functional and Topological Analysis of Yeast Acyl-CoA:Diacylglycerol Acyltransferase 2, an Endoplasmic Reticulum Enzyme Essential for Triacylglycerol Biosynthesis

Liu, Qin; Siloto, Rodrigo M.P.; Snyder, Crystal L.; Weselake, Randall J.

doi:10.1074/jbc.m110.204412

Cited by 87 publications

(121 citation statements)

References 39 publications

Supporting

Mentioning

116

Contrasting

Order By: Relevance

“…The amino acid sequences of JcDGAT1 and JcDGAT2 are 67 and 56% similar to the corresponding orthologues from Arabidopsis (At2 g19450 and At3 g51520, see Stone et al 2006;Liu et al 2011). Genomic structure analysis showed that JcDGAT1 (JQ319812) contains 16 exons and 15 introns, while JcDGAT2 (JQ319813) contains nine exons and eight introns.…”

Section: Cloning Gene Structure and Phylogenetic Analysis Of Jcdgat1mentioning

confidence: 96%

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

Yang

Wang

et al. 2014

Functional Plant Biol.

View full text Add to dashboard Cite

Abstract. Diacylglycerol acyltransferases (DGATs) catalyse the final step of triacylglycerol (TAG) biosynthesis of the Kennedy pathway, and play a critical role during TAG accumulation in developing oleaginous seeds. In this study, the molecular cloning and characterisation of two DGAT genes, JcDGAT1 and JcDGAT2, from jatropha (Jatropha curcas L., a potential biodiesel plant) is presented. Using heterogonous overexpression techniques, both JcDGAT1 and JcDGAT2 were able to restore TAG biosynthesis in a yeast mutant H1246 strain, and enhance the quantity of TAG biosynthesis by 16.6 and 14.3%, respectively, in strain INVSc1. In transgenic tobacco, overexpression of JcDGAT1 and JcDGAT2 resulted in an increase in seed oil content of, respectively, 32.8 and 31.8%. Further, the functional divergence of JcDGAT1 and JcDGAT2 in TAG biosynthesis was demonstrated by comparing the fatty acid compositions in both the transgenic yeast and tobacco systems. In particular, JcDGAT2 incorporated a 2.5-fold higher linoleic acid content into TAG than JcDGAT1 in transgenic yeast and exhibited a significant linoleic acid substrate preference in both yeast and tobacco. This study provides new insights in understanding the molecular mechanisms of DGAT genes underlying the biosynthesis of linoleic acids and TAG in plants.

show abstract

Section: Cloning Gene Structure and Phylogenetic Analysis Of Jcdgat1mentioning

confidence: 96%

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

Yang

Wang

et al. 2014

Functional Plant Biol.

View full text Add to dashboard Cite

show abstract

“…1). Pathways i-iii have been claimed to occur at the lumenal side of the secretory pathway based on biochemical data and bioinformatic analysis of the location of conserved motifs [27,29,[40][41][42][43]. This report makes it likely that also the degradation via alkaline ceramidases occurs lumenally.…”

mentioning

confidence: 94%

“…After 10 min on icewater the derivatization of target proteins was visualized by SDS-PAGE and Western blotting using anti-V5, anti-FLAG and anti-Kar2p antibodies for detection of, Ypc1p, Gpi8p and Kar2p, respectively. Alternatively, we performed SCAM TM assays using NEM adapting protocols described previously [27,28]. For this, microsomes (100 g of protein) were incubated with NEM for 30 min on ice-water unless indicated otherwise.…”

Section: Methodsmentioning

confidence: 99%

“…For this, microsomes (containing the proteins in their native state) are exposed to the membrane-permeating NEM so that cysteines on both sides of the membrane are alkylated. One then removes residual NEM and adds a mass-tagged maleimide derivative (UBImal or PEG-mal) in presence of the denaturating detergent SDS [27,28]. Those cysteines that are not alkylated by NEM when the protein is in the native state, but which get derivatized by a mass-tagged maleimide after denaturation, are classified as "buried" in the native protein structure.…”

Section: Cysteine 271 Is the Only Accessible Cys In Wild Type Ypc1pmentioning

confidence: 99%

See 1 more Smart Citation

Membrane topology of yeast alkaline ceramidase YPC1

Ramachandra

Conzelmann

2013

Biochemical Journal

View full text Add to dashboard Cite

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...

show abstract

“…In addition, more than 40% of the total amino acid residues are hydrophobic (Table 3). Yeast was the preferred host for DGAT expression (Bouvier-Nave et al, 2000;Burgal et al, 2008;Cao et al, 2010;He et al, 2004;Kalscheuer et al, 2004;Kalscheuer & Steinbuchel 2003;Kroon et al, 2006;Liu et al, 2011;Liu et al, 2010;ManasFernandez et al, 2009;Mavraganis et al, 2010;Milcamps et al, 2005;Nykiforuk et al, 2002;Quittnat et al, 2004;Shockey et al, 2006;Siloto et al, 2009;Wagner et al, 2010;Xu et al, 2008;Yu et al, 2008) followed by insect cells (Buszczak et al, 2002;Cases et al, 1998;Cases et al, 2001;Lardizabal et al, 2001). A limited number of reports used other host cells including E. coli (Saha et al, 2006;Siloto et al, 2008;Weselake et al, 2006) and human cells (Cheng et al, 2001).…”

Section: Recombinant Dgat Expression Updatementioning

confidence: 99%

Bioengineering Recombinant Diacylglycerol Acyltransferases

Cao¹

2011

Progress in Molecular and Environmental Bioengineering - From Analysis and Modeling to Technology Applications

View full text Add to dashboard Cite

Functional and Topological Analysis of Yeast Acyl-CoA:Diacylglycerol Acyltransferase 2, an Endoplasmic Reticulum Enzyme Essential for Triacylglycerol Biosynthesis

Cited by 87 publications

References 39 publications

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

Membrane topology of yeast alkaline ceramidase YPC1

Bioengineering Recombinant Diacylglycerol Acyltransferases

Contact Info

Product

Resources

About