title: cis-Prenyltransferase and Polymer Analysis from a Natural Rubber Perspective.
abstract: Dolichol and natural rubber are representative cis-polyisoprenoids in primary and secondary metabolism, respectively. Their biosynthesis is catalyzed by cis-prenyltransferase (CPT) by sequential condensations of isopentenyl diphosphates (IPPs) to a priming molecule. Although prokaryotic CPTs have been well characterized, the mechanism of eukaryotic CPTs in cis-polyisoprene biosynthesis was only recently revealed. It was shown that eukaryotes have evolved a unique protein complex, comprised of CPT and CPT-binding protein (CBP), to synthesize cis-polyisoprenoids. In the context of this new discovery, we found discrepancies in literature for CPT or CBP biochemical assays and in vivo CPT complementation using rer2 (yeast CPT) yeast mutant. Our study here shows that rer2 revertants occur at a frequency that cannot be disregarded and are likely accountable for the results that cannot be explained by the CPT/CBP heteroprotein complex model. To make a stable mutant, SRT1 gene (secondary CPT expressed at a basal level in yeast) was additionally deleted in the rer2Δ mutant background. This stable rer2Δ srt1Δ strain was then used to individually or simultaneously express Arabidopsis CPT1 (AtCPT1, At2g17570) and CBP (AtLEW1, At1G11755). We found that the simultaneous expression of Arabidopsis CPT1 and AtLEW1 effectively complements the rer2Δ srt1Δ strain, whereas the individual expression of AtCPT1 alone or AtLEW1 alone failed to rescue the yeast mutant. Microsomes from the dual expresser showed an efficient incorporation of IPPs into cis-polyisoprenoid (30% in 2h). These results showed that the CPT/CBP heteroprotein complex model is valid in Arabidopsis thaliana. Experimental details of these results are described in this methodology paper.
authors.affiliation: University of Calgary, Calgary, AB, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Kwon
authors.affiliation: University of Calgary, Calgary, AB, Canada.
authors.forename: E-J G
authors.initials: EJ
authors.lastname: Kwon
authors.affiliation: University of Calgary, Calgary, AB, Canada. Electronic address: daekyun.ro@ucalgary.ca.
authors.forename: D K
authors.initials: DK
authors.lastname: Ro
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D053898:Biosynthetic Pathways; D004122:Dimethylallyltranstransferase / Q000235:genetics* / Q000378:metabolism; D004286:Dolichols / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D045782:Hemiterpenes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009943:Organophosphorus Compounds / Q000378:metabolism; D012408:Rubber / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D064210:Secondary Metabolism; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D004286:Dolichols; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D029701:Saccharomyces cerevisiae Proteins; C004809:isopentenyl pyrophosphate; D012408:Rubber; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C511732:Srt1 protein, S cerevisiae; C106821:cis-prenyl transferase; D004122:Dimethylallyltranstransferase; C485360:RER2 protein, S cerevisiae
keywords: Dolichol; Heteroprotein complex; Natural rubber; Revertant; cis-Polyisoprene; cis-Prenyltransferase