title: A lettuce (Lactuca sativa) homolog of human Nogo-B receptor interacts with cis-prenyltransferase and is necessary for natural rubber biosynthesis.
abstract: Natural rubber (cis-1,4-polyisoprene) is an indispensable biopolymer used to manufacture diverse consumer products. Although a major source of natural rubber is the rubber tree (Hevea brasiliensis), lettuce (Lactuca sativa) is also known to synthesize natural rubber. Here, we report that an unusual cis-prenyltransferase-like 2 (CPTL2) that lacks the conserved motifs of conventional cis-prenyltransferase is required for natural rubber biosynthesis in lettuce. CPTL2, identified from the lettuce rubber particle proteome, displays homology to a human NogoB receptor and is predominantly expressed in latex. Multiple transgenic lettuces expressing CPTL2-RNAi constructs showed that a decrease of CPTL2 transcripts (3-15% CPTL2 expression relative to controls) coincided with the reduction of natural rubber as low as 5%. We also identified a conventional cis-prenyltransferase 3 (CPT3), exclusively expressed in latex. In subcellular localization studies using fluorescent proteins, cytosolic CPT3 was relocalized to endoplasmic reticulum by co-occurrence of CPTL2 in tobacco and yeast at the log phase. Furthermore, yeast two-hybrid data showed that CPTL2 and CPT3 interact. Yeast microsomes containing CPTL2/CPT3 showed enhanced synthesis of short cis-polyisoprenes, but natural rubber could not be synthesized in vitro. Intriguingly, a homologous pair CPTL1/CPT1, which displays ubiquitous expressions in lettuce, showed a potent dolichol biosynthetic activity in vitro. Taken together, our data suggest that CPTL2 is a scaffolding protein that tethers CPT3 on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta, but yeast-expressed CPTL2 and CPT3 alone could not synthesize high molecular weight natural rubber in vitro.
authors.affiliation: From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Yang
authors.initials: Y
authors.lastname: Qu
authors.affiliation: From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Romit
authors.initials: R
authors.lastname: Chakrabarty
authors.affiliation: From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Hue T
authors.initials: HT
authors.lastname: Tran
authors.affiliation: From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Eun-Joo G
authors.initials: EJ
authors.lastname: Kwon
authors.affiliation: From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Moonhyuk
authors.initials: M
authors.lastname: Kwon
authors.affiliation: From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Trinh-Don
authors.initials: TD
authors.lastname: Nguyen
authors.affiliation: From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada daekyun.ro@ucalgary.ca.
authors.forename: Dae-Kyun
authors.initials: DK
authors.lastname: Ro
mesh_terms: D060054:Agrobacterium / Q000378:metabolism; D000595:Amino Acid Sequence; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D028482:Hevea; D007840:Latex / Q000737:chemistry; D018545:Lactuca / Q000378:metabolism*; D018613:Microscopy, Confocal; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010641:Phenotype; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012408:Rubber / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000378:metabolism
chemical_list: D007840:Latex; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D049452:Green Fluorescent Proteins; D012408:Rubber; D004247:DNA; D014166:Transferases; C106821:cis-prenyl transferase
keywords: Enzyme Catalysis; Lettuce; Natural Rubber; Plant Biochemistry; Protein Complex; Proteomics; Terpenoid; cis-Prenyltransferase, RNA Interference