Publications

• Precise bacterial polyprenol length control fails in Saccharomyces cerevisiae.

  Poznanski J., Szkopinska A.

  A comparison of amino acid sequences of yeast Rer2p and Srt1p Z-prenyltransferases shows that the spatial organization of their substrate tunnels agrees with that determined by X-ray for the E. coli undecaprenyl diphosphate synthase (UPPs). The observed trend in the maxima of product length distri ... >> More

  A comparison of amino acid sequences of yeast Rer2p and Srt1p Z-prenyltransferases shows that the spatial organization of their substrate tunnels agrees with that determined by X-ray for the E. coli undecaprenyl diphosphate synthase (UPPs). The observed trend in the maxima of product length distribution shifted from C(55) in UPPs to C(80) in Rer2p and to C(110) in Srt1p. This suggests a significant increase in the size of the enzyme hydrophobic tunnel from approximately 1000 A(3) of E. coli UPPs to approximately 1300 A(3) required to accommodate C(80) in Rer2p and to 1700 A(3) for C(110) in Srt1p. Moreover, Srt1p products reaching C(290) indicate the failure of a strict bacterial-like chain length control. On the basis of E. coli UPPs crystallographic structure the yeast Rer2p model was constructed. In the model three amino acid residues inserted into the sequence corresponding to the "floor" region of the tunnel extends the bottom loop what results in the required increase of the tunnel volume. Moreover, thermal fluctuations of this loop occasionally create a hole in the tunnel floor, making escape of polyprenol omega end out of the tunnel possible what switches off the control mechanism of product length thereby allowing a practically unlimited elongation process leading to an exponential distribution of longer chain polyprenols. << Less

  Biopolymers 86:155-164(2007) [PubMed] [EuropePMC]

• Yeast Saccharomyces cerevisiae has two cis-prenyltransferases with different properties and localizations. Implication for their distinct physiological roles in dolichol synthesis.

  Sato M., Fujisaki S., Sato K., Nishimura Y., Nakano A.

  <h4>Background</h4>Dolichol is a family of long-chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER). We have identified a key enzyme of the dolichol synthesis, cis-prenyltransferase, as Rer2p from Saccharomyces cerevisiae. We have also ... >> More

  <h4>Background</h4>Dolichol is a family of long-chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER). We have identified a key enzyme of the dolichol synthesis, cis-prenyltransferase, as Rer2p from Saccharomyces cerevisiae. We have also isolated a multicopy suppressor of an rer2 mutant and named it SRT1. It encodes a protein similar to Rer2p but its function has not been established.<h4>Results</h4>The cis-prenyltransferase activity of Srt1p has been proved biochemically in the lysate of yeast cells lacking Rer2p. The polyprenol product of Srt1p is longer in chain length than that of Rer2p and is not sufficiently converted to dolichol and dolichyl phosphate, unlike that of Rer2p. The subcellular localization of these two isozymes has been examined by immunofluorescence microscopy and by the use of GFP fusion proteins. Whereas GFP-Rer2p is localized to the continuous ER and some dots associated with the ER, GFP-Srt1p shows only punctate localization patterns. Immunofluorescence double staining with Erg6p, a marker of lipid particles in yeast, indicates that Srt1p is mainly localized to lipid particles (lipid bodies). RER2 is mainly expressed in the early logarithmic phase, while the expression of SRT1 is induced in the stationary phase.<h4>Conclusions</h4>We have shown that yeast has two active cis-prenyltransferases with different properties. This result implies that the two isozymes have different physiological roles during the life cycle of the yeast. << Less

  Genes Cells 6:495-506(2001) [PubMed] [EuropePMC]

• The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis.

  Sato M., Sato K., Nishikawa S., Hirata A., Kato J., Nakano A.

  As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mfalpha1p fusion protein from the ER to later compartments of the secretory pathway (S. Nishikawa and A. Nakano, Proc. Natl. Acad. Sci. U ... >> More

  As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mfalpha1p fusion protein from the ER to later compartments of the secretory pathway (S. Nishikawa and A. Nakano, Proc. Natl. Acad. Sci. USA 90:8179-8183, 1993). The temperature-sensitive rer2 mutant mislocalizes different types of ER membrane proteins, suggesting that RER2 is involved in correct localization of ER proteins in general. The rer2 mutant shows several other characteristic phenotypes: slow growth, defects in N and O glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and the Golgi membranes. RER2 and SRT1, a gene whose overexpression suppresses rer2, encode novel proteins similar to each other, and their double disruption is lethal. RER2 homologues are found not only in eukaryotes but also in many prokaryote species and thus constitute a large gene family which has been well conserved during evolution. Taking a hint from the phenotype of newly established mutants of the Rer2p homologue of Escherichia coli, we discovered that the rer2 mutant is deficient in the activity of cis-prenyltransferase, a key enzyme of dolichol synthesis. This and other lines of evidence let us conclude that members of the RER2 family of genes encode cis-prenyltransferase itself. The difference in phenotypes between the rer2 mutant and previously obtained glycosylation mutants suggests a novel, as-yet-unknown role of dolichol. << Less

  Mol. Cell. Biol. 19:471-483(1999) [PubMed] [EuropePMC]

• Mutation of Nogo-B receptor, a subunit of cis-prenyltransferase, causes a congenital disorder of glycosylation.

  Park E.J., Grabinska K.A., Guan Z., Stranecky V., Hartmannova H., Hodanova K., Baresova V., Sovova J., Jozsef L., Ondruskova N., Hansikova H., Honzik T., Zeman J., Hulkova H., Wen R., Kmoch S., Sessa W.C.

  Dolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not bee ... >> More

  Dolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice, and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provide a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation. << Less

  Cell Metab. 20:448-457(2014) [PubMed] [EuropePMC]

• Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana. Implications in rubber biosynthesis.

  Oh S.K., Han K.H., Ryu S.B., Kang H.

  cis-Prenyltransferase catalyzes the sequential condensation of isopentenyl diphosphate with allylic diphosphate to synthesize polyprenyl diphosphates that play vital roles in cellular activity. Despite potential significance of cis-prenyltransferase in plant growth and development, no gene of the ... >> More

  cis-Prenyltransferase catalyzes the sequential condensation of isopentenyl diphosphate with allylic diphosphate to synthesize polyprenyl diphosphates that play vital roles in cellular activity. Despite potential significance of cis-prenyltransferase in plant growth and development, no gene of the enzyme has been cloned from higher plants. Using sequence information of the conserved region of cis-prenyltransferase cloned recently from Escherichia coli, Micrococcus luteus, and yeast, we have isolated and characterized the first plant cis-prenyltransferase from Arabidopsis thaliana. Sequence analysis revealed that the protein is highly homologous in several conserved regions to cis-prenyltransferases from M. luteus, E. coli, and yeast. In vitro analyses using the recombinant protein overexpressed in E. coli revealed that the enzyme catalyzed the formation of polyprenyl diphosphates ranging in carbon number from 100 to 130 with a predominance of C(120). The enzyme exhibited a higher affinity for farnesyl diphosphate than for geranylgeranyl diphosphate, with the K(m) values being 0.13 and 3.62 micrometer, respectively, but a lower affinity for isopentenyl diphosphate, with a K(m) value of 23 micrometer. In vitro rubber biosynthesis analysis indicated that the Arabidopsis cis-prenyltransferase itself could not catalyze the formation of higher molecular weight polyprenyl diphosphates similar to natural rubber. A reverse transcriptase-polymerase chain reaction analysis showed that the gene was expressed at low levels in Arabidopsis plant, in which expression of the cis-prenyltransferase in leaf and root was higher than that in stem, flower, and silique. These results indicate the tissue-specific expression of cis-prenyltransferase and suggest a potential role and significance of the enzyme in the polyisoprenoid biosynthesis in plants. << Less

  J. Biol. Chem. 275:18482-18488(2000) [PubMed] [EuropePMC]

• Characterization of dehydrodolichyl diphosphate synthase of Arabidopsis thaliana, a key enzyme in dolichol biosynthesis.

  Cunillera N., Arro M., Fores O., Manzano D., Ferrer A.

  The enzyme dehydrodolichyl diphosphate (dedol-PP) synthase is a cis-prenyltransferase that catalyzes the synthesis of dedol-PP, the long-chain polyprenyl diphosphate used as a precursor for the synthesis of dolichyl phosphate. Here we report the cloning and characterization of a cDNA from Arabidop ... >> More

  The enzyme dehydrodolichyl diphosphate (dedol-PP) synthase is a cis-prenyltransferase that catalyzes the synthesis of dedol-PP, the long-chain polyprenyl diphosphate used as a precursor for the synthesis of dolichyl phosphate. Here we report the cloning and characterization of a cDNA from Arabidopsis thaliana encoding dedol-PP synthase. The identity of the cloned enzyme was confirmed by functional complementation of a yeast mutant strain defective in dedol-PP synthase activity together with the detection of high levels of dedol-PP synthase activity in the transformed yeast mutant. The A. thaliana dedol-PP synthase mRNA was detected at high levels in roots but was hardly detected in flowers, leaves, stems and in A. thaliana suspension-cultured cells. << Less

  FEBS Lett. 477:170-174(2000) [PubMed] [EuropePMC]