Abstract
The maize regulatory protein Opaque-2 (O2) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of O2 to reporter protein beta-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of O2 containing 135 and 149 amino acids were identified that were able to redirect GUS to the nucleus in both systems. A quantitative biochemical analysis of GUS in nuclei isolated from transgenic tobacco plants revealed that the second region was more efficient than the first one. The precise location of nuclear localization signals (NLSs) was determined using an onion transformation system. The first NLS was located between residues 101 and 135 and had the structure of a simian virus 40 NLS. The second NLS was located in the basic, DNA binding domain (between residues 223 and 254) and had a bipartite structure. The presence of one of the O2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basic/leucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino- versus carboxy-terminal GUS fusions is discussed.
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- Angel P., Allegretto E. A., Okino S. T., Hattori K., Boyle W. J., Hunter T., Karin M. Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature. 1988 Mar 10;332(6160):166–171. doi: 10.1038/332166a0. [DOI] [PubMed] [Google Scholar]
- Bednarek S. Y., Wilkins T. A., Dombrowski J. E., Raikhel N. V. A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco. Plant Cell. 1990 Dec;2(12):1145–1155. doi: 10.1105/tpc.2.12.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bonner W. M. Protein migration into nuclei. II. Frog oocyte nuclei accumulate a class of microinjected oocyte nuclear proteins and exclude a class of microinjected oocyte cytoplasmic proteins. J Cell Biol. 1975 Feb;64(2):431–437. doi: 10.1083/jcb.64.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carrington J. C., Freed D. D., Leinicke A. J. Bipartite signal sequence mediates nuclear translocation of the plant potyviral NIa protein. Plant Cell. 1991 Sep;3(9):953–962. doi: 10.1105/tpc.3.9.953. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chrispeels M. J., Raikhel N. V. Short peptide domains target proteins to plant vacuoles. Cell. 1992 Feb 21;68(4):613–616. doi: 10.1016/0092-8674(92)90134-x. [DOI] [PubMed] [Google Scholar]
- Citovsky V., Zupan J., Warnick D., Zambryski P. Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science. 1992 Jun 26;256(5065):1802–1805. doi: 10.1126/science.1615325. [DOI] [PubMed] [Google Scholar]
- Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
- Dingwall C., Sharnick S. V., Laskey R. A. A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell. 1982 Sep;30(2):449–458. doi: 10.1016/0092-8674(82)90242-2. [DOI] [PubMed] [Google Scholar]
- Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dworetzky S. I., Lanford R. E., Feldherr C. M. The effects of variations in the number and sequence of targeting signals on nuclear uptake. J Cell Biol. 1988 Oct;107(4):1279–1287. doi: 10.1083/jcb.107.4.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garcia-Bustos J., Heitman J., Hall M. N. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. doi: 10.1016/0304-4157(91)90013-m. [DOI] [PubMed] [Google Scholar]
- Goff S. A., Klein T. M., Roth B. A., Fromm M. E., Cone K. C., Radicella J. P., Chandler V. L. Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. EMBO J. 1990 Aug;9(8):2517–2522. doi: 10.1002/j.1460-2075.1990.tb07431.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guiltinan M. J., Marcotte W. R., Jr, Quatrano R. S. A plant leucine zipper protein that recognizes an abscisic acid response element. Science. 1990 Oct 12;250(4978):267–271. doi: 10.1126/science.2145628. [DOI] [PubMed] [Google Scholar]
- Hall M. N., Hereford L., Herskowitz I. Targeting of E. coli beta-galactosidase to the nucleus in yeast. Cell. 1984 Apr;36(4):1057–1065. doi: 10.1016/0092-8674(84)90055-2. [DOI] [PubMed] [Google Scholar]
- Hinnebusch A. G. Evidence for translational regulation of the activator of general amino acid control in yeast. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6442–6446. doi: 10.1073/pnas.81.20.6442. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Howard E. A., Zupan J. R., Citovsky V., Zambryski P. C. The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells. Cell. 1992 Jan 10;68(1):109–118. doi: 10.1016/0092-8674(92)90210-4. [DOI] [PubMed] [Google Scholar]
- Jenuwein T., Müller R. Structure-function analysis of fos protein: a single amino acid change activates the immortalizing potential of v-fos. Cell. 1987 Feb 27;48(4):647–657. doi: 10.1016/0092-8674(87)90243-1. [DOI] [PubMed] [Google Scholar]
- Kalderon D., Richardson W. D., Markham A. F., Smith A. E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. doi: 10.1038/311033a0. [DOI] [PubMed] [Google Scholar]
- Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
- Keegstra K. Transport and routing of proteins into chloroplasts. Cell. 1989 Jan 27;56(2):247–253. doi: 10.1016/0092-8674(89)90898-2. [DOI] [PubMed] [Google Scholar]
- Kodrzycki R., Boston R. S., Larkins B. A. The opaque-2 mutation of maize differentially reduces zein gene transcription. Plant Cell. 1989 Jan;1(1):105–114. doi: 10.1105/tpc.1.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
- Lassner M. W., Jones A., Daubert S., Comai L. Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal. Plant Mol Biol. 1991 Aug;17(2):229–234. doi: 10.1007/BF00039497. [DOI] [PubMed] [Google Scholar]
- Moreland R. B., Langevin G. L., Singer R. H., Garcea R. L., Hereford L. M. Amino acid sequences that determine the nuclear localization of yeast histone 2B. Mol Cell Biol. 1987 Nov;7(11):4048–4057. doi: 10.1128/mcb.7.11.4048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oeda K., Salinas J., Chua N. H. A tobacco bZip transcription activator (TAF-1) binds to a G-box-like motif conserved in plant genes. EMBO J. 1991 Jul;10(7):1793–1802. doi: 10.1002/j.1460-2075.1991.tb07704.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Restrepo M. A., Freed D. D., Carrington J. C. Nuclear transport of plant potyviral proteins. Plant Cell. 1990 Oct;2(10):987–998. doi: 10.1105/tpc.2.10.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roux P., Blanchard J. M., Fernandez A., Lamb N., Jeanteur P., Piechaczyk M. Nuclear localization of c-Fos, but not v-Fos proteins, is controlled by extracellular signals. Cell. 1990 Oct 19;63(2):341–351. doi: 10.1016/0092-8674(90)90167-d. [DOI] [PubMed] [Google Scholar]
- Schmidt R. J., Ketudat M., Aukerman M. J., Hoschek G. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes. Plant Cell. 1992 Jun;4(6):689–700. doi: 10.1105/tpc.4.6.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simcox P. D., Reid E. E., Canvin D. T., Dennis D. T. Enzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinus communis L. Plant Physiol. 1977 Jun;59(6):1128–1132. doi: 10.1104/pp.59.6.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soltis D. E., Soltis P. S., Clegg M. T., Durbin M. rbcL sequence divergence and phylogenetic relationships in Saxifragaceae sensu lato. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4640–4644. doi: 10.1073/pnas.87.12.4640. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Struhl K. Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eukaryotic transcriptional regulatory proteins. Trends Biochem Sci. 1989 Apr;14(4):137–140. doi: 10.1016/0968-0004(89)90145-X. [DOI] [PubMed] [Google Scholar]
- Tabata T., Takase H., Takayama S., Mikami K., Nakatsuka A., Kawata T., Nakayama T., Iwabuchi M. A protein that binds to a cis-acting element of wheat histone genes has a leucine zipper motif. Science. 1989 Sep 1;245(4921):965–967. doi: 10.1126/science.2772648. [DOI] [PubMed] [Google Scholar]
- Tsuneoka M., Imamoto N. S., Uchida T. Monoclonal antibody against non-histone chromosomal protein high mobility group 1 Co-migrates with high mobility group 1 into the nucleus. J Biol Chem. 1986 Feb 5;261(4):1829–1834. [PubMed] [Google Scholar]
- Varagona M. J., Schmidt R. J., Raikhel N. V. Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants. Plant Cell. 1991 Feb;3(2):105–113. doi: 10.1105/tpc.3.2.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
- Wilkins T. A., Bednarek S. Y., Raikhel N. V. Role of propeptide glycan in post-translational processing and transport of barley lectin to vacuoles in transgenic tobacco. Plant Cell. 1990 Apr;2(4):301–313. doi: 10.1105/tpc.2.4.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Willmitzer L., Wagner K. G. The isolation of nuclei from tissue-cultured plant cells. Exp Cell Res. 1981 Sep;135(1):69–77. doi: 10.1016/0014-4827(81)90300-1. [DOI] [PubMed] [Google Scholar]
- Xia Y. P., Yeh C. T., Ou J. H., Lai M. M. Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex. J Virol. 1992 Feb;66(2):914–921. doi: 10.1128/jvi.66.2.914-921.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van Straaten F., Müller R., Curran T., Van Beveren C., Verma I. M. Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3183–3187. doi: 10.1073/pnas.80.11.3183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van der Krol A. R., Chua N. H. The basic domain of plant B-ZIP proteins facilitates import of a reporter protein into plant nuclei. Plant Cell. 1991 Jul;3(7):667–675. doi: 10.1105/tpc.3.7.667. [DOI] [PMC free article] [PubMed] [Google Scholar]