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ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe

Nature Biotechnology volume 24, pages 841–847 (2006)Cite this article

A Corrigendum to this article was published on 01 August 2006

Abstract

Cloning of the entire set of an organism's protein-coding open reading frames (ORFs), or 'ORFeome', is a means of connecting the genome to downstream 'omics' applications. Here we report a proteome-scale study of the fission yeast Schizosaccharomyces pombe based on cloning of the ORFeome. Taking advantage of a recombination-based cloning system, we obtained 4,910 ORFs in a form that is readily usable in various analyses. First, we evaluated ORF prediction in the fission yeast genome project by expressing each ORF tagged at the 3′ terminus. Next, we determined the localization of 4,431 proteins, corresponding to ∼90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. Furthermore, using leptomycin B, an inhibitor of the nuclear export protein Crm1, we identified 285 proteins whose localization is regulated by Crm1.

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Figure 1: Cloning and expression of the fission yeast ORFeome.
Figure 2: Microscopic analysis of S. pombe strains expressing ORF-YFP fusions.
Figure 3: Comparative analysis of localization pattern of S. pombe proteins.
Figure 4: Regulation of protein localization by Crm1-mediated nuclear export.

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Acknowledgements

We thank Megumi Takase, Kunio Watanabe and Ryuji Yoshinari for technical assistance and the members of Chemical Genetics Laboratory, RIKEN, for discussions. We also thank Daniel McCollum for critical reading of the manuscript, and Jürg Bähler for useful comments. This work was supported in part by the CREST Research Project, Japan Science and Technology Agency, The Strategic Research Programs for R&D, RIKEN and a Grant-in-Aid for Scientific Research on Priority Area 'Cancer' from The Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Author notes

  1. Makiko Hamamoto

    Present address: School of Agriculture, Meiji University, Tama-ku, Kanagawa, 214-8571, Japan

  2. Akihisa Matsuyama, Ritsuko Arai and Yoko Yashiroda: These authors contributed equally to this work.

Authors and Affiliations

  1. Chemical Genetics Laboratory, RIKEN, Wako, 351-0198, Saitama, Japan

    Akihisa Matsuyama, Ritsuko Arai, Yoko Yashiroda, Atsuko Shirai, Ayako Kamata, Shigeko Sekido, Yumiko Kobayashi, Atsushi Hashimoto, Makiko Hamamoto & Minoru Yoshida

  2. CREST Research Project, JST, Kawaguchi, 332-0012, Saitama, Japan

    Akihisa Matsuyama, Ritsuko Arai, Yoko Yashiroda, Atsuko Shirai, Ayako Kamata, Yasushi Hiraoka & Minoru Yoshida

  3. Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, 113-8657, Bunkyo-ku, Japan

    Atsuko Shirai, Ayako Kamata, Sueharu Horinouchi & Minoru Yoshida

  4. Kansai Advanced Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492, Hyogo, Japan

    Yasushi Hiraoka

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Contributions

The order of listing of the authors A.M., R.A. and Y.Y. in no way reflects their relative contribution to this work. M.Y. is responsible for the project planning and experimental design with support from Y.H. and S.H.; A.M., Y.Y. and A.H. carried out cloning and sequencing; R.A., Y.Y. and M.H. collected the localizome data; R.A. and A.K. carried out the chemical genetic screen; S.S. and Y.K. generated plasmids and transformants; and A.M. and A.S. carried out reverse array and statistic analyses.

Note: Supplementary information is available on the Nature Biotechnology website.

Corresponding author

Correspondence to Minoru Yoshida.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Distribution of the protein expression levels. (PDF 735 kb)

Supplementary Fig. 2

Comparison of the fission yeast localizome with the GO_cellular component categories. (PDF 873 kb)

Supplementary Fig. 3

Distribution pattern of proteins in cellular compartments in budding yeast. (PDF 831 kb)

Supplementary Table 1

Summary of S. pombe expression library construction. (PDF 75 kb)

Supplementary Table 2

ORFs that showed disagreement with the existing genome database. (PDF 77 kb)

Supplementary Table 3

Functional classification of 'non-SPB' nuclear dots proteins. (PDF 62 kb)

Supplementary Table 4

Screening of Crm1-dependently transported proteins. (PDF 68 kb)

Supplementary Table 5

Functional classification of Crm1-dependently transported proteins. (PDF 96 kb)

Supplementary Table 6

Sequence information and localizome data for the entry clones. (XLS 1736 kb)

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Matsuyama, A., Arai, R., Yashiroda, Y. et al. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol 24, 841–847 (2006). https://doi.org/10.1038/nbt1222

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