Temperature-sensitive control of protein activity by conditionally splicing inteins

Nature Biotechnology volume 22, pages871–876(2004)Cite this article

Abstract

Conditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function. Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems. However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use. Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins. We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80. We show that Gal80-inteinTS is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster. Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.

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Figure 1: Generation of temperature-sensitive Gal4 molecules using conditionally splicing inteins.
Figure 3: Temperature-dependent control of Notch activity in wing imaginal discs.
Figure 2: Activity of inteinTS in Gal80 in yeast and D. melanogaster.

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Acknowledgements

We would like to especially thank Francine Perler for introducing us to the Intein system. We would also like to thank Aimée Dudley, Craig Kaplan, Erica Larschan and Mary Bryk as well as other members of the Winston lab for yeast strains and much technical advice. In addition we would like to thank Maggie Chang and Christians Villalta for technical assistance; Craig Micchelli for constructive suggestions and for fly stocks; Gyeong-Hun Baeg and Matthew Gibson for fly stocks; Susan Smith, Dan Curtis, Konrad Basler and Daisuke Yamamoto for plasmids; and Hugo Bellen and James Hopper for antibodies. M.P.Z. was a Special Fellow of the Leukemia and Lymphoma Society and is currently supported by the Emmy Noether program of the Deutsche Forschungsgemeinschaft. C.T. is supported by the Charles King Medical Foundation. N.P. is a Howard Hughes Investigator.

Author information

Author notes
    • Martin P Zeidler
    •  & Sabine Häder

    Present address: Department of Molecular Developmental Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany

    • Yohanns Bellaiche

    Present address: Institut Curie, UMR 144, 12 rue Lhomond, 75005, Paris, France

    • Urte Gayko

    Present address: Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320, USA

  4. Martin P Zeidler and Change Tan: These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA
    • Martin P Zeidler
    • , Change Tan
    • , Yohanns Bellaiche
    • , Sara Cherry
    • , Urte Gayko
    •  & Norbert Perrimon
  2. Department of Molecular Developmental Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany
    • Yohanns Bellaiche
    •  & Urte Gayko
  3. Institut Curie, UMR 144, 12 rue Lhomond, 75005, Paris, France
    • Martin P Zeidler
    • , Sabine Häder
    •  & Urte Gayko
  4. Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320, USA
    • Martin P Zeidler
    • , Yohanns Bellaiche
    •  & Sabine Häder
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Corresponding author

Correspondence to Norbert Perrimon.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Mutations in inteinTS alleles. (PDF 108 kb)

Supplementary Table 1

Insertion context of inteinTS alleles. (PDF 37 kb)

Supplementary Table 2

Oligonucleotides used. (PDF 25 kb)

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Zeidler, M., Tan, C., Bellaiche, Y. et al. Temperature-sensitive control of protein activity by conditionally splicing inteins. Nat Biotechnol 22, 871–876 (2004). https://doi.org/10.1038/nbt979

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