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Functional inactivation of the conserved Sem1p in yeast by intrabodies

Mirka Reinman1, Jussi Jäntti, Kaija Alfthan

  • 1VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland. mirka.reinman@vtt.fi

Yeast (Chichester, England)
|September 10, 2003
PubMed

Insights

Intrabodies targeting Saccharomyces cerevisiae Sem1 protein revealed its nuclear function. Nuclear-localized intrabodies inhibited yeast growth, indicating Sem1 protein inactivation and its role in nuclear transport.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Yeast Genetics

Background:

  • The Sem1 protein in Saccharomyces cerevisiae and its mammalian homologue DSS1 are highly conserved.
  • DSS1 interacts with the tumor suppressor BRCA2 in mammalian cells, suggesting a conserved biological role.
  • Intrabody technology offers a tool to study intracellular protein function and localization.

Purpose of the Study:

  • To characterize the function and intracellular localization of Saccharomyces cerevisiae Sem1 protein using intrabody technology.
  • To investigate the conserved function of Sem1p/DSS1 across species.
  • To establish a yeast system for studying Sem1p/DSS1 function.

Main Methods:

  • Phage display library was used to isolate a Sem1p-specific antibody.
  • Antibody fragments (intrabodies) were expressed intracellularly in yeast, targeted to the cytosol or nucleus.
  • The efficiency of different antibody fragments (e.g., Fab) as intrabodies was analyzed.
  • Yeast growth inhibition assays were performed to assess intrabody function.

Main Results:

  • The Fab intrabody fragment demonstrated the highest expression efficiency.
  • Nuclear-targeted anti-Sem1p Fab intrabodies significantly inhibited yeast growth, mimicking SEM1 gene deletion.
  • In vivo interaction between Fab intrabodies and Sem1p was confirmed, leading to Sem1p inactivation.
  • Sem1p mediated the nuclear transport of the intrabody, even without a nuclear localization signal.

Conclusions:

  • Sem1 protein plays a crucial role in yeast cell function, partly manifested in the nucleus.
  • Intrabody technology is effective for studying conserved protein functions in vivo.
  • Sem1 protein is involved in the nuclear transport mechanism within yeast cells.

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