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Tandem affinity purification in transgenic mouse embryonic stem cells identifies DDOST as a novel PPP1CC2 interacting
Graham MacLeod1, Susannah Varmuza
1Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada.
Abstract:
Members of the PP1 family of protein phosphatases achieve functional diversity through numerous and varied protein-protein interactions. In mammals, there are four PP1 isoforms, the ubiquitously expressed PPP1CA, PPP1CB, and PPP1CC1, and the testis specific splice isoform PPP1CC2. When the mouse Ppp1cc gene is deleted, the only phenotypic consequence is a failure of spermatogenesis in homozygous males. To elucidate the function of the Ppp1cc gene, we sought to identify novel protein-protein interactions. To this end, we have created SBP-3XFLAG-PPP1CC1 and SBP-3XFLAG-PPP1CC2 knock-in mouse embryonic stem cell lines using a gene-trap-based system. Tandem affinity purification using our knock-in cell lines identified 11 significant protein-protein interactions, including nine known PP1 interacting proteins and two additional proteins (ATP5C1 and DDOST). Reciprocal in vitro sedimentation assays confirmed the interaction between PPP1CC2 and DDOST that may have physiological implications in spermatogenesis. Immunolocalization studies revealed that DDOST localized to the nuclear envelope in dissociated spermatogenic cells and persists throughout spermatogenesis. The knock-in system described in this paper can be applied in creating tandem affinity-tagged knock-in embryonic stem cell lines with any gene for which a compatible gene-trap line is available.
Insights
Protein phosphatase 1 (PP1) isoforms, particularly PPP1CC2, are crucial for male fertility. This study identifies novel protein interactions, including DDOST, essential for spermatogenesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Reproductive Biology
Background:
- Protein phosphatases, specifically the PP1 family, exhibit functional diversity through protein-protein interactions.
- Mammals possess four PP1 isoforms: PPP1CA, PPP1CB, PPP1CC1 (ubiquitous), and PPP1CC2 (testis-specific).
- Deletion of the mouse Ppp1cc gene results in male-specific spermatogenesis failure.
Purpose of the Study:
- To elucidate the function of the Ppp1cc gene by identifying novel protein-protein interactions.
- To characterize the interactions of PPP1CC1 and PPP1CC2 isoforms.
Main Methods:
- Creation of SBP-3XFLAG-PPP1CC1 and SBP-3XFLAG-PPP1CC2 knock-in mouse embryonic stem cell lines using a gene-trap system.
- Tandem affinity purification to identify interacting proteins.
- Reciprocal in vitro sedimentation assays and immunolocalization studies.
Main Results:
- Identified 11 significant protein-protein interactions for PP1 isoforms.
- Discovered two novel interacting proteins: ATP5C1 and DDOST.
- Confirmed PPP1CC2 interaction with DDOST, which localizes to the nuclear envelope during spermatogenesis.
Conclusions:
- The Ppp1cc gene plays a critical role in spermatogenesis, potentially through its interaction with DDOST.
- The developed knock-in system is a versatile tool for studying protein interactions of various genes.
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