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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
Interaction of the endoplasmic reticulum alpha 1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system
1McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada.
Journal of Cell Science
|January 17, 2002
Summary
The alpha1,2-mannosidase Mns1p requires Rer1p for endoplasmic reticulum localization, and these proteins interact in vivo. This study confirms Mns1p
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mns1p is an alpha1,2-mannosidase in Saccharomyces cerevisiae's N-glycosidic pathway.
- Mns1p is a type II membrane protein localized to the endoplasmic reticulum.
- Rer1p is involved in retrieving Mns1p from the Golgi.
Purpose of the Study:
- To investigate the interaction between Mns1p and Rer1p in vivo.
- To determine if the transmembrane domain of Mns1p is sufficient for Rer1p-dependent localization.
- To confirm the role of Rer1p in Mns1p localization.
Main Methods:
- Utilized a chimera of Mns1p's transmembrane domain and Kre2p's catalytic domain.
- Employed indirect immunofluorescence for protein localization.
- Applied the split-ubiquitin system to detect protein-protein interactions in vivo.
- Performed western blot analysis and beta-galactosidase assays.
Main Results:
- The Mns1p chimera localized to the endoplasmic reticulum, indicating its transmembrane domain mediates Rer1p-dependent localization.
- The split-ubiquitin assay confirmed an in vivo interaction between Mns1p and Rer1p.
- Sec12p also interacted with Rer1p, while Ost1p did not, suggesting specificity.
- A weak interaction was observed between Alg5p and Rer1p.
Conclusions:
- The transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization.
- Mns1p and Rer1p directly interact in vivo.
- The C-terminal of Rer1p is accessible to the cytosol, as shown by the split-ubiquitin system.
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