scholarly journals Analysis of protein-protein proximities using the split-ubiquitin system

2002 ◽  
Vol 1 (3) ◽  
pp. 230-238 ◽  
Author(s):  
N. Lehming
Keyword(s):  
Ubiquitin System ◽  
Split Ubiquitin

Interaction of the endoplasmic reticulum α1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system

2001 ◽  
Vol 114 (24) ◽  
pp. 4629-4635
Author(s):  
Michel J. Massaad ◽  
Annette Herscovics
Keyword(s):  
Endoplasmic Reticulum ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Protein A ◽  
Yeast Cells ◽  
Type Ii ◽  
Ubiquitin System ◽  
Endoplasmic Reticulum Protein ◽  
Split Ubiquitin

The α1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum. The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p. A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi α1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Δpep4 cells and in the vacuoles of rer1/Δpep4 by indirect immunofluorescence. The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo. Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of β-galactosidase activity. Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction. A weak interaction was observed between Alg5p and Rer1p. These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact. Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol.

scholarly journals Intra- and Intermolecular Interactions in Sucrose Transporters at the Plasma Membrane Detected by the Split-Ubiquitin System and Functional Assays

Structure ◽  
2002 ◽  
Vol 10 (6) ◽  
pp. 763-772 ◽  
Author(s):  
Anke Reinders ◽  
Waltraud Schulze ◽  
Safia Thaminy ◽  
Igor Stagljar ◽  
Wolf B. Frommer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intermolecular Interactions ◽  
Functional Assays ◽  
Sucrose Transporters ◽  
Ubiquitin System ◽  
Split Ubiquitin

Identification of VKORC1 interaction partners by split-ubiquitin system and coimmunoprecipitation

2011 ◽  
Vol 105 (02) ◽  
pp. 285-294 ◽  
Author(s):  
Anne Schaafhausen ◽  
Simone Rost ◽  
Johannes Oldenburg ◽  
Clemens Müller
Keyword(s):  
Vitamin K ◽  
Potential Interaction ◽  
Chondrodysplasia Punctata ◽  
Ubiquitin System ◽  
Anticoagulant Drugs ◽  
Interaction Partners ◽  
Endoplasmic Reticulum Protein ◽  
Key Enzyme ◽  
Split Ubiquitin

SummarySince the discovery of vitamin K epoxide reductase complex subunit 1 (VKORC1), the key enzyme for the regeneration of vitamin KH2, numerous studies have addressed the role of VKORC1 in the posttranslational modification of vitamin K-dependent proteins. VKORC1 is also the target protein of anticoagulant drugs of the coumarin type (e.g. warfarin). Genetic variants in VKORC1 have recently been shown to significantly affect the coumarin dose and international normalised ratio level. In the present study, we have used the split-ubiquitin yeast two-hybrid system to identify potential interaction partners of VKORC1. With this system we could identify 90 candidates. Out of these, we focused on VKORC1 itself, its paralog VKORC1L1, emopamil binding protein (EBP) and stress-associated endoplasmic reticulum protein 1 (SERP1). By coimmunprecipitation and colocalisation experiments, we were able to demonstrate evidence for the interaction of these proteins. Mutations in the EBP gene cause X-linked dominant chondrodysplasia punctata (CDPX2) which can be considered as a phenocopy of warfarin embryo-pathy. The interaction could be a link between these phenotypes. SERP1 represents an oxidative stress-associated endoplasmatic reticulum protein with chaperon-like functions. Antioxidant capacities have been described for vitamin K hydroquinone, the substrate of VKORC1. Both VKORC1 and SERP1, might have a synergistic function in eliminating reactive oxygen species generated during the VKOR redox process. Further studies are needed to investigate the role of these proteins in the vitamin K pathway.

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