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Isolation of a point-mutated p47 lacking binding affinity to p97ATPase
Yayoi Kaneko1, Kaori Tamura, Go Totsukawa
1Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Abstract:
p47, a p97-binding protein, functions in Golgi membrane fusion together with p97 and VCIP135, another p97-binding protein. We have succeeded in creating p47 with a point mutation, F253S, which lacks p97-binding affinity. p47 mapping experiments revealed that p47 had two p97-binding regions and the F253S mutation occurred in the first p97-binding site. p47(F253S) could not form a complex with p97 and did not caused any cisternal regrowth in an in vitro Golgi reassembly assay. In addition, mutation corresponding to the p47 F253S mutation in p37 and ufd1 also abolished their binding ability to p97.
Insights
Researchers created a mutated p47 protein, F253S, that cannot bind to p97. This mutation disrupted Golgi membrane fusion and cisternal regrowth, highlighting p47
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Interactions
Background:
- p47 is a p97-binding protein crucial for Golgi membrane fusion.
- It collaborates with p97 and VCIP135 in this process.
Purpose of the Study:
- To investigate the role of p97-binding affinity in p47's function.
- To elucidate the specific p97-binding regions of p47.
Main Methods:
- Site-directed mutagenesis to create p47 F253S mutant.
- Co-immunoprecipitation to assess p97-p47 complex formation.
- In vitro Golgi reassembly assay to evaluate cisternal regrowth.
Main Results:
- The F253S mutation in p47 abolished its binding affinity to p97.
- p47(F253S) failed to form a complex with p97 and did not induce cisternal regrowth.
- Similar mutations in p37 and Ufd1 also impaired their p97-binding ability.
Conclusions:
- p97-binding is essential for p47's role in Golgi membrane fusion and reassembly.
- The F253S mutation identifies a critical p97-binding site in p47.
- This finding has implications for understanding the broader p97-dependent protein complex functions.
