Abstract
Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications.
Original language | American English |
---|---|
Pages (from-to) | 764-779.e8 |
Journal | Structure |
Volume | 31 |
Issue number | 7 |
DOIs | |
State | Published - 6 Jul 2023 |
Bibliographical note
Funding Information:We are grateful for the financial support from the Israel Science Foundation (grant number: 1537/18 for DR and 1466/18 for DS), Human Frontier Science program ( CDA00064/2014 ), the US- Israel Binational Science Foundation (grant number: 2015056 ), Israel Ministry of Science and Technology (grant number: 80802 ) and the Azrieli Foundation for supporting MR. We are tremendously thankful to Prof. Maya Schuldiner for providing us with the UPRE-GFP plasmid and for stimulating discussions, to Adi Levy for her assistance with the cell imaging, to Dr. Hadar Amartely for the assistance with ITC and protein purification, and to Yael Sidkiyahu for her assistance with figure preparation.
Funding Information:
We are grateful for the financial support from the Israel Science Foundation (grant number: 1537/18 for DR and 1466/18 for DS), Human Frontier Science program (CDA00064/2014), the US-Israel Binational Science Foundation (grant number: 2015056), Israel Ministry of Science and Technology (grant number: 80802) and the Azrieli Foundation for supporting MR. We are tremendously thankful to Prof. Maya Schuldiner for providing us with the UPRE-GFP plasmid and for stimulating discussions, to Adi Levy for her assistance with the cell imaging, to Dr. Hadar Amartely for the assistance with ITC and protein purification, and to Yael Sidkiyahu for her assistance with figure preparation. Conceptualization: T. O. M. R. M. B. D. S-D. and D. R.; Data curation: T. O. M. R. E. S. B. and D. R.; Formal Analysis: T. O. M. R. M. B. E. S. B. D. S-D. and D. R.; Funding acquisition: D. S-D. and D. R.; Investigation: T. O. M. R. E. S. B. O. Y. E. G. and Y. Y.; Methodology: T. O. M. R. M. B. T. R. D. S-D. and D. R.; Resources: T. R. D. S-D. and D. R.; Software: M. B. E. S. B. and D. S-D.; Supervision: D. S-D. and D. R.; Writing - original draft: T. O. M. R. D. S-D. and D. R.; Writing - review & editing: T. O. M. R. M. B. D. S-D. and D. R. The authors state that no competing financial interests exist. We support inclusive, diverse, and equitable conduct of research.
Publisher Copyright:
© 2023 Elsevier Ltd
Keywords
- CDC48
- CL-MS
- NPL4
- UFD1
- crosslinking coupled with MS (XL-MS)
- integrative modeling
- protein quality control
- structural mass spectrometry