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Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1

Gomez, Tara A. and Kolawa, Natalie and Gee, Marvin and Sweredoski, Michael J. and Deshaies, Raymond J. (2011) Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1. BMC Biology, 9 . Art. No. 33. ISSN 1741-7007. PMCID PMC3126750.

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Background: The proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined. Results: To shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A. Conclusions: These studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms.

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URLURL TypeDescription CentralArticle
Sweredoski, Michael J.0000-0003-0878-3831
Deshaies, Raymond J.0000-0002-3671-9354
Additional Information:© 2011 Gomez et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 10 March 2011 Accepted: 31 May 2011 Published: 31 May 2011. For reagents, we thank M. Vidal, H. Rao, D. Finley, W. Tansey, H. Yokosawa, M. Funakoshi, H. Fu, R. Baker, D. Raveh, J. Gerst, C. Gordon, M. Glickman, K. Tanaka and K. Madura. We thank the Caltech Proteome Exploration Laboratory for mass spectrometry expertise. We are grateful to Caitlin Rugani and Derek Tu for technical assistance. We thank all members of the Deshaies lab, especially K.J. Chang, G. Kleiger, J.E. Lee, R. Oania and R. Verma for valuable comments and reagents. TAG was supported with funding from the Gordon Ross Medical Foundation and an NSF pre-doctoral fellowship. RJD is an investigator of the HHMI, which supported this study. Authors’ contributions: RJD and TAG designed and interpreted all of the experiments and wrote the paper. TAG also carried out all of the experiments. NK performed the mass spectrometry. MJS analyzed the mass spectrometry data. MG designed and performed the first repetition of the experiment in Figure 4. All authors read and approved the final manuscript.
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Gordon Ross Medical FoundationUNSPECIFIED
NSF Predoctoral FellowshipUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
PubMed Central ID:PMC3126750
Record Number:CaltechAUTHORS:20110705-093954428
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Official Citation:Gomez et al.: Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1. BMC Biology 2011 9:33.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:24289
Deposited By: Tony Diaz
Deposited On:27 Oct 2011 18:30
Last Modified:03 Oct 2019 02:55

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