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Published May 31, 2011 | Supplemental Material + Published
Journal Article Open

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


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.

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 (http://creativecommons.org/licenses/by/2.0), 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.

Attached Files

Published - Gomez2011p14391Bmc_Biol.pdf

Supplemental Material - Figure_S1.pdf

Supplemental Material - Figure_S2.pdf

Supplemental Material - Figure_S3.pdf

Supplemental Material - Figure_S4.pdf

Supplemental Material - Table_S1.pdf


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