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

Tara A Gomez,Natalie Kolawa,Raymond J Deshaies,Marvin Gee,Michael J Sweredoski

doi:10.1186/1741-7007-9-33

Tara A Gomez, Natalie Kolawa + Show 3 more

Open Access

https://doi.org/10.1186/1741-7007-9-33

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Abstract

BackgroundThe 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.ResultsTo 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.ConclusionsThese 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.

Highlights

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
Rpn1391 to 642 interacts with UBL domain proteins To screen for mutations in Rpn1 that disrupt binding to Ub association domain (UBA)-UBL proteins, we engineered a reverse yeast twohybrid system that reports on association between a fragment of Rpn1, a fragment including regions previously shown to be necessary and sufficient for UBA-UBL binding and four distinct UBLcontaining proteins (Rad23, Dsk2, Ddi1, and the deubiquitinase Ubp6) known to interact with the proteasome [15,34,36,37,38]
Productive binding between Rpn1391 to 642 and UBL proteins was expected to drive expression of HIS3 and URA3, resulting in growth on 3-aminotriazole (3-AT) and inability to grow on 5-fluoroorotic acid (5FOA) (Figure 1A)

Summary

Introduction

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 Rpn, Rpn, or Rpt, or are shuttled to the proteasome by Rad, Dsk, or Ddi. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn, Rpn, or Rpt, or are shuttled to the proteasome by Rad, Dsk, 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. Once a protein substrate has been modified by a chain of at least four ubiquitins, it is degraded by the 26S proteasome in an ATP-dependent manner [3,4].

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