UBL/UBA Ubiquitin Receptor Proteins Bind a Common Tetraubiquitin Chain

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The ubiquitin–proteasome pathway is essential throughout the life cycle of a cell. This system employs an astounding number of proteins to ubiquitylate and to deliver protein substrates to the proteasome for their degradation. At the heart of this process is the large and growing family of ubiquitin receptor proteins. Within this family is an intensely studied group that contains both ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains: Rad23, Ddi1 and Dsk2. Although UBL/UBA family members are reported to regulate the degradation of other proteins, their individual roles in ubiquitin-mediated protein degradation has proven difficult to resolve due to their overlapping functional roles and interaction with each other and other ubiquitin family members. Here, we use a combination of NMR spectroscopy and molecular biology to reveal that Rad23 and Ddi1 interact with each other by using UBL/UBA domain interactions in a manner that does not preclude their interaction with ubiquitin. We demonstrate that UBL/UBA proteins can bind a common tetraubiquitin molecule and thereby provide strong evidence for a model in which chains adopt an opened structure to bind multiple receptor proteins. Altogether our results suggest a mechanism through which UBL/UBA proteins could protect chains from premature de-ubiquitylation and unnecessary elongation during their transit to the proteasome.

Introduction

Ubiquitin signaling regulates an astounding array of cellular events and remains essential throughout the life cycle of a cell. In its most established role ubiquitylation targets proteins for degradation by the 26 S proteasome,1 a process important for controlling the lifespan of regulatory proteins, removing misfolded proteins,2 producing immunocompetent peptides,3 activating and repressing transcription,4, 5 and regulating cell cycle progression.6 In addition ubiquitylation can signal proteasome-independent events including endocytic sorting7, 8 and DNA repair.9, 10 Ubiquitylation is connected to proteasome-mediated protein degradation by an intricate network of ubiquitin recognition proteins. Elucidating this network remains a difficult albeit active area of research, as it is clouded by redundancy and cooperation between the large and growing ubiquitin receptor protein family. Among these proteins exists a group that harbors both ubiquitin-associated (UBA) and ubiquitin-like (UBL) domains (Figure 1(a)).

UBL/UBA proteins have attracted much attention for their ability to regulate the lifespans of other proteins. In Saccharomyces cerevisiae, Rad23 (hHR23a/b in humans), Dsk2 (hPLIC-1/2 in humans) and Ddi1 are UBL/UBA proteins that recruit ubiquitylated substrates to the proteasome for their degradation11, 12, 13, 14, 15, 16 via UBA domain interactions with ubiquitin17, 18, 19 and UBL domain interactions with the proteasome.20, 21, 22, 23 Depending on their protein levels, UBL/UBA-containing proteins can also inhibit the degradation of ubiquitylated substrates.16 Such inhibition occurs because UBA domains sequester K48-linked polyubiquitin chains to in turn prevent their elongation and de-ubiquitylation.16, 24, 25 In a perhaps related role, C-terminal UBA domains are reported to protect Rad23/hHR23a, Ddi1 and Dsk2 from their own degradation via the proteasome.26

Adding to the complexity of the ubiquitin family network, Rad23 is reported to interact with Ddi1 and Dsk2, and yeast two-hybrid experiments implicate UBA/UBA domain interactions as essential for such dimerization.27, 28 However, in previous work on hHR23a we found no such UBA/UBA domain interactions but instead found that the UBL domain of hHR23a interacts dynamically with each of its UBA domains.29 Furthermore, whereas Rad23 is reported to dimerize,18, 27, 28 hHR23a does not.29

To resolve these ambiguities we used NMR spectroscopy to determine the mechanism by which Rad23 binds Ddi1 and itself. Yeast two-hybrid experiments are unable to discriminate between direct interactions and those that are mediated by other proteins, both of which, as we report here, are available to Rad23 and Ddi1. Here, we reveal that UBL/UBA and not UBA/UBA domain interactions result in heterodimerization of Rad23 and Ddi1. These findings demonstrate the first published example for UBL/UBA domain interactions mediating heterodimerization. According to its crystal structure, the ubiquitin moieties of K48-linked tetraubiquitin are packed against each other with only the most distal moiety available for binding a UBA domain.30 This structure suggests that K48-linked tetraubiquitin, which is the smallest chain length that signals for proteasome degradation31 is able to bind only one ubiquitin receptor protein. In addition, Rad23 has two UBA domains, the C-terminal of which is reported to sandwich between the two ubiquitin subunits of diubiquitin.32 Surprisingly, we have found that K48-linked tetraubiquitin can bind simultaneously to two Rad23 molecules as well as to Rad23 and Ddi1. This finding illustrates that K48-linked tetraubiquitin adopts an opened structure when bound to its receptors and leads to a working model for how ubiquitylated substrates are transferred to the proteasome.

Section snippets

Rad23 heterodimerizes with Ddi1 via UBL/UBA domain contacts

Yeast two-hybrid experiments suggest that Rad23 interacts with itself, Dsk2 and Ddi1 by using UBA/UBA domain interactions.27, 28 These in vivo analyses, however, do not exclude the possibility of indirect associations through bridging molecules such as ubiquitin chains, which are known to interact with UBA domains.18 To test whether Rad23 and Ddi1 interact in their purified forms we performed [1H,15N] heteronuclear single quantum coherence (HSQC) experiments on 15N-labeled Rad23 or Ddi1 alone

Discussion

Perhaps the most impressive attribute of ubiquitin receptor proteins and polyubiquitin is their adaptability. The ubiquitin moieties of K48-linked tetra-30 and diubiquitin34 pack against each other to form a closed structure. Recent literature, however, suggests a flexible model for polyubiquitin structure,35, 36 and diubiquitin forms an open structure to bind the C-terminal UBA domain of hHR23a.32 Here, we demonstrate that tetraubiquitin can bind more than one ubiquitin receptor protein and

Sample preparation

For NMR spectroscopy Rad23, Ddi1 and single-domain constructs of the Rad23 UBL, UBA1 and UBA2 domains as well as of Ddi1's UBA domain were each cloned into the pET15b expression vector (Novagen) in-frame with the N-terminal histidine tag. The plasmids containing these genes were each transformed into Escherichia coli BL21 (DE3) cells and grown at 37 °C in M9 minimal medium or in Luria broth containing ampicillin (100 μg/ml). The cells were harvested 3 h after protein expression was induced with 0.4

Acknowledgements

We are grateful to Casey Litchke and Jeannette Zinggeler for assisting with the sample preparation. We also thank Dr Leonard Banaszak for allowing us to use his dynamic light-scattering instrument. NMR data were acquired at the NMR facility of the University of Minnesota and we thank Dr David Live and Dr Beverly Ostrowsky for their technical assistance. NMR instrumentation was provided with funds from the NSF (BIR-961477), the University of Minnesota Medical School, and the Minnesota Medical

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