Journal of Molecular Biology
Volume 365, Issue 4, 26 January 2007, Pages 1093-1101
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Ubiquitin Receptor Proteins hHR23a and hPLIC2 Interact

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Abstract

Ubiquitin receptor proteins play an important role in delivering ubiquitylated protein substrates to the proteasome for degradation. HHR23a and hPLIC2 are two such ubiquitin receptors that contain ubiquitin-like (UBL) domains, which interact with the proteasome, and ubiquitin-associated (UBA) domains, which interact with ubiquitin. Depending on their abundance UBL/UBA family members can either promote or inhibit the degradation of other proteins, which suggests their participation in the delivery of substrates to the proteasome is highly regulated. In previous work, we determined UBL/UBA domain interactions to promote intramolecular interactions in hHR23a that are abrogated with the addition of either ubiquitin or the proteasome component S5a. In yeast, we determined the hHR23a ortholog (Rad23) to interact with another UBL/UBA family member (Ddi1) and to bind a common tetraubiquitin chain. Here, we use NMR spectroscopy to reveal that hHR23a interacts with hPLIC2 via UBL/UBA domain interactions and to map their binding surfaces. In addition, we demonstrate that these two proteins associate in mammalian cells. Intriguingly, inhibition of the proteasome mitigates hHR23a/hPLIC2 interaction.

Introduction

The ubiquitin-proteasome pathway plays a key regulatory role in an astounding number of cellular events, including the removal of misfolded proteins,1 production of immunocompetent peptides,2 activation or repression of transcription,3., 4. and regulation of cell-cycle progression.5 Through it, proteins are ubiquitylated and consequently delivered to the 26 S proteasome for degradation.6 The moieties of a polyubiquitin chain are linked by isopeptide bonds between the carbonyl carbon of the C-terminal glycine and a lysine side-chain NH group. Ubiquitin contains seven lysine residues, and among the factors that determine whether ubiquitylation leads to proteasomal degradation is the chain length and lysine used to link the ubiquitin subunits.7 Another key factor is the recognition of the ubiquitylated protein by a ubiquitin receptor family member. In particular, proteins with attached K48-linked chains are degraded by the proteasome; however, distinct receptor pathways are required to funnel them there.8., 9.

Within the ubiquitin receptor family of proteins is a group that can connect ubiquitylated proteins directly to the proteasome as their UBL domains bind the proteasome,10., 11., 12., 13. and ubiquitin-associated (UBA) domains bind ubiquitin14., 15., 16. simultaneously.16 Depending on their levels, ubiquitin-like (UBL)/UBA-containing proteins can promote or inhibit the degradation of ubiquitylated substrates.9 The inhibition is caused by the UBA domains, which sequester the polyubiquitin chain to in turn prevent deubiquitylation.9., 17., 18.

Whether ubiquitin receptor proteins act independently or collaboratively has yet to be determined. However, in yeast, UBL/UBA family members interact with each other,19., 20. via UBL/UBA domain interactions.21 No such intermolecular interaction has been reported yet in humans; however, the N-terminal UBL domain of the human homolog of Rad23 (hHR23a) interacts dynamically with its internal and C-terminal UBA domains.22 Evidence exists for such intramolecular interactions in the yeast Dsk2 protein. In particular, a single domain protein construct of its UBA domain interacts with itself and its UBL domain when crystallized.23

Here, we used NMR spectroscopy to demonstrate that hHR23a binds in a bidentate manner to human homolog 2 of protein linking integrin-associated protein and cytoskeleton (hPLIC2), a human ortholog of Dsk2, which functions in spindle body duplication24 and, like hHR23a's ortholog Rad23, has been implicated in endoplasmic reticulum-associated degradation of certain substrates.25 Our studies indicate that the UBA domain of hPLIC2 binds to the hHR23a UBL domain, and that its UBL domain binds the hHR23a C-terminal UBA domain. In contrast to these interactions, the hPLIC2 UBL domain does not demonstrate binding to the internal UBA domain of hHR23a, even when at twofold molar excess and millimolar protein concentrations. Our data provide an explanation for the ability of hPLIC2 to bind the hHR23a C-terminal, but not internal UBA domain. Finally, we use immunoprecipitation experiments on endogenous hHR23a and hPLIC2 proteins to demonstrate that they associate in mammalian cells. Intriguingly, proteasome inhibition reduces the interaction of these two proteins substantially. This finding provides insights into the functional significance of their interaction.

Section snippets

HHR23a and hPLIC2 interact via UBL/UBA domain interactions

Since the hHR23a UBL domain interacts with its own UBA domains,22 we tested whether it binds that of hPLIC2 (Figure 1(a)). We acquired [1H,15N] heteronuclear single quantum coherence (HSQC) experiments on 15N-labeled hHR23a with or without unlabeled hPLIC2 UBA domain (Figure 1(b)). [1H,15N] HSQC experiments detect all hydrogen atoms that are attached to nitrogen, and their chemical shift values are sensitive to their chemical environment.26 We determined that addition of equimolar quantities of

Discussion

Here, we demonstrate the two human UBL/UBA family members hHR23a and hPLIC2 to interact in vitro as well as in vivo. Our study complements others, which demonstrate that the ubiquitin-proteasome pathway is plagued with packing problems, as the same β-sheet surface of ubiquitin and ubiquitin-like domains is required for intra- and intermolecular interactions.10., 11., 22., 27., 29., 30. Indeed, critical to understanding the contribution of these interactions to ubiquitin-mediated protein

Sample preparation

Dr Peter Howley generously provided us with the plasmids for the expression of GST tagged hPLIC2 UBL domain [pGEX-2T-hPLIC2 (26–103)] and GST tagged hPLIC2 UBA domain [pGEX-2T-hPLIC2 (574–624)]. The plasmids containing these genes were each transformed into BL21 (DE3) cells and grown at 37 °C in M9 minimal medium or in Luria broth containing 100 μg/ml of ampicillin. The cells were harvested 3 h after protein expression was induced with 0.4 mM isopropyl β-d-thiogalactoside (IPTG). The proteins

Acknowledgements

We are grateful to Dr Peter M. Howley and Dr Maurits Kleijnen for useful discussions and for providing us with anti-hPLIC2 antibody and hPLIC2 DNA constructs. NMR data were acquired in the NMR facility of the UMN and we thank Dr David Live and Dr Beverly Ostrowsky for their technical assistance. Data processing and visualization occurred in the Minnesota Supercomputing Institute Basic Sciences Computing Lab. This work was funded by grants from the National Institutes of Health CA097004 (to

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