UBL/UBA Ubiquitin Receptor Proteins Bind a Common Tetraubiquitin Chain

Yang, Kang; Vossler, Rebecca A.; Díaz-Martínez, Laura A.; Winter, Nathan S.; Clarke, Duncan J.; Walters, Kylie J.

doi:10.1016/j.jmb.2005.12.001

Cited by 76 publications

(100 citation statements)

References 39 publications

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“…In hHR23a, the human homologue of Rad23, the UbL domain engages in a transient intramolecular interaction with both UBA domains (Walters et al, 2003). NMR spectroscopic analysis of yeast Rad23 has revealed that although the first UBA domain can participate in an intramolecular interaction with the UbL, the carboxy-terminal UBA domain does not (Kang et al, 2006). Intramolecular interactions could result in inhibition of intermolecular activity such as proteasome and ubiquitin chain binding.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, when overexpressed, Rad23 and Dsk2 are capable of inhibiting proteolysis by binding a polyubiquitin chain on a substrate and inhibiting the ligation of additional ubiquitin molecules (Kleijnen et al, 2000;Ortolan et al, 2000). Even the stoichiometry of receptor-ubiquitin chain interactions is potentially complex given that Rad23 can form homodimers as well as heterodimers with the UbL/UBA protein Ddi1 (Bertolaet et al, 2001a,b;Kang et al, 2006) and that one polyubiquitin chain may simultaneously capture two receptor molecules (Kang et al, 2006;Lowe et al, 2006). Finally, intramolecular interactions between UbL and UBA domains may function in regulating UbL/UBA protein activity (Ryu et al, 2003;Walters et al, 2003;Raasi et al, 2004).…”

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confidence: 99%

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A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

Ghaboosi

Deshaies

2007

MBoC

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E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin-proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo. INTRODUCTIONActivation of ubiquitin by ubiquitin-activating enzyme (E1) is the requisite first step in all ubiquitin-dependent pathways, including regulated proteolysis. The process begins with an ATP-dependent reaction in which the ubiquitin moiety forms a high-energy thioester bond with the active site cysteine of E1. E1 can then transfer the activated ubiquitin to a conjugating enzyme (E2), which acts alone or in conjunction with a ubiquitin ligase (E3) to covalently link ubiquitin to lysine residues on specific target proteins (Haas and Siepmann, 1997). Through successive ligation reactions, a polyubiquitin chain can form on the substrate and serve as a signal for targeting to the multisubunit 26S proteasome. Composed of a cylindrical 20S proteolytic core complex capped at one or both ends by 19S regulatory complexes, the proteasome deubiquitinates and unfolds substrates before translocating them into its core for proteolysis (Amerik and Hochstrasser, 2004;Pickart and Cohen, 2004).The existence of mammalian cell lines that carry temperature-sensitive alleles of E1 has been of great importance to the study of the functions of the ubiquitin system in animal cells Finley et al., 1984;Kulka et al., 1988;Salvat et al., 2000). Ironically, no tight, rapid-acting conditional mutation has been described for the budding yeast UBA1 gene that encodes E1 despite the availability of sophisticated molecular genetic techniques in this organism.Although a temperature-sensitive allele of UBA1 exists, this allele results in only moderate stabilization of tested substrates and possible defects in ubiquitination were not examined (McGrath, 1991;Gandre and Kahana, 2002). A second allele resulting from a transposon insertion upstream of the UBA1 coding sequence reduced wild-type Uba1 protein function, causing inefficient degradation of some proteins (Swanson and Hochstrasser, 2000). Additional alleles were later en...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

Ghaboosi

Deshaies

2007

MBoC

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“…All of them are unique in that they contain an N-terminal Ub-like (UBL) domain that binds to the proteasome via Rpn1 (42,52,53) and a C-terminal Ubassociated (UBA) domain that binds to Ub chains (54,55). These UBL-UBA proteins have been seen to cycle on and off the proteasome while delivering the ubiquitinated cargo from the cytoplasm and the nucleus to the proteasome, which is why they are also known as shuttle proteins (56,57).…”

Section: Rub1 Is Structurally Similar To Ubiquitin-although Rub1 Ismentioning

confidence: 99%

Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System

Singh

Zerath

Kleifeld

et al. 2012

Molecular & Cellular Proteomics

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Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of ubiquitin. We show via NMR that structurally, Rub1 and ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/ Nedd8 and ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or ubiquitin or both, we examined whether ubiquitin receptors can differentiate between Rub1 and ubiquitin. Surprisingly, Rub1 interacts with proteasome ubiquitin-shuttle proteins comparably to ubiquitin but binds more weakly to a proteasomal ubiquitin receptor Rpn10. We identified Rub1-ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-ubiquitin heterodimers are recognized by various proteasomal ubiquitin shuttles and receptors comparably to K48-linked ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deubiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into ubiquitin pathways, it is recognized essentially like The selective degradation of many proteins in eukaryotic cells is a highly specific and irreversible process required to perform vital cellular functions such as cell cycle progression (1, 2), differentiation and development (3, 4), and transcriptional control (5). One of the major pathways involved in this selective degradation is the ubiquitin-proteasome pathway. In this pathway, ubiquitin (Ub), a 76-amino-acid protein, is attached to the substrate, and this is followed by the recognition and degradation of the substrate by a protein complex collectively known as proteasome (6 -8).The attachment of Ub (ubiquitination) to a substrate protein is achieved via a cascade of enzymatic reactions (involving E1, E2, and E3 enzymes) that results in the formation of an isopeptide bond between the C-terminal glycine of Ub and a lysine residue of the substrate (9 -11). Sequential repetition of this cascade can result in the attachment of a chain of Ub molecules (polyubiquitin) to the substrate protein. The length and topology of the polyubiquitin (polyUb) tag decide the fate of the substrate protein. For example, we and others have shown that a K48-linked tetraubiquitin (tetraUb) chain that acts as a proteasomal degradation signal forms a "closed" structure (12, 13), whereas a K63-linked Ub...

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“…Although UBA domains are found in many proteins within eukaryotic proteomes, the arrangement of linked kinase and UBA domains only is observed in the MARKs and their homologs within the AMPK/Snf1 family of protein kinases. Many functions have been attributed to UBA domains in other proteins, including dimerization (32,33), the binding of Ub-like (UBL) domains (34,35), monoUb (15,16), and K48-linked and K63-linked polyUb (16). In the present work, we have demonstrated that the hMARK3 UBA domain stably interacts with the N-lobe of the kinase domain in the hMARK3 (residues 48-370) crystal structure (average B factors for atoms in the kinase versus UBA domains were 33.05 versus 33.72, respectively) via a series of hydrophobic interactions.…”

Section: Conformational Instability Modifies the Ligand-binding Propementioning

confidence: 99%

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

Murphy

Korzhnev

Ceccarelli

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for monoubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the Nand C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.catalytic domain ͉ dynamics ͉ Par-1 ͉ relaxation ͉ ubiquitin-associated

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UBL/UBA Ubiquitin Receptor Proteins Bind a Common Tetraubiquitin Chain

Cited by 76 publications

References 39 publications

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

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