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C-terminal UBA domains protect ubiquitin receptors by preventing initiation of protein degradation.

Heinen C, Acs K, Hoogstraten D, Dantuma NP - Nat Commun (2011)

Bottom Line: We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains.Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains.Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers väg 3, S-17177 Stockholm, Sweden. nico.dantuma@ki.se

ABSTRACT
The ubiquitin receptors Rad23 and Dsk2 deliver polyubiquitylated substrates to the proteasome for destruction. The C-terminal ubiquitin-associated (UBA) domain of Rad23 functions as a cis-acting stabilization signal that protects this protein from proteasomal degradation. Here, we provide evidence that the C-terminal UBA domains guard ubiquitin receptors from destruction by preventing initiation of degradation at the proteasome. We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains. Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains. Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

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Model for UBA-mediated protection from proteasomal degradation.(a) The schematic drawing shows an ubiquitin receptor (Rad23/Dsk2) that delivers a ubiquitylated substrate to the 26S proteasome. The ubiquitin receptor binds the proteasome through its N-terminal UbL domain that directly interacts with the Rpn1 subunit of the proteasome, thereby delivering the polyubiquitylated substrate bound to the UBA domain. Wild-type Rad23/Dsk2 contains a C-terminal UBA domain that resists initiation of protein degradation, resulting in release of the ubiquitin receptor from the proteasome. (b) In case the ubiquitin receptor has a mutant UBA domain or is provided with a polypeptide that can function as an unstructured initiation site, the ubiquitin receptor itself is translocated into the proteasome and hydrolyzed in the proteolytic chamber.
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f8: Model for UBA-mediated protection from proteasomal degradation.(a) The schematic drawing shows an ubiquitin receptor (Rad23/Dsk2) that delivers a ubiquitylated substrate to the 26S proteasome. The ubiquitin receptor binds the proteasome through its N-terminal UbL domain that directly interacts with the Rpn1 subunit of the proteasome, thereby delivering the polyubiquitylated substrate bound to the UBA domain. Wild-type Rad23/Dsk2 contains a C-terminal UBA domain that resists initiation of protein degradation, resulting in release of the ubiquitin receptor from the proteasome. (b) In case the ubiquitin receptor has a mutant UBA domain or is provided with a polypeptide that can function as an unstructured initiation site, the ubiquitin receptor itself is translocated into the proteasome and hydrolyzed in the proteolytic chamber.

Mentions: Our data strongly suggest that C-terminal UBA domains protect ubiquitin receptors from proteasomal degradation by preventing the generation of initiation sites for degradation, which are required for proper engagement of the inherent unfolding machinery of the proteasome4 (Fig. 8). The molecular mechanism that is proposed in this study is fundamentally different from the inhibition of ubiquitin chain elongation by which the UIM domain has been suggested to protect Met4 from degradation23. The implication of distinct mechanisms may seem surprising given that the UIM and UBA domain are structurally unrelated and have little in common except for their ability to selectively bind polyubiquitin chains. However, it is noteworthy that several properties of the involved proteins already argued against a shared mode of action. First, whereas the UIM prevents binding of Met4 to the proteasome23, Rad23 and Dsk2 need to physically interact with the proteasome in order to deliver their polyubiquitylated cargo14. Second, the UbL domains of Rad23 and Dsk2 bind directly to the Rpn1 subunit of the proteasome without the need for polyubiquitin modifications3132, suggesting that blocking of polyubiquitylation will be inadequate to prevent binding of Rad23 and Dsk2 to the proteasome. Third, we recently showed that the structural stability rather than the ubiquitin binding properties of UBA domains is a critical determinant for their ability to protect from proteasomal degradation29. Fourth, the UBA domains in Rad23 and Dsk2 bind polyubiquitylated proteins designated for delivery at the proteasome14, which is hard to reconcile with a role of the very same domains in preventing proteasomal degradation by inhibiting ubiquitylation of the ubiquitin receptors themselves, as this would require simultaneous binding of ubiquitin chains conjugated to the ubiquitin receptor (to prevent chain elongation) and polyubiquitylated proteins (for delivery to the proteasome).


C-terminal UBA domains protect ubiquitin receptors by preventing initiation of protein degradation.

Heinen C, Acs K, Hoogstraten D, Dantuma NP - Nat Commun (2011)

Model for UBA-mediated protection from proteasomal degradation.(a) The schematic drawing shows an ubiquitin receptor (Rad23/Dsk2) that delivers a ubiquitylated substrate to the 26S proteasome. The ubiquitin receptor binds the proteasome through its N-terminal UbL domain that directly interacts with the Rpn1 subunit of the proteasome, thereby delivering the polyubiquitylated substrate bound to the UBA domain. Wild-type Rad23/Dsk2 contains a C-terminal UBA domain that resists initiation of protein degradation, resulting in release of the ubiquitin receptor from the proteasome. (b) In case the ubiquitin receptor has a mutant UBA domain or is provided with a polypeptide that can function as an unstructured initiation site, the ubiquitin receptor itself is translocated into the proteasome and hydrolyzed in the proteolytic chamber.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105319&req=5

f8: Model for UBA-mediated protection from proteasomal degradation.(a) The schematic drawing shows an ubiquitin receptor (Rad23/Dsk2) that delivers a ubiquitylated substrate to the 26S proteasome. The ubiquitin receptor binds the proteasome through its N-terminal UbL domain that directly interacts with the Rpn1 subunit of the proteasome, thereby delivering the polyubiquitylated substrate bound to the UBA domain. Wild-type Rad23/Dsk2 contains a C-terminal UBA domain that resists initiation of protein degradation, resulting in release of the ubiquitin receptor from the proteasome. (b) In case the ubiquitin receptor has a mutant UBA domain or is provided with a polypeptide that can function as an unstructured initiation site, the ubiquitin receptor itself is translocated into the proteasome and hydrolyzed in the proteolytic chamber.
Mentions: Our data strongly suggest that C-terminal UBA domains protect ubiquitin receptors from proteasomal degradation by preventing the generation of initiation sites for degradation, which are required for proper engagement of the inherent unfolding machinery of the proteasome4 (Fig. 8). The molecular mechanism that is proposed in this study is fundamentally different from the inhibition of ubiquitin chain elongation by which the UIM domain has been suggested to protect Met4 from degradation23. The implication of distinct mechanisms may seem surprising given that the UIM and UBA domain are structurally unrelated and have little in common except for their ability to selectively bind polyubiquitin chains. However, it is noteworthy that several properties of the involved proteins already argued against a shared mode of action. First, whereas the UIM prevents binding of Met4 to the proteasome23, Rad23 and Dsk2 need to physically interact with the proteasome in order to deliver their polyubiquitylated cargo14. Second, the UbL domains of Rad23 and Dsk2 bind directly to the Rpn1 subunit of the proteasome without the need for polyubiquitin modifications3132, suggesting that blocking of polyubiquitylation will be inadequate to prevent binding of Rad23 and Dsk2 to the proteasome. Third, we recently showed that the structural stability rather than the ubiquitin binding properties of UBA domains is a critical determinant for their ability to protect from proteasomal degradation29. Fourth, the UBA domains in Rad23 and Dsk2 bind polyubiquitylated proteins designated for delivery at the proteasome14, which is hard to reconcile with a role of the very same domains in preventing proteasomal degradation by inhibiting ubiquitylation of the ubiquitin receptors themselves, as this would require simultaneous binding of ubiquitin chains conjugated to the ubiquitin receptor (to prevent chain elongation) and polyubiquitylated proteins (for delivery to the proteasome).

Bottom Line: We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains.Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains.Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers väg 3, S-17177 Stockholm, Sweden. nico.dantuma@ki.se

ABSTRACT
The ubiquitin receptors Rad23 and Dsk2 deliver polyubiquitylated substrates to the proteasome for destruction. The C-terminal ubiquitin-associated (UBA) domain of Rad23 functions as a cis-acting stabilization signal that protects this protein from proteasomal degradation. Here, we provide evidence that the C-terminal UBA domains guard ubiquitin receptors from destruction by preventing initiation of degradation at the proteasome. We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains. Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains. Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

Show MeSH