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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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The UBA domain of Dsk2 functions as a stabilization signal.(a) Steady-state levels of FLAG-tagged Dsk2 determined by western blotting with a FLAG-specific antibody. FLAGDsk2, FLAGDsk2L368,369A and FLAGDsk2ΔUbL/L368,369A were ectopically expressed from a GAL1 promoter. Yeast was grown until early log phase. Wherever indicated, proteasome inhibitor was added to a final concentration of 50 μM, 2 h before harvesting. β-Actin is shown as loading control. Molecular weight markers are indicated. Asterisk marks a non-specific band. (b) Turnover of FLAGDsk2 (closed circles), FLAGDsk2L368,369A (open circles) and FLAGDsk2ΔUbL/L368,369A (closed squares). Samples were taken at the indicated time points and probed with a FLAG-specific antibody. Densitometric quantification of the western blot is shown. (c) Turnover of FLAGDsk2 (closed circles), FLAGDsk2L368,369A (open circles) and FLAGDsk2ΔUbL/L368,369A (closed squares) in the presence of 50 μM proteasome inhibitor MG132, as shown in b.
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f1: The UBA domain of Dsk2 functions as a stabilization signal.(a) Steady-state levels of FLAG-tagged Dsk2 determined by western blotting with a FLAG-specific antibody. FLAGDsk2, FLAGDsk2L368,369A and FLAGDsk2ΔUbL/L368,369A were ectopically expressed from a GAL1 promoter. Yeast was grown until early log phase. Wherever indicated, proteasome inhibitor was added to a final concentration of 50 μM, 2 h before harvesting. β-Actin is shown as loading control. Molecular weight markers are indicated. Asterisk marks a non-specific band. (b) Turnover of FLAGDsk2 (closed circles), FLAGDsk2L368,369A (open circles) and FLAGDsk2ΔUbL/L368,369A (closed squares). Samples were taken at the indicated time points and probed with a FLAG-specific antibody. Densitometric quantification of the western blot is shown. (c) Turnover of FLAGDsk2 (closed circles), FLAGDsk2L368,369A (open circles) and FLAGDsk2ΔUbL/L368,369A (closed squares) in the presence of 50 μM proteasome inhibitor MG132, as shown in b.

Mentions: Dsk2 shares with Rad23 not only its ability to deliver polyubiquitylated proteins to the proteasome14 but also its long half-life1516, which encouraged us to investigate whether the UBA domain of Dsk2 has a role in protecting this ubiquitin receptor from proteasomal degradation analogous to what has been observed for Rad23 (ref. 17). To this end, we substituted two conserved leucine residues in the UBA domain of Dsk2 for alanine residues in FLAG-tagged Dsk2 (FLAGDsk2L368,369A). Notably, structural analysis has revealed that the leucine at position 369 is part of a hydrophobic patch that is important for the typically tightly packed α-helical fold of UBA domains18, and simultaneous substitution of these two adjacent leucine residues abolishes ubiquitin binding19. These amino-acid substitutions resulted in a strongly reduced steady-state level of the mutant protein, as compared with its wild-type counterpart (Fig. 1a). To test whether the reduced steady-state levels of FLAGDsk2L368,369A were due to proteasomal degradation, we administrated the proteasome inhibitor MG132 to the yeast in a specific medium that allows efficient uptake of this compound20. Inhibitor treatment caused a striking accumulation of FLAGDsk2L368,369A, in line with efficient proteasomal degradation being responsible for the reduced levels (Fig. 1a). FLAGDsk2L368,369A had a short half-life (Fig. 1b) and was stabilized by administration of proteasome inhibitor, consistent with efficient proteasomal degradation of the mutant Dsk2 (Fig. 1c). Importantly, deletion of the UbL domain also resulted in stabilization of the mutant Dsk2, demonstrating that its degradation is largely dependent on its proteasome-interacting domain (Fig. 1b).


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

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

The UBA domain of Dsk2 functions as a stabilization signal.(a) Steady-state levels of FLAG-tagged Dsk2 determined by western blotting with a FLAG-specific antibody. FLAGDsk2, FLAGDsk2L368,369A and FLAGDsk2ΔUbL/L368,369A were ectopically expressed from a GAL1 promoter. Yeast was grown until early log phase. Wherever indicated, proteasome inhibitor was added to a final concentration of 50 μM, 2 h before harvesting. β-Actin is shown as loading control. Molecular weight markers are indicated. Asterisk marks a non-specific band. (b) Turnover of FLAGDsk2 (closed circles), FLAGDsk2L368,369A (open circles) and FLAGDsk2ΔUbL/L368,369A (closed squares). Samples were taken at the indicated time points and probed with a FLAG-specific antibody. Densitometric quantification of the western blot is shown. (c) Turnover of FLAGDsk2 (closed circles), FLAGDsk2L368,369A (open circles) and FLAGDsk2ΔUbL/L368,369A (closed squares) in the presence of 50 μM proteasome inhibitor MG132, as shown in b.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: The UBA domain of Dsk2 functions as a stabilization signal.(a) Steady-state levels of FLAG-tagged Dsk2 determined by western blotting with a FLAG-specific antibody. FLAGDsk2, FLAGDsk2L368,369A and FLAGDsk2ΔUbL/L368,369A were ectopically expressed from a GAL1 promoter. Yeast was grown until early log phase. Wherever indicated, proteasome inhibitor was added to a final concentration of 50 μM, 2 h before harvesting. β-Actin is shown as loading control. Molecular weight markers are indicated. Asterisk marks a non-specific band. (b) Turnover of FLAGDsk2 (closed circles), FLAGDsk2L368,369A (open circles) and FLAGDsk2ΔUbL/L368,369A (closed squares). Samples were taken at the indicated time points and probed with a FLAG-specific antibody. Densitometric quantification of the western blot is shown. (c) Turnover of FLAGDsk2 (closed circles), FLAGDsk2L368,369A (open circles) and FLAGDsk2ΔUbL/L368,369A (closed squares) in the presence of 50 μM proteasome inhibitor MG132, as shown in b.
Mentions: Dsk2 shares with Rad23 not only its ability to deliver polyubiquitylated proteins to the proteasome14 but also its long half-life1516, which encouraged us to investigate whether the UBA domain of Dsk2 has a role in protecting this ubiquitin receptor from proteasomal degradation analogous to what has been observed for Rad23 (ref. 17). To this end, we substituted two conserved leucine residues in the UBA domain of Dsk2 for alanine residues in FLAG-tagged Dsk2 (FLAGDsk2L368,369A). Notably, structural analysis has revealed that the leucine at position 369 is part of a hydrophobic patch that is important for the typically tightly packed α-helical fold of UBA domains18, and simultaneous substitution of these two adjacent leucine residues abolishes ubiquitin binding19. These amino-acid substitutions resulted in a strongly reduced steady-state level of the mutant protein, as compared with its wild-type counterpart (Fig. 1a). To test whether the reduced steady-state levels of FLAGDsk2L368,369A were due to proteasomal degradation, we administrated the proteasome inhibitor MG132 to the yeast in a specific medium that allows efficient uptake of this compound20. Inhibitor treatment caused a striking accumulation of FLAGDsk2L368,369A, in line with efficient proteasomal degradation being responsible for the reduced levels (Fig. 1a). FLAGDsk2L368,369A had a short half-life (Fig. 1b) and was stabilized by administration of proteasome inhibitor, consistent with efficient proteasomal degradation of the mutant Dsk2 (Fig. 1c). Importantly, deletion of the UbL domain also resulted in stabilization of the mutant Dsk2, demonstrating that its degradation is largely dependent on its proteasome-interacting domain (Fig. 1b).

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