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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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C-terminal unstructured polypeptides abrogate protection.(a) Turnover of Ub-R-GFP, Ub-R-GFP-UBA2, Ub-R-GFP-UBA2L392A and Ub-R-GFP-UBA2-V5His. Samples were taken at the indicated time points after switching off the expression and probed with a GFP-specific antibody. (b) Steady-state levels of FLAGRad23, FLAGRad23-V5His and FLAGRad23-V5HisΔUbL in the absence or presence of the proteasome inhibitor MG132. β-Actin is shown as loading control. Molecular weight marker is indicated. (c) Turnover of FLAGRad23, FLAGRad23-V5His and FLAGRad23-V5HisΔUbL. Samples were collected at the indicated time points and analysed with a FLAG-specific antibody. Asterisk indicates a non-specific band. (d) Steady-state levels of FLAGRad23+15aa, FLAGRad23+20aa, FLAGRad23+25aa, FLAGRad23-V5His (33aa) and FLAGRad23-V5HisΔUbL in the absence or presence of the proteasome inhibitor MG132. The proteins were detected with a FLAG-specific antibody. β-Actin is shown as loading control. Molecular weight marker is indicated.
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f5: C-terminal unstructured polypeptides abrogate protection.(a) Turnover of Ub-R-GFP, Ub-R-GFP-UBA2, Ub-R-GFP-UBA2L392A and Ub-R-GFP-UBA2-V5His. Samples were taken at the indicated time points after switching off the expression and probed with a GFP-specific antibody. (b) Steady-state levels of FLAGRad23, FLAGRad23-V5His and FLAGRad23-V5HisΔUbL in the absence or presence of the proteasome inhibitor MG132. β-Actin is shown as loading control. Molecular weight marker is indicated. (c) Turnover of FLAGRad23, FLAGRad23-V5His and FLAGRad23-V5HisΔUbL. Samples were collected at the indicated time points and analysed with a FLAG-specific antibody. Asterisk indicates a non-specific band. (d) Steady-state levels of FLAGRad23+15aa, FLAGRad23+20aa, FLAGRad23+25aa, FLAGRad23-V5His (33aa) and FLAGRad23-V5HisΔUbL in the absence or presence of the proteasome inhibitor MG132. The proteins were detected with a FLAG-specific antibody. β-Actin is shown as loading control. Molecular weight marker is indicated.

Mentions: The importance of the C-terminal position of the UBA2 domain, combined with our earlier observation that the structural stability of mutant UBA domains of human p62 correlates with their protective effect29, raised the question of whether these domains can interfere with protein unfolding. Using designed substrates, it has previously been shown that proteasomes, when interacting with protein complexes, display a strong preference for those proteins that contain unstructured initiation sites30. Consistent with a critical role for preventing initiation of protein degradation, we found that introduction of a 34-amino-acid-long polypeptide, which is sufficiently long to function as an initiation site for proteasomal degradation1011, strongly reduced the stabilizing effect of the UBA2 domain, resulting in low steady-state levels and accumulation of the reporter substrate in the presence of the proteasome inhibitor MG132 (Supplementary Fig. S1). Promoter shut-off experiments showed that the half-life of the fusion harbouring the extension was substantially reduced (Fig. 5a).


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

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

C-terminal unstructured polypeptides abrogate protection.(a) Turnover of Ub-R-GFP, Ub-R-GFP-UBA2, Ub-R-GFP-UBA2L392A and Ub-R-GFP-UBA2-V5His. Samples were taken at the indicated time points after switching off the expression and probed with a GFP-specific antibody. (b) Steady-state levels of FLAGRad23, FLAGRad23-V5His and FLAGRad23-V5HisΔUbL in the absence or presence of the proteasome inhibitor MG132. β-Actin is shown as loading control. Molecular weight marker is indicated. (c) Turnover of FLAGRad23, FLAGRad23-V5His and FLAGRad23-V5HisΔUbL. Samples were collected at the indicated time points and analysed with a FLAG-specific antibody. Asterisk indicates a non-specific band. (d) Steady-state levels of FLAGRad23+15aa, FLAGRad23+20aa, FLAGRad23+25aa, FLAGRad23-V5His (33aa) and FLAGRad23-V5HisΔUbL in the absence or presence of the proteasome inhibitor MG132. The proteins were detected with a FLAG-specific antibody. β-Actin is shown as loading control. Molecular weight marker is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: C-terminal unstructured polypeptides abrogate protection.(a) Turnover of Ub-R-GFP, Ub-R-GFP-UBA2, Ub-R-GFP-UBA2L392A and Ub-R-GFP-UBA2-V5His. Samples were taken at the indicated time points after switching off the expression and probed with a GFP-specific antibody. (b) Steady-state levels of FLAGRad23, FLAGRad23-V5His and FLAGRad23-V5HisΔUbL in the absence or presence of the proteasome inhibitor MG132. β-Actin is shown as loading control. Molecular weight marker is indicated. (c) Turnover of FLAGRad23, FLAGRad23-V5His and FLAGRad23-V5HisΔUbL. Samples were collected at the indicated time points and analysed with a FLAG-specific antibody. Asterisk indicates a non-specific band. (d) Steady-state levels of FLAGRad23+15aa, FLAGRad23+20aa, FLAGRad23+25aa, FLAGRad23-V5His (33aa) and FLAGRad23-V5HisΔUbL in the absence or presence of the proteasome inhibitor MG132. The proteins were detected with a FLAG-specific antibody. β-Actin is shown as loading control. Molecular weight marker is indicated.
Mentions: The importance of the C-terminal position of the UBA2 domain, combined with our earlier observation that the structural stability of mutant UBA domains of human p62 correlates with their protective effect29, raised the question of whether these domains can interfere with protein unfolding. Using designed substrates, it has previously been shown that proteasomes, when interacting with protein complexes, display a strong preference for those proteins that contain unstructured initiation sites30. Consistent with a critical role for preventing initiation of protein degradation, we found that introduction of a 34-amino-acid-long polypeptide, which is sufficiently long to function as an initiation site for proteasomal degradation1011, strongly reduced the stabilizing effect of the UBA2 domain, resulting in low steady-state levels and accumulation of the reporter substrate in the presence of the proteasome inhibitor MG132 (Supplementary Fig. S1). Promoter shut-off experiments showed that the half-life of the fusion harbouring the extension was substantially reduced (Fig. 5a).

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
Related in: MedlinePlus