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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 UBA domains of Rad23 can shield an internal unstructured polypeptide.(a) Schematic drawing of the UbLRad23-GFP fusions. (b) Flow cytometric quantification of the mean fluorescence intensities of yeast expressing UbLRad23-GFP, UbLRad23-GFP-V5His, UbLRad23-GFP-V5His-UBA2 and UbLRad23-GFP-V5His-UBA1. UbLRad23GFP was standardized as 100%. Values are means and standard deviations (n=3). **P<0.01 (Student's t-test). (c) Steady-state levels of UbLRad23-GFP, UbLRad23-GFP-V5His, UbLRad23-GFP-V5His-UBA2 and UbLRad23-GFP-V5His-UBA1 in the absence or presence of the proteasome inhibitor MG132. β-Actin is shown as loading control. Molecular weight markers are indicated. (d) Turnover of UbLRad23-GFP (closed circles), UbLRad23-GFP-V5His (open circles), UbLRad23-GFP-V5His-UBA2 (closed squares) and UbLRad23-GFP-V5His-UBA1 (open squares). Samples were collected at the indicated time points and probed with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right.
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f6: C-terminal UBA domains of Rad23 can shield an internal unstructured polypeptide.(a) Schematic drawing of the UbLRad23-GFP fusions. (b) Flow cytometric quantification of the mean fluorescence intensities of yeast expressing UbLRad23-GFP, UbLRad23-GFP-V5His, UbLRad23-GFP-V5His-UBA2 and UbLRad23-GFP-V5His-UBA1. UbLRad23GFP was standardized as 100%. Values are means and standard deviations (n=3). **P<0.01 (Student's t-test). (c) Steady-state levels of UbLRad23-GFP, UbLRad23-GFP-V5His, UbLRad23-GFP-V5His-UBA2 and UbLRad23-GFP-V5His-UBA1 in the absence or presence of the proteasome inhibitor MG132. β-Actin is shown as loading control. Molecular weight markers are indicated. (d) Turnover of UbLRad23-GFP (closed circles), UbLRad23-GFP-V5His (open circles), UbLRad23-GFP-V5His-UBA2 (closed squares) and UbLRad23-GFP-V5His-UBA1 (open squares). Samples were collected at the indicated time points and probed with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right.

Mentions: We observed that efficient proteasomal degradation of an UbL–GFP fusion (Fig. 6a) is strictly dependent on the presence of an unstructured polypeptide (Fig. 6b,c). Interestingly, positioning of the UBA2 domain at the C-terminal end of the extension resulted in a strong inhibition of proteasomal degradation of the UbLRad23 fusion (Fig. 6b,c). Whereas the UBA2 domain blocked degradation of the fusion, the UBA1 domain did not have any noteworthy effect on its stability. Turnover experiments clearly showed that the C-terminal positioning of the UBA2 domain, but not the UBA1 domain, resulted in an extended half-life similar to the fusion that lacked an unstructured polypeptide (Fig. 6d). Similarly, we found that the UBA domain of Dsk2 protected the UbLDsk2–GFP carrying an unstructured polypeptide from proteasomal degradation (Supplementary Fig. S2). The above data strongly suggest that UBA domains hinder the generation of an unstructured initiation site for degradation and provide an explanation for the importance of the C-terminal position for UBA-mediated protection from proteasomal degradation.


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 UBA domains of Rad23 can shield an internal unstructured polypeptide.(a) Schematic drawing of the UbLRad23-GFP fusions. (b) Flow cytometric quantification of the mean fluorescence intensities of yeast expressing UbLRad23-GFP, UbLRad23-GFP-V5His, UbLRad23-GFP-V5His-UBA2 and UbLRad23-GFP-V5His-UBA1. UbLRad23GFP was standardized as 100%. Values are means and standard deviations (n=3). **P<0.01 (Student's t-test). (c) Steady-state levels of UbLRad23-GFP, UbLRad23-GFP-V5His, UbLRad23-GFP-V5His-UBA2 and UbLRad23-GFP-V5His-UBA1 in the absence or presence of the proteasome inhibitor MG132. β-Actin is shown as loading control. Molecular weight markers are indicated. (d) Turnover of UbLRad23-GFP (closed circles), UbLRad23-GFP-V5His (open circles), UbLRad23-GFP-V5His-UBA2 (closed squares) and UbLRad23-GFP-V5His-UBA1 (open squares). Samples were collected at the indicated time points and probed with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right.
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Related In: Results  -  Collection

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f6: C-terminal UBA domains of Rad23 can shield an internal unstructured polypeptide.(a) Schematic drawing of the UbLRad23-GFP fusions. (b) Flow cytometric quantification of the mean fluorescence intensities of yeast expressing UbLRad23-GFP, UbLRad23-GFP-V5His, UbLRad23-GFP-V5His-UBA2 and UbLRad23-GFP-V5His-UBA1. UbLRad23GFP was standardized as 100%. Values are means and standard deviations (n=3). **P<0.01 (Student's t-test). (c) Steady-state levels of UbLRad23-GFP, UbLRad23-GFP-V5His, UbLRad23-GFP-V5His-UBA2 and UbLRad23-GFP-V5His-UBA1 in the absence or presence of the proteasome inhibitor MG132. β-Actin is shown as loading control. Molecular weight markers are indicated. (d) Turnover of UbLRad23-GFP (closed circles), UbLRad23-GFP-V5His (open circles), UbLRad23-GFP-V5His-UBA2 (closed squares) and UbLRad23-GFP-V5His-UBA1 (open squares). Samples were collected at the indicated time points and probed with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right.
Mentions: We observed that efficient proteasomal degradation of an UbL–GFP fusion (Fig. 6a) is strictly dependent on the presence of an unstructured polypeptide (Fig. 6b,c). Interestingly, positioning of the UBA2 domain at the C-terminal end of the extension resulted in a strong inhibition of proteasomal degradation of the UbLRad23 fusion (Fig. 6b,c). Whereas the UBA2 domain blocked degradation of the fusion, the UBA1 domain did not have any noteworthy effect on its stability. Turnover experiments clearly showed that the C-terminal positioning of the UBA2 domain, but not the UBA1 domain, resulted in an extended half-life similar to the fusion that lacked an unstructured polypeptide (Fig. 6d). Similarly, we found that the UBA domain of Dsk2 protected the UbLDsk2–GFP carrying an unstructured polypeptide from proteasomal degradation (Supplementary Fig. S2). The above data strongly suggest that UBA domains hinder the generation of an unstructured initiation site for degradation and provide an explanation for the importance of the C-terminal position for UBA-mediated protection from proteasomal degradation.

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