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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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Protective UBA domains cannot function as initiation sites.(a) Schematic drawing of the UbL-GFP fusions. (b) Flow cytometric quantification of the mean fluorescence intensities of yeast expressing UbLRad23-GFP-UBA2, UbLRad23-GFP-UBA2L392A, UbLDsk2-GFP-UBA or UbLDsk2-GFP-UBAL368,369A. UbLRad23-GFP-UBA2 and UbLDsk2-GFP-UBA were standardized as 100%. Values are means and standard deviations (n=3). **P<0.01 (Student's t-test). (c) Turnover of UbLRad23GFP-UBA2 (closed circles) and, UbLRad23GFP-UBA2L392A (open circles). Samples were collected at the indicated time points and detected with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right. (d) Turnover of UbLDsk2-GFP-UBA (closed squares) and UbLDsk2-GFP-UBAL368,369A (open squares). Samples were collected at the indicated time points and analysed by western blotting with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right.
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f7: Protective UBA domains cannot function as initiation sites.(a) Schematic drawing of the UbL-GFP fusions. (b) Flow cytometric quantification of the mean fluorescence intensities of yeast expressing UbLRad23-GFP-UBA2, UbLRad23-GFP-UBA2L392A, UbLDsk2-GFP-UBA or UbLDsk2-GFP-UBAL368,369A. UbLRad23-GFP-UBA2 and UbLDsk2-GFP-UBA were standardized as 100%. Values are means and standard deviations (n=3). **P<0.01 (Student's t-test). (c) Turnover of UbLRad23GFP-UBA2 (closed circles) and, UbLRad23GFP-UBA2L392A (open circles). Samples were collected at the indicated time points and detected with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right. (d) Turnover of UbLDsk2-GFP-UBA (closed squares) and UbLDsk2-GFP-UBAL368,369A (open squares). Samples were collected at the indicated time points and analysed by western blotting with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right.

Mentions: Our observations that Rad23 variants, in which the C-terminal UBA2 domain has been mutated, are subjected to proteasomal degradation suggest that mutant UBA domains not only fail to protect but can also function as initiation sites. We tested this possibility by providing the UbLRad23–GFP with the UBA2 or UBA2L392A domain of Rad23 and the UbLDsk2–GFP with the UBA or UBAL368,369A of Dsk2 (Fig. 7a). Consistent with this model, we found by flow cytometric (Fig. 7b) and western blot analysis (Fig. 7c,d) that the UBA domains could be clearly divided into two groups: (i) the mutant UBA2L392A and UBAL368,369A domains, which facilitated degradation of the UbL-targeted proteins, and (ii) the protective UBA domains of Rad23 and Dsk2, which did not cause degradation of UbL-targeted proteins. These findings suggest that protective UBA domains prevent protein degradation through their inability to function as unstructured initiation sites for protein 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)

Protective UBA domains cannot function as initiation sites.(a) Schematic drawing of the UbL-GFP fusions. (b) Flow cytometric quantification of the mean fluorescence intensities of yeast expressing UbLRad23-GFP-UBA2, UbLRad23-GFP-UBA2L392A, UbLDsk2-GFP-UBA or UbLDsk2-GFP-UBAL368,369A. UbLRad23-GFP-UBA2 and UbLDsk2-GFP-UBA were standardized as 100%. Values are means and standard deviations (n=3). **P<0.01 (Student's t-test). (c) Turnover of UbLRad23GFP-UBA2 (closed circles) and, UbLRad23GFP-UBA2L392A (open circles). Samples were collected at the indicated time points and detected with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right. (d) Turnover of UbLDsk2-GFP-UBA (closed squares) and UbLDsk2-GFP-UBAL368,369A (open squares). Samples were collected at the indicated time points and analysed by western blotting with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Protective UBA domains cannot function as initiation sites.(a) Schematic drawing of the UbL-GFP fusions. (b) Flow cytometric quantification of the mean fluorescence intensities of yeast expressing UbLRad23-GFP-UBA2, UbLRad23-GFP-UBA2L392A, UbLDsk2-GFP-UBA or UbLDsk2-GFP-UBAL368,369A. UbLRad23-GFP-UBA2 and UbLDsk2-GFP-UBA were standardized as 100%. Values are means and standard deviations (n=3). **P<0.01 (Student's t-test). (c) Turnover of UbLRad23GFP-UBA2 (closed circles) and, UbLRad23GFP-UBA2L392A (open circles). Samples were collected at the indicated time points and detected with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right. (d) Turnover of UbLDsk2-GFP-UBA (closed squares) and UbLDsk2-GFP-UBAL368,369A (open squares). Samples were collected at the indicated time points and analysed by western blotting with a GFP-specific antibody. Densitometric quantification of the blot is shown to the right.
Mentions: Our observations that Rad23 variants, in which the C-terminal UBA2 domain has been mutated, are subjected to proteasomal degradation suggest that mutant UBA domains not only fail to protect but can also function as initiation sites. We tested this possibility by providing the UbLRad23–GFP with the UBA2 or UBA2L392A domain of Rad23 and the UbLDsk2–GFP with the UBA or UBAL368,369A of Dsk2 (Fig. 7a). Consistent with this model, we found by flow cytometric (Fig. 7b) and western blot analysis (Fig. 7c,d) that the UBA domains could be clearly divided into two groups: (i) the mutant UBA2L392A and UBAL368,369A domains, which facilitated degradation of the UbL-targeted proteins, and (ii) the protective UBA domains of Rad23 and Dsk2, which did not cause degradation of UbL-targeted proteins. These findings suggest that protective UBA domains prevent protein degradation through their inability to function as unstructured initiation sites for protein 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