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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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Different stabilizing potentials of UBA and UIM domains.(a) Ub-R-GFP-UBA2 and Ub-R-GFP-UBA2L392A were expressed in yeast together with His-tagged ubiquitin. Ubiquitylated proteins were precipitated using Ni beads and probed with a GFP-specific antibody. Input (I) and precipitated (P) samples are shown. Ub-R-GFP-UBA2 and Ub-R-GFP-UBA2L392A are indicated. Note that the upper band in the Ub-R-GFP-UBA2 corresponds with diubiquitylated Ub-R-GFP-UBA2. Molecular weight markers are indicated. (b) Relative fluorescence levels of yeast expressing the Ub-M-GFP, Ub-R-GFP, Ub-R-GFP-UBA2Rad23, Ub-R-GFP-UBADsk2 and Ub-R-GFP-UIMMet4 analysed by flow cytometry. Ub-M-GFP was standardized as 100%. Values are means and standard deviations (n=3). *P<0.05, **P<0.01 (Student's t-test). (c) Western blot analysis with GFP-specific antibody of steady-state levels of Ub-M-GFP, Ub-R-GFP, Ub-R-GFP-UBA2Rad23, Ub-R-GFP-UBADsk2 and Ub-R-GFP-UIMMet4 in the absence or presence of 50 μM proteasome inhibitor MG132. Note that the upper bands correspond with diubiquitylated Ub-R-GFP-UBA2Rad23 and putative monoubiquitylated Ub-R-GFP-UBADsk2. β-Actin is shown as loading control. Molecular weight markers are indicated. Specific bands are indicated with arrowheads.
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f2: Different stabilizing potentials of UBA and UIM domains.(a) Ub-R-GFP-UBA2 and Ub-R-GFP-UBA2L392A were expressed in yeast together with His-tagged ubiquitin. Ubiquitylated proteins were precipitated using Ni beads and probed with a GFP-specific antibody. Input (I) and precipitated (P) samples are shown. Ub-R-GFP-UBA2 and Ub-R-GFP-UBA2L392A are indicated. Note that the upper band in the Ub-R-GFP-UBA2 corresponds with diubiquitylated Ub-R-GFP-UBA2. Molecular weight markers are indicated. (b) Relative fluorescence levels of yeast expressing the Ub-M-GFP, Ub-R-GFP, Ub-R-GFP-UBA2Rad23, Ub-R-GFP-UBADsk2 and Ub-R-GFP-UIMMet4 analysed by flow cytometry. Ub-M-GFP was standardized as 100%. Values are means and standard deviations (n=3). *P<0.05, **P<0.01 (Student's t-test). (c) Western blot analysis with GFP-specific antibody of steady-state levels of Ub-M-GFP, Ub-R-GFP, Ub-R-GFP-UBA2Rad23, Ub-R-GFP-UBADsk2 and Ub-R-GFP-UIMMet4 in the absence or presence of 50 μM proteasome inhibitor MG132. Note that the upper bands correspond with diubiquitylated Ub-R-GFP-UBA2Rad23 and putative monoubiquitylated Ub-R-GFP-UBADsk2. β-Actin is shown as loading control. Molecular weight markers are indicated. Specific bands are indicated with arrowheads.

Mentions: More recently, it has been shown that a ubiquitin-interacting motif (UIM) in the transcription factor Met4, whose activity is regulated in a non-proteolytic manner21 by the conjugation of Lys48-linked polyubiquitin chains22, prevents proteasomal degradation of Met4 (ref. 23). It has been proposed that the UIM domain protects Met4 by inhibiting ubiquitin chain assembly, allowing only formation of polyubiquitin chains that are below the critical length required for proteasomal degradation23. To test whether a similar phenomenon could be responsible for the protective effect of UBA domains, we compared the effects of the wild-type UBA2 domain and the mutant UBA2L392A domain, which lacks protective activity17, on ubiquitylation of a well-characterized N-end rule green fluorescent protein (GFP) substrate, ubiquitin–arginine–GFP (Ub-R-GFP)2425. Ubi-quitylated proteins were precipitated under reducing conditions to avoid contaminations with polyubiquitylated proteins bound to the UBA domain. We observed a very similar polyubiquitylation pattern for the reporter substrates, irrespective of the nature of the UBA domain (Fig. 2a). As the polyubiquitylation pattern was comparable in substrates carrying either the protective or mutant UBA domain, inhibition of ubiquitin chain elongation cannot fully explain the ability of UBA domains to protect substrates 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)

Different stabilizing potentials of UBA and UIM domains.(a) Ub-R-GFP-UBA2 and Ub-R-GFP-UBA2L392A were expressed in yeast together with His-tagged ubiquitin. Ubiquitylated proteins were precipitated using Ni beads and probed with a GFP-specific antibody. Input (I) and precipitated (P) samples are shown. Ub-R-GFP-UBA2 and Ub-R-GFP-UBA2L392A are indicated. Note that the upper band in the Ub-R-GFP-UBA2 corresponds with diubiquitylated Ub-R-GFP-UBA2. Molecular weight markers are indicated. (b) Relative fluorescence levels of yeast expressing the Ub-M-GFP, Ub-R-GFP, Ub-R-GFP-UBA2Rad23, Ub-R-GFP-UBADsk2 and Ub-R-GFP-UIMMet4 analysed by flow cytometry. Ub-M-GFP was standardized as 100%. Values are means and standard deviations (n=3). *P<0.05, **P<0.01 (Student's t-test). (c) Western blot analysis with GFP-specific antibody of steady-state levels of Ub-M-GFP, Ub-R-GFP, Ub-R-GFP-UBA2Rad23, Ub-R-GFP-UBADsk2 and Ub-R-GFP-UIMMet4 in the absence or presence of 50 μM proteasome inhibitor MG132. Note that the upper bands correspond with diubiquitylated Ub-R-GFP-UBA2Rad23 and putative monoubiquitylated Ub-R-GFP-UBADsk2. β-Actin is shown as loading control. Molecular weight markers are indicated. Specific bands are indicated with arrowheads.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Different stabilizing potentials of UBA and UIM domains.(a) Ub-R-GFP-UBA2 and Ub-R-GFP-UBA2L392A were expressed in yeast together with His-tagged ubiquitin. Ubiquitylated proteins were precipitated using Ni beads and probed with a GFP-specific antibody. Input (I) and precipitated (P) samples are shown. Ub-R-GFP-UBA2 and Ub-R-GFP-UBA2L392A are indicated. Note that the upper band in the Ub-R-GFP-UBA2 corresponds with diubiquitylated Ub-R-GFP-UBA2. Molecular weight markers are indicated. (b) Relative fluorescence levels of yeast expressing the Ub-M-GFP, Ub-R-GFP, Ub-R-GFP-UBA2Rad23, Ub-R-GFP-UBADsk2 and Ub-R-GFP-UIMMet4 analysed by flow cytometry. Ub-M-GFP was standardized as 100%. Values are means and standard deviations (n=3). *P<0.05, **P<0.01 (Student's t-test). (c) Western blot analysis with GFP-specific antibody of steady-state levels of Ub-M-GFP, Ub-R-GFP, Ub-R-GFP-UBA2Rad23, Ub-R-GFP-UBADsk2 and Ub-R-GFP-UIMMet4 in the absence or presence of 50 μM proteasome inhibitor MG132. Note that the upper bands correspond with diubiquitylated Ub-R-GFP-UBA2Rad23 and putative monoubiquitylated Ub-R-GFP-UBADsk2. β-Actin is shown as loading control. Molecular weight markers are indicated. Specific bands are indicated with arrowheads.
Mentions: More recently, it has been shown that a ubiquitin-interacting motif (UIM) in the transcription factor Met4, whose activity is regulated in a non-proteolytic manner21 by the conjugation of Lys48-linked polyubiquitin chains22, prevents proteasomal degradation of Met4 (ref. 23). It has been proposed that the UIM domain protects Met4 by inhibiting ubiquitin chain assembly, allowing only formation of polyubiquitin chains that are below the critical length required for proteasomal degradation23. To test whether a similar phenomenon could be responsible for the protective effect of UBA domains, we compared the effects of the wild-type UBA2 domain and the mutant UBA2L392A domain, which lacks protective activity17, on ubiquitylation of a well-characterized N-end rule green fluorescent protein (GFP) substrate, ubiquitin–arginine–GFP (Ub-R-GFP)2425. Ubi-quitylated proteins were precipitated under reducing conditions to avoid contaminations with polyubiquitylated proteins bound to the UBA domain. We observed a very similar polyubiquitylation pattern for the reporter substrates, irrespective of the nature of the UBA domain (Fig. 2a). As the polyubiquitylation pattern was comparable in substrates carrying either the protective or mutant UBA domain, inhibition of ubiquitin chain elongation cannot fully explain the ability of UBA domains to protect substrates 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