Limits...
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.

Show MeSH
A C-terminal UBA1 domain does not protect from degradation.(a) Schematic drawing of the positions of the UBA1 and UBA2 domains in wild-type Rad23 (Rad23UBA1/UBA2), Rad23UBA1/UBA1 and Rad23UBA2/UBA1. (b) Steady-state levels of FLAG-tagged Rad23UBA1/UBA2 (wild-type), Rad23UBA1/UBA2* (Rad23L392A), Rad23UBA1/UBA1 and Rad23UBA2/UBA1 were determined with a FLAG-specific antibody. Arrows indicateFLAGRad23 bands. β-Actin is shown as loading control. Molecular weight marker is indicated. Asterisk indicates a non-specific band.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105319&req=5

f3: A C-terminal UBA1 domain does not protect from degradation.(a) Schematic drawing of the positions of the UBA1 and UBA2 domains in wild-type Rad23 (Rad23UBA1/UBA2), Rad23UBA1/UBA1 and Rad23UBA2/UBA1. (b) Steady-state levels of FLAG-tagged Rad23UBA1/UBA2 (wild-type), Rad23UBA1/UBA2* (Rad23L392A), Rad23UBA1/UBA1 and Rad23UBA2/UBA1 were determined with a FLAG-specific antibody. Arrows indicateFLAGRad23 bands. β-Actin is shown as loading control. Molecular weight marker is indicated. Asterisk indicates a non-specific band.

Mentions: The protective UBA domains of Rad23 and Dsk2 are localized at the C termini of these proteins. We wondered whether the positioning of the UBA domains was of significance for their ability to protect substrates from proteasomal degradation. To address this issue, we swapped the internal UBA1 domain and C-terminal UBA2 domain within the Rad23 protein (Fig. 3a). We first replaced the C-terminal UBA2 domain with the UBA1 domain, giving rise to a Rad23 with two UBA1 domains (Rad23UBA1/UBA1). Next, we replaced the internal UBA1 domain with the UBA2 domain to generate Rad23UBA2/UBA1, in which the two domains had been swapped. Expression of wild-type Rad23UBA1/UBA2, and the two variants Rad23UBA1/UBA1 and Rad23UBA2/UBA1, in yeast revealed that the UBA1 domain is unable to protect even when placed at the same C-terminal position as the UBA2 domain (Fig. 3b). Furthermore, placing the UBA2 domain at the internal position inactivated its stabilizing potential, as evidenced by the efficient proteasomal degradation of the Rad23UBA2/UBA1 variant. Taken together, there are two important conclusions from this experiment. First, it confirmed, now in the context of Rad23, our earlier conclusion17 that the UBA1 domain is unable to function as a stabilization signal. Second, these data show that the UBA2 domain can only protect from proteasomal degradation when positioned at the C terminus, which corresponds with its native location.


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

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

A C-terminal UBA1 domain does not protect from degradation.(a) Schematic drawing of the positions of the UBA1 and UBA2 domains in wild-type Rad23 (Rad23UBA1/UBA2), Rad23UBA1/UBA1 and Rad23UBA2/UBA1. (b) Steady-state levels of FLAG-tagged Rad23UBA1/UBA2 (wild-type), Rad23UBA1/UBA2* (Rad23L392A), Rad23UBA1/UBA1 and Rad23UBA2/UBA1 were determined with a FLAG-specific antibody. Arrows indicateFLAGRad23 bands. β-Actin is shown as loading control. Molecular weight marker is indicated. Asterisk indicates a non-specific band.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: A C-terminal UBA1 domain does not protect from degradation.(a) Schematic drawing of the positions of the UBA1 and UBA2 domains in wild-type Rad23 (Rad23UBA1/UBA2), Rad23UBA1/UBA1 and Rad23UBA2/UBA1. (b) Steady-state levels of FLAG-tagged Rad23UBA1/UBA2 (wild-type), Rad23UBA1/UBA2* (Rad23L392A), Rad23UBA1/UBA1 and Rad23UBA2/UBA1 were determined with a FLAG-specific antibody. Arrows indicateFLAGRad23 bands. β-Actin is shown as loading control. Molecular weight marker is indicated. Asterisk indicates a non-specific band.
Mentions: The protective UBA domains of Rad23 and Dsk2 are localized at the C termini of these proteins. We wondered whether the positioning of the UBA domains was of significance for their ability to protect substrates from proteasomal degradation. To address this issue, we swapped the internal UBA1 domain and C-terminal UBA2 domain within the Rad23 protein (Fig. 3a). We first replaced the C-terminal UBA2 domain with the UBA1 domain, giving rise to a Rad23 with two UBA1 domains (Rad23UBA1/UBA1). Next, we replaced the internal UBA1 domain with the UBA2 domain to generate Rad23UBA2/UBA1, in which the two domains had been swapped. Expression of wild-type Rad23UBA1/UBA2, and the two variants Rad23UBA1/UBA1 and Rad23UBA2/UBA1, in yeast revealed that the UBA1 domain is unable to protect even when placed at the same C-terminal position as the UBA2 domain (Fig. 3b). Furthermore, placing the UBA2 domain at the internal position inactivated its stabilizing potential, as evidenced by the efficient proteasomal degradation of the Rad23UBA2/UBA1 variant. Taken together, there are two important conclusions from this experiment. First, it confirmed, now in the context of Rad23, our earlier conclusion17 that the UBA1 domain is unable to function as a stabilization signal. Second, these data show that the UBA2 domain can only protect from proteasomal degradation when positioned at the C terminus, which corresponds with its native location.

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