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Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates.

Wang X, Herr RA, Rabelink M, Hoeben RC, Wiertz EJ, Hansen TH - J. Cell Biol. (2009)

Bottom Line: Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD).In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates.However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD). Until recently, ubiquitination of substrates was thought to occur only via isopeptide bonds, typically to lysine residues. Several recent studies suggest that Ub can also be coupled to nonlysine residues by ester/thiolester bonds; however, the molecular basis for these novel modifications remains elusive. To probe the mechanism and importance of nonlysine ubiquitination, we have studied the viral ligase murine K3 (mK3), which facilitates the polyubiquitination of hydroxylated amino acids serine/threonine on its ERAD substrate. In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates. However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

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Membrane-bound E2s support the ubiquitination of the HC by mK3. (A) Mouse WT3 cells coexpressing WT Ld and mK3 were treated briefly with digitonin. The cytosolic proteins (CP) in solution and membrane proteins in the pellet were separated by centrifugation. The pellet was then suspended with reaction buffer containing an ATP-regenerating system. Three aliquots were made from this suspension and incubated with E1 only, E1 + HA-Ub (Ub*), or rabbit reticulocyte FII + Ub*, respectively, and followed by centrifugation. Samples from supernatant (S) and pellet (P) of each group as well as the cytosolic protein fraction were blotted by the antibodies indicated to verify the depletion of the cytosolic proteins and the maintenance of membrane proteins in the pellet. It should be noted that FII likely contains most soluble E2s; thus, the supernatant fraction with FII added is Ube2k and Ube2n positive. (B) Precipitations of Ld HCs from the supernatant or the pellet fractions of three reaction groups described in A were performed using anti-Ld mAbs. The precipitates were blotted for newly formed Ub-Ld conjugates with anti-HA. An anti-Ld blot was included to show similar input in each group and as an Endo H treatment control. Data shown are representative of three independent experiments. (A and B) Molecular mass is indicated in kilodaltons. IB, immunoblot; IP, immunoprecipitation.
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fig1: Membrane-bound E2s support the ubiquitination of the HC by mK3. (A) Mouse WT3 cells coexpressing WT Ld and mK3 were treated briefly with digitonin. The cytosolic proteins (CP) in solution and membrane proteins in the pellet were separated by centrifugation. The pellet was then suspended with reaction buffer containing an ATP-regenerating system. Three aliquots were made from this suspension and incubated with E1 only, E1 + HA-Ub (Ub*), or rabbit reticulocyte FII + Ub*, respectively, and followed by centrifugation. Samples from supernatant (S) and pellet (P) of each group as well as the cytosolic protein fraction were blotted by the antibodies indicated to verify the depletion of the cytosolic proteins and the maintenance of membrane proteins in the pellet. It should be noted that FII likely contains most soluble E2s; thus, the supernatant fraction with FII added is Ube2k and Ube2n positive. (B) Precipitations of Ld HCs from the supernatant or the pellet fractions of three reaction groups described in A were performed using anti-Ld mAbs. The precipitates were blotted for newly formed Ub-Ld conjugates with anti-HA. An anti-Ld blot was included to show similar input in each group and as an Endo H treatment control. Data shown are representative of three independent experiments. (A and B) Molecular mass is indicated in kilodaltons. IB, immunoblot; IP, immunoprecipitation.

Mentions: To test whether the cellular E2 supporting mK3 ubiquitination of HC is membrane associated, a permeabilized cell system was used (Shamu et al., 1999). WT3 cells expressing mK3 ligase and its substrate Ld HCs were treated briefly with digitonin and then centrifuged to separate cytosolic proteins in solution from membrane proteins in the pellet. Next, pellets of permeabilized cells were resuspended with an energy-regenerating system (Feldman et al., 1997) and buffer containing E1, E1 + HA-Ub, or fraction II (FII; which contains E1, most E2s, some E3s, and the proteasome) + HA-Ub. After incubation, the reaction mixture was separated into the supernatant and pellet fractions. Samples from the supernatant and pellet fractions of these three experimental groups as well as samples from the cytosolic protein fraction were tested by immunoblot to verify the elimination of cytosolic proteins (Ube2n and Ube2k) and the retention of membrane proteins such as mK3 (Fig. 1 A). The de novo Ub conjugates on the HCs in these supernatant and pellet fractions were identified by immunoprecipitation of the HC and blotting for HA. To clearly distinguish the ubiquitinated HC from nonspecific bands, half of the HC precipitates were treated with endoglycosidase H (Endo H). This treatment cleaves the high mannose glycan moieties from immature HCs (nonubiquitinated as well as ubiquitinated), resulting in a down shift of corresponding HC bands in the gel. From the experiment shown in Fig. 1 B, four important conclusions can be drawn. First, Endo H–sensitive ubiquitinated HC bands were only observed in the samples incubated with HA-tagged Ub (lanes 7, 8, 11, and 12) but not in the samples without added HA-Ub (lanes 1–4). Thus, only newly ubiquitinated HCs were detected in the HA blots. Second, the pattern of the de novo ubiquitinated HCs in the permeabilized cells is the same as previously seen with nonpermeabilized cells (Wang et al., 2007), demonstrating recapitulation of what is observed in vivo. Third, the ubiquitinated HCs were only detected in the pellet, indicating that they were membrane bound, which is consistent with in vivo observations (Wang et al., 2007). Fourth, profound ubiquitination of HC was detected in the samples with only E1 (lanes 7 and 8), supporting the hypothesis that a cognate E2 of mK3 is membrane bound. Consistent with this conclusion, samples with added FII fraction (likely containing most soluble E2s) gave less robust ubiquitination of HC (lanes 11 and 12). Furthermore, by using the cells expressing a catalytically inactive RING mutant of mK3, we confirmed that ubiquitination in the permeabilized system was indeed mediated by mK3 (Fig. S1). Collectively, these data validate the approach and further suggest that a membrane-bound E2 can support mK3-mediated ubiquitination of HC.


Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates.

Wang X, Herr RA, Rabelink M, Hoeben RC, Wiertz EJ, Hansen TH - J. Cell Biol. (2009)

Membrane-bound E2s support the ubiquitination of the HC by mK3. (A) Mouse WT3 cells coexpressing WT Ld and mK3 were treated briefly with digitonin. The cytosolic proteins (CP) in solution and membrane proteins in the pellet were separated by centrifugation. The pellet was then suspended with reaction buffer containing an ATP-regenerating system. Three aliquots were made from this suspension and incubated with E1 only, E1 + HA-Ub (Ub*), or rabbit reticulocyte FII + Ub*, respectively, and followed by centrifugation. Samples from supernatant (S) and pellet (P) of each group as well as the cytosolic protein fraction were blotted by the antibodies indicated to verify the depletion of the cytosolic proteins and the maintenance of membrane proteins in the pellet. It should be noted that FII likely contains most soluble E2s; thus, the supernatant fraction with FII added is Ube2k and Ube2n positive. (B) Precipitations of Ld HCs from the supernatant or the pellet fractions of three reaction groups described in A were performed using anti-Ld mAbs. The precipitates were blotted for newly formed Ub-Ld conjugates with anti-HA. An anti-Ld blot was included to show similar input in each group and as an Endo H treatment control. Data shown are representative of three independent experiments. (A and B) Molecular mass is indicated in kilodaltons. IB, immunoblot; IP, immunoprecipitation.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806592&req=5

fig1: Membrane-bound E2s support the ubiquitination of the HC by mK3. (A) Mouse WT3 cells coexpressing WT Ld and mK3 were treated briefly with digitonin. The cytosolic proteins (CP) in solution and membrane proteins in the pellet were separated by centrifugation. The pellet was then suspended with reaction buffer containing an ATP-regenerating system. Three aliquots were made from this suspension and incubated with E1 only, E1 + HA-Ub (Ub*), or rabbit reticulocyte FII + Ub*, respectively, and followed by centrifugation. Samples from supernatant (S) and pellet (P) of each group as well as the cytosolic protein fraction were blotted by the antibodies indicated to verify the depletion of the cytosolic proteins and the maintenance of membrane proteins in the pellet. It should be noted that FII likely contains most soluble E2s; thus, the supernatant fraction with FII added is Ube2k and Ube2n positive. (B) Precipitations of Ld HCs from the supernatant or the pellet fractions of three reaction groups described in A were performed using anti-Ld mAbs. The precipitates were blotted for newly formed Ub-Ld conjugates with anti-HA. An anti-Ld blot was included to show similar input in each group and as an Endo H treatment control. Data shown are representative of three independent experiments. (A and B) Molecular mass is indicated in kilodaltons. IB, immunoblot; IP, immunoprecipitation.
Mentions: To test whether the cellular E2 supporting mK3 ubiquitination of HC is membrane associated, a permeabilized cell system was used (Shamu et al., 1999). WT3 cells expressing mK3 ligase and its substrate Ld HCs were treated briefly with digitonin and then centrifuged to separate cytosolic proteins in solution from membrane proteins in the pellet. Next, pellets of permeabilized cells were resuspended with an energy-regenerating system (Feldman et al., 1997) and buffer containing E1, E1 + HA-Ub, or fraction II (FII; which contains E1, most E2s, some E3s, and the proteasome) + HA-Ub. After incubation, the reaction mixture was separated into the supernatant and pellet fractions. Samples from the supernatant and pellet fractions of these three experimental groups as well as samples from the cytosolic protein fraction were tested by immunoblot to verify the elimination of cytosolic proteins (Ube2n and Ube2k) and the retention of membrane proteins such as mK3 (Fig. 1 A). The de novo Ub conjugates on the HCs in these supernatant and pellet fractions were identified by immunoprecipitation of the HC and blotting for HA. To clearly distinguish the ubiquitinated HC from nonspecific bands, half of the HC precipitates were treated with endoglycosidase H (Endo H). This treatment cleaves the high mannose glycan moieties from immature HCs (nonubiquitinated as well as ubiquitinated), resulting in a down shift of corresponding HC bands in the gel. From the experiment shown in Fig. 1 B, four important conclusions can be drawn. First, Endo H–sensitive ubiquitinated HC bands were only observed in the samples incubated with HA-tagged Ub (lanes 7, 8, 11, and 12) but not in the samples without added HA-Ub (lanes 1–4). Thus, only newly ubiquitinated HCs were detected in the HA blots. Second, the pattern of the de novo ubiquitinated HCs in the permeabilized cells is the same as previously seen with nonpermeabilized cells (Wang et al., 2007), demonstrating recapitulation of what is observed in vivo. Third, the ubiquitinated HCs were only detected in the pellet, indicating that they were membrane bound, which is consistent with in vivo observations (Wang et al., 2007). Fourth, profound ubiquitination of HC was detected in the samples with only E1 (lanes 7 and 8), supporting the hypothesis that a cognate E2 of mK3 is membrane bound. Consistent with this conclusion, samples with added FII fraction (likely containing most soluble E2s) gave less robust ubiquitination of HC (lanes 11 and 12). Furthermore, by using the cells expressing a catalytically inactive RING mutant of mK3, we confirmed that ubiquitination in the permeabilized system was indeed mediated by mK3 (Fig. S1). Collectively, these data validate the approach and further suggest that a membrane-bound E2 can support mK3-mediated ubiquitination of HC.

Bottom Line: Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD).In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates.However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD). Until recently, ubiquitination of substrates was thought to occur only via isopeptide bonds, typically to lysine residues. Several recent studies suggest that Ub can also be coupled to nonlysine residues by ester/thiolester bonds; however, the molecular basis for these novel modifications remains elusive. To probe the mechanism and importance of nonlysine ubiquitination, we have studied the viral ligase murine K3 (mK3), which facilitates the polyubiquitination of hydroxylated amino acids serine/threonine on its ERAD substrate. In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates. However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

Show MeSH
Related in: MedlinePlus