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Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.

Plafker SM, Plafker KS, Weissman AM, Macara IG - J. Cell Biol. (2004)

Bottom Line: We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11.This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes.Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. scott-plafker@ouhsc.edu

ABSTRACT
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

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Endogenous UbcH6 and UbcM2 are resident nuclear enzymes. (A) Mouse embryonic fibroblasts, from a 12.5-d mouse embryo, and HeLa cells were fixed, permeabilized, and immunostained for endogenous UbcH6 using an anti-UbcH6 antibody and a goat anti–rabbit-Alexa546nm secondary antibody (a and e). The specificity of the immunostaining was verified by blocking the anti-UbcH6 antibody with recombinant UbcH6 (c and g). (B) HeLa cells stained with an anti-UbcM2 antibody (i). Nuclei were counterstained with DAPI (b, d, f, h, and j). (C) HeLa whole-cell extracts probed with an anti-UbcM2–specific antibody followed by a goat anti–rabbit-HRP conjugate and ECL. The antibody detects a primary band at the estimated size for UbcM2 and a faint, slower migrating band.
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fig6: Endogenous UbcH6 and UbcM2 are resident nuclear enzymes. (A) Mouse embryonic fibroblasts, from a 12.5-d mouse embryo, and HeLa cells were fixed, permeabilized, and immunostained for endogenous UbcH6 using an anti-UbcH6 antibody and a goat anti–rabbit-Alexa546nm secondary antibody (a and e). The specificity of the immunostaining was verified by blocking the anti-UbcH6 antibody with recombinant UbcH6 (c and g). (B) HeLa cells stained with an anti-UbcM2 antibody (i). Nuclei were counterstained with DAPI (b, d, f, h, and j). (C) HeLa whole-cell extracts probed with an anti-UbcM2–specific antibody followed by a goat anti–rabbit-HRP conjugate and ECL. The antibody detects a primary band at the estimated size for UbcM2 and a faint, slower migrating band.

Mentions: To determine if endogenous class III E2s are resident nuclear proteins, we examined the subcellular distribution of UbcH6 and UbcM2. HeLa and 12-d mouse embryonic fibroblasts were fixed, permeabilized, and exposed to anti-UbcH6 antibodies and an Alexa546nm-conjugated anti–rabbit secondary antibody. The distribution of UbcH6 was then assessed by fluorescence microscopy. In both cell types, UbcH6 immunostaining revealed a nuclear distribution (Fig. 6 A, a and e), and this nuclear signal was ablated by coincubation of the antibody with recombinant UbcH6 (Fig. 6 A, c and g). Similarly, immunostaining of HeLa cells with an antibody against the unique NH2-terminal extension of UbcM2 revealed that this enzyme also resides in the nucleus (Fig. 6 B, i and j).


Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.

Plafker SM, Plafker KS, Weissman AM, Macara IG - J. Cell Biol. (2004)

Endogenous UbcH6 and UbcM2 are resident nuclear enzymes. (A) Mouse embryonic fibroblasts, from a 12.5-d mouse embryo, and HeLa cells were fixed, permeabilized, and immunostained for endogenous UbcH6 using an anti-UbcH6 antibody and a goat anti–rabbit-Alexa546nm secondary antibody (a and e). The specificity of the immunostaining was verified by blocking the anti-UbcH6 antibody with recombinant UbcH6 (c and g). (B) HeLa cells stained with an anti-UbcM2 antibody (i). Nuclei were counterstained with DAPI (b, d, f, h, and j). (C) HeLa whole-cell extracts probed with an anti-UbcM2–specific antibody followed by a goat anti–rabbit-HRP conjugate and ECL. The antibody detects a primary band at the estimated size for UbcM2 and a faint, slower migrating band.
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fig6: Endogenous UbcH6 and UbcM2 are resident nuclear enzymes. (A) Mouse embryonic fibroblasts, from a 12.5-d mouse embryo, and HeLa cells were fixed, permeabilized, and immunostained for endogenous UbcH6 using an anti-UbcH6 antibody and a goat anti–rabbit-Alexa546nm secondary antibody (a and e). The specificity of the immunostaining was verified by blocking the anti-UbcH6 antibody with recombinant UbcH6 (c and g). (B) HeLa cells stained with an anti-UbcM2 antibody (i). Nuclei were counterstained with DAPI (b, d, f, h, and j). (C) HeLa whole-cell extracts probed with an anti-UbcM2–specific antibody followed by a goat anti–rabbit-HRP conjugate and ECL. The antibody detects a primary band at the estimated size for UbcM2 and a faint, slower migrating band.
Mentions: To determine if endogenous class III E2s are resident nuclear proteins, we examined the subcellular distribution of UbcH6 and UbcM2. HeLa and 12-d mouse embryonic fibroblasts were fixed, permeabilized, and exposed to anti-UbcH6 antibodies and an Alexa546nm-conjugated anti–rabbit secondary antibody. The distribution of UbcH6 was then assessed by fluorescence microscopy. In both cell types, UbcH6 immunostaining revealed a nuclear distribution (Fig. 6 A, a and e), and this nuclear signal was ablated by coincubation of the antibody with recombinant UbcH6 (Fig. 6 A, c and g). Similarly, immunostaining of HeLa cells with an antibody against the unique NH2-terminal extension of UbcM2 revealed that this enzyme also resides in the nucleus (Fig. 6 B, i and j).

Bottom Line: We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11.This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes.Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. scott-plafker@ouhsc.edu

ABSTRACT
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

Show MeSH
Related in: MedlinePlus