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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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The active site cysteine of UbcM2 is required for the enzyme to be efficiently imported in the nucleus. BHK cells were micro-injected into the cytoplasm with a mixture of TRITC-labeled dextran (Inj marker; 1 mg/ml) plus either wt, C145S, or C145A UbcM2-GGH6 (9 μM). (A) Aliquots of the injection mixtures were solubilized, separated by SDS-PAGE, and the proteins were detected by CBB staining. Full-length proteins are denoted with an asterisk, and the migration of molecular size markers is indicated on the left. (B) After injection, cells were incubated for 20 min at 37°C and then imaged live by fluorescence microscopy. 50–75 cells were injected with each protein and representative cells are shown for wt (a and b), C145S (c and d), and C145A (e and f). Bar, 10 μm. (C) WT and C145S UbcM2-GGH6 were injected into cells maintained at 37°C on a heated stage and were imaged by time-lapse fluorescence microscopy for the time course indicated.
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fig2: The active site cysteine of UbcM2 is required for the enzyme to be efficiently imported in the nucleus. BHK cells were micro-injected into the cytoplasm with a mixture of TRITC-labeled dextran (Inj marker; 1 mg/ml) plus either wt, C145S, or C145A UbcM2-GGH6 (9 μM). (A) Aliquots of the injection mixtures were solubilized, separated by SDS-PAGE, and the proteins were detected by CBB staining. Full-length proteins are denoted with an asterisk, and the migration of molecular size markers is indicated on the left. (B) After injection, cells were incubated for 20 min at 37°C and then imaged live by fluorescence microscopy. 50–75 cells were injected with each protein and representative cells are shown for wt (a and b), C145S (c and d), and C145A (e and f). Bar, 10 μm. (C) WT and C145S UbcM2-GGH6 were injected into cells maintained at 37°C on a heated stage and were imaged by time-lapse fluorescence microscopy for the time course indicated.

Mentions: The data from Fig. 1 suggested that the C145S and C145A mutants would not be imported into the nuclei of cells by importin-11. We tested this prediction using a micro-injection assay. BHK cells were injected in the cytoplasm with equal amounts (Fig. 2 A) of either wt, C145S, or C145A UbcM2-GGH6, and after a 20-min incubation at 37°C the intracellular localization of the reporter proteins was examined in live cells by GFP fluorescence microscopy. Wt UbcM2 localized efficiently to the nuclei of injected cells (Fig. 2 B, a and b), whereas the mutants remained largely in the cytoplasm (Fig. 2 B, c–f). These findings were further confirmed in a parallel experiment using time-lapse fluorescence microscopy to compare the transport kinetics of wt UbcM2 and the C145S active site mutant (Fig. 2 C).


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)

The active site cysteine of UbcM2 is required for the enzyme to be efficiently imported in the nucleus. BHK cells were micro-injected into the cytoplasm with a mixture of TRITC-labeled dextran (Inj marker; 1 mg/ml) plus either wt, C145S, or C145A UbcM2-GGH6 (9 μM). (A) Aliquots of the injection mixtures were solubilized, separated by SDS-PAGE, and the proteins were detected by CBB staining. Full-length proteins are denoted with an asterisk, and the migration of molecular size markers is indicated on the left. (B) After injection, cells were incubated for 20 min at 37°C and then imaged live by fluorescence microscopy. 50–75 cells were injected with each protein and representative cells are shown for wt (a and b), C145S (c and d), and C145A (e and f). Bar, 10 μm. (C) WT and C145S UbcM2-GGH6 were injected into cells maintained at 37°C on a heated stage and were imaged by time-lapse fluorescence microscopy for the time course indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172591&req=5

fig2: The active site cysteine of UbcM2 is required for the enzyme to be efficiently imported in the nucleus. BHK cells were micro-injected into the cytoplasm with a mixture of TRITC-labeled dextran (Inj marker; 1 mg/ml) plus either wt, C145S, or C145A UbcM2-GGH6 (9 μM). (A) Aliquots of the injection mixtures were solubilized, separated by SDS-PAGE, and the proteins were detected by CBB staining. Full-length proteins are denoted with an asterisk, and the migration of molecular size markers is indicated on the left. (B) After injection, cells were incubated for 20 min at 37°C and then imaged live by fluorescence microscopy. 50–75 cells were injected with each protein and representative cells are shown for wt (a and b), C145S (c and d), and C145A (e and f). Bar, 10 μm. (C) WT and C145S UbcM2-GGH6 were injected into cells maintained at 37°C on a heated stage and were imaged by time-lapse fluorescence microscopy for the time course indicated.
Mentions: The data from Fig. 1 suggested that the C145S and C145A mutants would not be imported into the nuclei of cells by importin-11. We tested this prediction using a micro-injection assay. BHK cells were injected in the cytoplasm with equal amounts (Fig. 2 A) of either wt, C145S, or C145A UbcM2-GGH6, and after a 20-min incubation at 37°C the intracellular localization of the reporter proteins was examined in live cells by GFP fluorescence microscopy. Wt UbcM2 localized efficiently to the nuclei of injected cells (Fig. 2 B, a and b), whereas the mutants remained largely in the cytoplasm (Fig. 2 B, c–f). These findings were further confirmed in a parallel experiment using time-lapse fluorescence microscopy to compare the transport kinetics of wt UbcM2 and the C145S active site mutant (Fig. 2 C).

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