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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.

Show MeSH
The enzymatic activity of E1 is required for the interaction of UbcM2 with importin-11. (A) 35S-importin-11–expressing reticulocyte lysates were pretreated with buffer (+ energy) or an ATP depletion mixture (hexo/glucose) for 30 min before being mixed with glutathione Sepharose beads and either GST-UbcM2 (lanes 1 and 2), GST (lanes 3 and 4), or GST-Ran (Q69L) (lanes 5 and 6). GST-Ran is marked with an asterisk. Bound and unbound proteins were resolved by SDS-PAGE and detected by CBB staining (GST fusions) or fluorography (35S-importin-11). (B) 35S-importin-11–expressing reticulocyte lysates were immunodepleted (lanes 1–4) or mock depleted (lanes 5–8) of E1 before being mixed with myc-UbcM2-H6 (UbcM2; lanes 1, 4, 5, and 8), Ran (Q69L)-H6 (Ran; lanes 2 and 6), or no protein (lanes 3 and 7) and Ni2+-agarose beads. Purified E1 was added back (lanes 4 and 8) to restore the binding interaction. Bead-associated proteins and unbound 35S-importin-11 were analyzed by SDS-PAGE and detected by CBB staining (UbcM2, Ran) or fluorography (35S-importin-11). (C) Anti-E1 Western blot of the E1 remaining (3% of lysate) in 35S-importin-11–expressing reticulocyte lysates after immunodepletion (E1 depletion) or mock depletion (Mock depletion). Bands corresponding to E1 is indicated with an arrow, and molecular size markers are denoted to the right of the blot. (D) 35S-importin-11–expressing reticulocyte lysates were immunodepleted of E1 and then supplemented with either buffer (lanes 1 and 4), enzymatically inactivated E1 (Iodoacet.; lanes 2 and 5), or mock-inactivated E1 (Mock; lanes 3 and 6). GST-UbcM2 is immobilized on glutathione Sepharose beads (lanes 1–3) and Ran (Q69L)-H6 is on Ni2+-agarose beads (lanes 4–6). Bound proteins were detected as in B and a Western blot of the E1 added to each lysate is also shown. For A, B, and D, bound represents 50% of bead-associated proteins and unbound represents 10% of proteins remaining in lysate.
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fig3: The enzymatic activity of E1 is required for the interaction of UbcM2 with importin-11. (A) 35S-importin-11–expressing reticulocyte lysates were pretreated with buffer (+ energy) or an ATP depletion mixture (hexo/glucose) for 30 min before being mixed with glutathione Sepharose beads and either GST-UbcM2 (lanes 1 and 2), GST (lanes 3 and 4), or GST-Ran (Q69L) (lanes 5 and 6). GST-Ran is marked with an asterisk. Bound and unbound proteins were resolved by SDS-PAGE and detected by CBB staining (GST fusions) or fluorography (35S-importin-11). (B) 35S-importin-11–expressing reticulocyte lysates were immunodepleted (lanes 1–4) or mock depleted (lanes 5–8) of E1 before being mixed with myc-UbcM2-H6 (UbcM2; lanes 1, 4, 5, and 8), Ran (Q69L)-H6 (Ran; lanes 2 and 6), or no protein (lanes 3 and 7) and Ni2+-agarose beads. Purified E1 was added back (lanes 4 and 8) to restore the binding interaction. Bead-associated proteins and unbound 35S-importin-11 were analyzed by SDS-PAGE and detected by CBB staining (UbcM2, Ran) or fluorography (35S-importin-11). (C) Anti-E1 Western blot of the E1 remaining (3% of lysate) in 35S-importin-11–expressing reticulocyte lysates after immunodepletion (E1 depletion) or mock depletion (Mock depletion). Bands corresponding to E1 is indicated with an arrow, and molecular size markers are denoted to the right of the blot. (D) 35S-importin-11–expressing reticulocyte lysates were immunodepleted of E1 and then supplemented with either buffer (lanes 1 and 4), enzymatically inactivated E1 (Iodoacet.; lanes 2 and 5), or mock-inactivated E1 (Mock; lanes 3 and 6). GST-UbcM2 is immobilized on glutathione Sepharose beads (lanes 1–3) and Ran (Q69L)-H6 is on Ni2+-agarose beads (lanes 4–6). Bound proteins were detected as in B and a Western blot of the E1 added to each lysate is also shown. For A, B, and D, bound represents 50% of bead-associated proteins and unbound represents 10% of proteins remaining in lysate.

Mentions: First, we tested the ATP dependence of complex formation. The initial step in the enzymatic activation of Ub by E1 involves ATP hydrolysis and the subsequent adenylation of Ub (Ciechanover et al., 1981, 1982; Haas and Rose, 1982; Haas et al., 1982, 1983). ATP depletion will inhibit Ub activation by E1 and thereby preclude the charging of UbcM2 with Ub. 35S-importin-11–containing reticulocyte lysates were pretreated either with a hexokinase/glucose ATP depletion mixture or were mock treated before being mixed with glutathione Sepharose beads and either GST-UbcM2, GST, or GST-Ran. We previously demonstrated that importin-11 binds the Ran GTPase (S.M. Plafker and Macara, 2000), so the binding of importin-11 to GST-Ran was included as a control to ensure that treatments of the lysate did not adversely affect the function of importin-11. Bound and unbound proteins were resolved by SDS-PAGE and detected by CBB staining or fluorography. We found that complex formation between UbcM2 and importin-11 was ATP dependent (Fig. 3 A, compare lane 1 with lane 2), whereas Ran binding to importin-11 was not affected by ATP depletion (Fig. 3 A, compare lane 5 with lane 6). As expected, 35S-importin-11 was not precipitated by GST alone (Fig. 3 A, lanes 3 and 4). These results establish that ATP is required for the UbcM2/importin-11 interaction and are consistent with a requirement for E1 activity.


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 enzymatic activity of E1 is required for the interaction of UbcM2 with importin-11. (A) 35S-importin-11–expressing reticulocyte lysates were pretreated with buffer (+ energy) or an ATP depletion mixture (hexo/glucose) for 30 min before being mixed with glutathione Sepharose beads and either GST-UbcM2 (lanes 1 and 2), GST (lanes 3 and 4), or GST-Ran (Q69L) (lanes 5 and 6). GST-Ran is marked with an asterisk. Bound and unbound proteins were resolved by SDS-PAGE and detected by CBB staining (GST fusions) or fluorography (35S-importin-11). (B) 35S-importin-11–expressing reticulocyte lysates were immunodepleted (lanes 1–4) or mock depleted (lanes 5–8) of E1 before being mixed with myc-UbcM2-H6 (UbcM2; lanes 1, 4, 5, and 8), Ran (Q69L)-H6 (Ran; lanes 2 and 6), or no protein (lanes 3 and 7) and Ni2+-agarose beads. Purified E1 was added back (lanes 4 and 8) to restore the binding interaction. Bead-associated proteins and unbound 35S-importin-11 were analyzed by SDS-PAGE and detected by CBB staining (UbcM2, Ran) or fluorography (35S-importin-11). (C) Anti-E1 Western blot of the E1 remaining (3% of lysate) in 35S-importin-11–expressing reticulocyte lysates after immunodepletion (E1 depletion) or mock depletion (Mock depletion). Bands corresponding to E1 is indicated with an arrow, and molecular size markers are denoted to the right of the blot. (D) 35S-importin-11–expressing reticulocyte lysates were immunodepleted of E1 and then supplemented with either buffer (lanes 1 and 4), enzymatically inactivated E1 (Iodoacet.; lanes 2 and 5), or mock-inactivated E1 (Mock; lanes 3 and 6). GST-UbcM2 is immobilized on glutathione Sepharose beads (lanes 1–3) and Ran (Q69L)-H6 is on Ni2+-agarose beads (lanes 4–6). Bound proteins were detected as in B and a Western blot of the E1 added to each lysate is also shown. For A, B, and D, bound represents 50% of bead-associated proteins and unbound represents 10% of proteins remaining in lysate.
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Related In: Results  -  Collection

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fig3: The enzymatic activity of E1 is required for the interaction of UbcM2 with importin-11. (A) 35S-importin-11–expressing reticulocyte lysates were pretreated with buffer (+ energy) or an ATP depletion mixture (hexo/glucose) for 30 min before being mixed with glutathione Sepharose beads and either GST-UbcM2 (lanes 1 and 2), GST (lanes 3 and 4), or GST-Ran (Q69L) (lanes 5 and 6). GST-Ran is marked with an asterisk. Bound and unbound proteins were resolved by SDS-PAGE and detected by CBB staining (GST fusions) or fluorography (35S-importin-11). (B) 35S-importin-11–expressing reticulocyte lysates were immunodepleted (lanes 1–4) or mock depleted (lanes 5–8) of E1 before being mixed with myc-UbcM2-H6 (UbcM2; lanes 1, 4, 5, and 8), Ran (Q69L)-H6 (Ran; lanes 2 and 6), or no protein (lanes 3 and 7) and Ni2+-agarose beads. Purified E1 was added back (lanes 4 and 8) to restore the binding interaction. Bead-associated proteins and unbound 35S-importin-11 were analyzed by SDS-PAGE and detected by CBB staining (UbcM2, Ran) or fluorography (35S-importin-11). (C) Anti-E1 Western blot of the E1 remaining (3% of lysate) in 35S-importin-11–expressing reticulocyte lysates after immunodepletion (E1 depletion) or mock depletion (Mock depletion). Bands corresponding to E1 is indicated with an arrow, and molecular size markers are denoted to the right of the blot. (D) 35S-importin-11–expressing reticulocyte lysates were immunodepleted of E1 and then supplemented with either buffer (lanes 1 and 4), enzymatically inactivated E1 (Iodoacet.; lanes 2 and 5), or mock-inactivated E1 (Mock; lanes 3 and 6). GST-UbcM2 is immobilized on glutathione Sepharose beads (lanes 1–3) and Ran (Q69L)-H6 is on Ni2+-agarose beads (lanes 4–6). Bound proteins were detected as in B and a Western blot of the E1 added to each lysate is also shown. For A, B, and D, bound represents 50% of bead-associated proteins and unbound represents 10% of proteins remaining in lysate.
Mentions: First, we tested the ATP dependence of complex formation. The initial step in the enzymatic activation of Ub by E1 involves ATP hydrolysis and the subsequent adenylation of Ub (Ciechanover et al., 1981, 1982; Haas and Rose, 1982; Haas et al., 1982, 1983). ATP depletion will inhibit Ub activation by E1 and thereby preclude the charging of UbcM2 with Ub. 35S-importin-11–containing reticulocyte lysates were pretreated either with a hexokinase/glucose ATP depletion mixture or were mock treated before being mixed with glutathione Sepharose beads and either GST-UbcM2, GST, or GST-Ran. We previously demonstrated that importin-11 binds the Ran GTPase (S.M. Plafker and Macara, 2000), so the binding of importin-11 to GST-Ran was included as a control to ensure that treatments of the lysate did not adversely affect the function of importin-11. Bound and unbound proteins were resolved by SDS-PAGE and detected by CBB staining or fluorography. We found that complex formation between UbcM2 and importin-11 was ATP dependent (Fig. 3 A, compare lane 1 with lane 2), whereas Ran binding to importin-11 was not affected by ATP depletion (Fig. 3 A, compare lane 5 with lane 6). As expected, 35S-importin-11 was not precipitated by GST alone (Fig. 3 A, lanes 3 and 4). These results establish that ATP is required for the UbcM2/importin-11 interaction and are consistent with a requirement for E1 activity.

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