Limits...
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 active site cysteine of UbcM2 is required for the interaction with importin-11. (A) HF7c (MATa) yeast expressing the indicated bait proteins (on left) as GAL4-DBD fusions were mated with W303α (MATα) strains expressing VP16 TA domain fusions (across top). Diploid yeast were selected on Leu−/Trp− plates and replica-plated onto Leu−/Trp−/His− plates with (L,W,H− + 3-AT) or without (L,W,H−) 3-amino triazole (3-AT). (B) Wt (lane 1), C145S (lane 2), or C145A (lane 3) UbcM2 fused to two GFPs and 6× His tag (UbcM2-GGH6) were mixed with Ni2+-agarose beads and a reticuloctye lysate containing 35S-labeled importin-11. Bound proteins (50% of bound) were separated by SDS-PAGE and detected by CBB staining (UbcM2-GGH6) or fluorography (35S-importin-11).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172591&req=5

fig1: The active site cysteine of UbcM2 is required for the interaction with importin-11. (A) HF7c (MATa) yeast expressing the indicated bait proteins (on left) as GAL4-DBD fusions were mated with W303α (MATα) strains expressing VP16 TA domain fusions (across top). Diploid yeast were selected on Leu−/Trp− plates and replica-plated onto Leu−/Trp−/His− plates with (L,W,H− + 3-AT) or without (L,W,H−) 3-amino triazole (3-AT). (B) Wt (lane 1), C145S (lane 2), or C145A (lane 3) UbcM2 fused to two GFPs and 6× His tag (UbcM2-GGH6) were mixed with Ni2+-agarose beads and a reticuloctye lysate containing 35S-labeled importin-11. Bound proteins (50% of bound) were separated by SDS-PAGE and detected by CBB staining (UbcM2-GGH6) or fluorography (35S-importin-11).

Mentions: The difficulty in detecting a robust interaction of UbcM2 with importin-11 in the absence of cell lysates suggested that UbcM2 might require a post-translational modification in order to be recognized as a transport cargo. One obvious modification is the attachment of Ub to the active site cysteine of the enzyme. As for all E2s, this cysteine receives activated Ub from E1 by forming a thiolester linkage with the COOH-terminal glycine of Ub (for review see Glickman and Ciechanover, 2002). To examine whether the activation state and nuclear transport of UbcM2 are coupled, we first tested the sensitivity of the UbcM2/importin-11 interaction to the integrity of the active site residue (Cys145). Active site mutants of UbcM2, bearing either a serine (C145S) or alanine (C145A) substitution, were expressed as fusions with the transactivation domain of VP16 and assayed for their ability to interact with a GAL4 DNA-binding domain fusion of human importin-11 in a yeast two-hybrid assay. Wild-type (wt) UbcM2 interacted with importin-11 in this assay, whereas the active site mutants did not (Fig. 1 A). In addition, importin-11 showed selectivity among E2s, as it bound the class III E2, UbcM2, but not the human class I E2, UbcH7. Expression of all E2s in this assay was confirmed by interactions with the RING finger domains of the putative E3 ligases, RNF5 or Ariadne-2.


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 interaction with importin-11. (A) HF7c (MATa) yeast expressing the indicated bait proteins (on left) as GAL4-DBD fusions were mated with W303α (MATα) strains expressing VP16 TA domain fusions (across top). Diploid yeast were selected on Leu−/Trp− plates and replica-plated onto Leu−/Trp−/His− plates with (L,W,H− + 3-AT) or without (L,W,H−) 3-amino triazole (3-AT). (B) Wt (lane 1), C145S (lane 2), or C145A (lane 3) UbcM2 fused to two GFPs and 6× His tag (UbcM2-GGH6) were mixed with Ni2+-agarose beads and a reticuloctye lysate containing 35S-labeled importin-11. Bound proteins (50% of bound) were separated by SDS-PAGE and detected by CBB staining (UbcM2-GGH6) or fluorography (35S-importin-11).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172591&req=5

fig1: The active site cysteine of UbcM2 is required for the interaction with importin-11. (A) HF7c (MATa) yeast expressing the indicated bait proteins (on left) as GAL4-DBD fusions were mated with W303α (MATα) strains expressing VP16 TA domain fusions (across top). Diploid yeast were selected on Leu−/Trp− plates and replica-plated onto Leu−/Trp−/His− plates with (L,W,H− + 3-AT) or without (L,W,H−) 3-amino triazole (3-AT). (B) Wt (lane 1), C145S (lane 2), or C145A (lane 3) UbcM2 fused to two GFPs and 6× His tag (UbcM2-GGH6) were mixed with Ni2+-agarose beads and a reticuloctye lysate containing 35S-labeled importin-11. Bound proteins (50% of bound) were separated by SDS-PAGE and detected by CBB staining (UbcM2-GGH6) or fluorography (35S-importin-11).
Mentions: The difficulty in detecting a robust interaction of UbcM2 with importin-11 in the absence of cell lysates suggested that UbcM2 might require a post-translational modification in order to be recognized as a transport cargo. One obvious modification is the attachment of Ub to the active site cysteine of the enzyme. As for all E2s, this cysteine receives activated Ub from E1 by forming a thiolester linkage with the COOH-terminal glycine of Ub (for review see Glickman and Ciechanover, 2002). To examine whether the activation state and nuclear transport of UbcM2 are coupled, we first tested the sensitivity of the UbcM2/importin-11 interaction to the integrity of the active site residue (Cys145). Active site mutants of UbcM2, bearing either a serine (C145S) or alanine (C145A) substitution, were expressed as fusions with the transactivation domain of VP16 and assayed for their ability to interact with a GAL4 DNA-binding domain fusion of human importin-11 in a yeast two-hybrid assay. Wild-type (wt) UbcM2 interacted with importin-11 in this assay, whereas the active site mutants did not (Fig. 1 A). In addition, importin-11 showed selectivity among E2s, as it bound the class III E2, UbcM2, but not the human class I E2, UbcH7. Expression of all E2s in this assay was confirmed by interactions with the RING finger domains of the putative E3 ligases, RNF5 or Ariadne-2.

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