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).

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.

Class III E2s can access the nucleus by the same import pathway. BHK cells were microinjected in the cytoplasm with TRITC-labeled dextran (Inj marker; 1mg/ml), GFP-UbcH6-H₆ (A), or GFP-UBE2E2-H₆ (B), and a 20-molar excess of GST-UbcM2 (C145A) (a, b, e, and f) or GST-UbcM2 (wt) (c, d, g, and h). After injection, cells were incubated for 15 min at 37°C and analyzed live by fluorescence microscopy. 50–75 cells were injected for each condition. Bar, 10 μm. (C) Cells injected with GST-UbcM2 (C145A) (i–k) or GST-UbcM2 (wt) (l–n) were incubated for 30 min at 37°C and fixed. The localization of the GST fusions was determined by indirect immunofluorescence using an anti-GST antibody and a FITC-conjugated secondary antibody. DNA was stained with DAPI to mark the nuclear compartment for each cell.

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.

Importin-11 interacts in vivo with the Ub-charged forms of class III E2 enzymes. (A) Transfected HEK293T cells expressing HA³-importin-11 and myc-tagged UbcM2 (wt, C145A, or C145S) were harvested under nonreducing conditions and exposed to 12CA5 antibody and protein A–Sepharose beads to precipitate HA³-importin-11 and any associated myc-tagged UbcM2. Bead-associated and unbound proteins were separated by both nonreducing and reducing SDS-PAGE and detected by Western blotting with 12CA5-HRP (anti-HA blot) or anti-myc-HRP (anti-Myc blot) conjugates and ECL. Ub-charged UbcM2 migrates more slowly than its uncharged counterpart in nonreducing SDS-PAGE (lane 6). Under reducing conditions, Ub is readily removed from wt UbcM2, but not from the C145S mutant (wt, lanes 6 and 12 vs. C145S, lanes 5 and 11). The migration of molecular size markers is indicated to the right of the blots. (B) Lysates from transfected HEK cells expressing HA³-importin-11 were mixed with recombinant (C145S) UbcM2 not loaded (lanes 1 and 4) or preloaded with Ub (lanes 2, 3, 5, and 6). Precipitation of the HA³-importin-11 and any bound, recombinant (C145S) UbcM2 was then done as described in A, except that one lysate (lane 5) was spiked with (Q69L) Ran before 12CA5 addition. Samples were resolved by reducing SDS-PAGE, and HA³-importin-11 and (C145S) UbcM2 were detected with a 12CA5 antibody (Anti-HA blot) or an anti-UbcM2 antibody (Anti-UbcM2 blot), respectively. 75% of bound (lanes 4–6) and 5% of unbound (lanes 1–3) are shown. The migration of molecular size markers are indicated to the right. Ub-charged (C145S) UbcM2 (H-S-UbcM2 (C145S)~Ub) migrates more slowly than the uncharged enzyme (H-S-UbcM2 (C145S)). (C) Same experiment as in A, except that in place of the UbcM2 mutants, myc-tagged forms of UbcH6, UBE2E2, and UbcH7 were each coexpressed with HA³-importin-11. The Ub-charged form of each E2 is marked with an asterisk. For experiments A and C, bound represents 50% of total and unbound represents 10% of total.

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-Alexa_546nm 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.