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
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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172591&req=5

fig4: 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.

Mentions: We used coimmunoprecipitation to examine if importin-11 has a binding preference for Ub-loaded UbcM2 versus the unloaded enzyme. Human embryonic kidney (HEK) 293T cells were cotransfected with plasmids expressing HA3-tagged importin-11 and myc-tagged E2s (i.e., wt UbcM2, C145S UbcM2, and C145A UbcM2). Transfected cells were collected and lysed in a detergent-containing buffer lacking reducing agents. The resulting lysates were sequentially exposed to 12CA5 mAbs and protein A–Sepharose beads to precipitate HA3-importin-11 and any associated myc-tagged UbcM2. Fractions of the bead-bound and unbound tagged proteins were then analyzed in parallel by nonreducing and reducing SDS-PAGE followed by Western blotting. Nonreducing conditions were used to maintain the thiolester linkage between the active site cysteine of UbcM2 and the COOH-terminal glycine of Ub. Addition of β-mercaptoethanol (β-ME) reduces the thiolester bond and results in removal of Ub from the active site, as illustrated by collapse of the slower migrating 33-kD myc-UbcM2 band to the faster migrating 25-kD band (Fig. 4 A, compare lane 6 with lane 12).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: 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.
Mentions: We used coimmunoprecipitation to examine if importin-11 has a binding preference for Ub-loaded UbcM2 versus the unloaded enzyme. Human embryonic kidney (HEK) 293T cells were cotransfected with plasmids expressing HA3-tagged importin-11 and myc-tagged E2s (i.e., wt UbcM2, C145S UbcM2, and C145A UbcM2). Transfected cells were collected and lysed in a detergent-containing buffer lacking reducing agents. The resulting lysates were sequentially exposed to 12CA5 mAbs and protein A–Sepharose beads to precipitate HA3-importin-11 and any associated myc-tagged UbcM2. Fractions of the bead-bound and unbound tagged proteins were then analyzed in parallel by nonreducing and reducing SDS-PAGE followed by Western blotting. Nonreducing conditions were used to maintain the thiolester linkage between the active site cysteine of UbcM2 and the COOH-terminal glycine of Ub. Addition of β-mercaptoethanol (β-ME) reduces the thiolester bond and results in removal of Ub from the active site, as illustrated by collapse of the slower migrating 33-kD myc-UbcM2 band to the faster migrating 25-kD band (Fig. 4 A, compare lane 6 with lane 12).

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