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Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme.

Moutty MC, Sakin V, Melchior F - Mol. Biol. Cell (2011)

Bottom Line: Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells.Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible.These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany. f.melchior@zmbh.uni-heidelberg.de

ABSTRACT
SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

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Importin β binds to Aos1 and to Uba2 via the adaptor importin α. Immobilized Aos1 wt and the NLS mutant Aos1-KR195, 196A2 (A) or Uba2 and the Uba2 mutant Uba2-KR623, 624A2 (B) were incubated with recombinant import factors importin α, β, α/β, transportin, importin 5, 7, or 13. Bound proteins were eluted by SDS sample buffer and compared with 20% of the input by SDS–PAGE and silverstaining. To control for specificity of binding, experiments were also performed in the presence of RanGTP, which interferes with formation of receptor–cargo complexes.
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Figure 3: Importin β binds to Aos1 and to Uba2 via the adaptor importin α. Immobilized Aos1 wt and the NLS mutant Aos1-KR195, 196A2 (A) or Uba2 and the Uba2 mutant Uba2-KR623, 624A2 (B) were incubated with recombinant import factors importin α, β, α/β, transportin, importin 5, 7, or 13. Bound proteins were eluted by SDS sample buffer and compared with 20% of the input by SDS–PAGE and silverstaining. To control for specificity of binding, experiments were also performed in the presence of RanGTP, which interferes with formation of receptor–cargo complexes.

Mentions: To identify transport receptors capable of recognizing the SUMO E1 subunits, we performed pull-down assays with His-CFP-Aos1 and Uba2-YFP-His immobilized on CNBr activated sepharose, and a variety of soluble import receptors (Figure 3, A and B). To control for specificity of the detected interactions, binding assays were carried out in the absence or presence of RanGTP. Inhibition of binding in the presence of RanGTP is characteristic for bona fide import receptor/cargo complexes (Rexach and Blobel, 1995; Izaurralde et al., 1997). To test whether the interactions depend on the putative NLSs identified in Aos1 and Uba2, binding was tested for both wt E1 subunits and for the import-deficient double mutants (Aos1-KR195, 196A2 and Uba2-KR623, 624A2). Figure 3A shows the analysis of Aos1 interactions: Heterodimeric importin α/β (Gorlich et al., 1995), transportin (Pollard et al., 1996), and importin 13 (Mingot et al., 2001) interacted with Aos1 in a RanGTP-sensitive manner. Importin β alone did not bind. Although importin 13 bound to wt and to mutant Aos1, transportin and importin α/β did not interact with the import-incompetent variant. Taken together, these findings pointed to both transportin and importin α/β as potentially relevant for import of Aos1 in HeLa cells; moreover, importin 13 could contribute to Aos1 import, even if it would not play a major role in HeLa cells. However, neither transportin nor importin 13 was capable of mediating import of CFP-Aos1 in vitro (Supplemental Figures 3 and 4A). Finally, we tested whether importin 13 could synergize with importin α/β to make transport more efficiently. Precedence for cooperation of import receptors comes, for example, from the finding that histone H1 can be imported by an importin β/importin 7 heterodimer (Jäkel et al., 1999). However, importin 13 impaired, rather than stimulated, CFP-Aos1 import under rate-limiting concentrations of importin α/β (Supplemental Figure 4B).


Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme.

Moutty MC, Sakin V, Melchior F - Mol. Biol. Cell (2011)

Importin β binds to Aos1 and to Uba2 via the adaptor importin α. Immobilized Aos1 wt and the NLS mutant Aos1-KR195, 196A2 (A) or Uba2 and the Uba2 mutant Uba2-KR623, 624A2 (B) were incubated with recombinant import factors importin α, β, α/β, transportin, importin 5, 7, or 13. Bound proteins were eluted by SDS sample buffer and compared with 20% of the input by SDS–PAGE and silverstaining. To control for specificity of binding, experiments were also performed in the presence of RanGTP, which interferes with formation of receptor–cargo complexes.
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Related In: Results  -  Collection

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Figure 3: Importin β binds to Aos1 and to Uba2 via the adaptor importin α. Immobilized Aos1 wt and the NLS mutant Aos1-KR195, 196A2 (A) or Uba2 and the Uba2 mutant Uba2-KR623, 624A2 (B) were incubated with recombinant import factors importin α, β, α/β, transportin, importin 5, 7, or 13. Bound proteins were eluted by SDS sample buffer and compared with 20% of the input by SDS–PAGE and silverstaining. To control for specificity of binding, experiments were also performed in the presence of RanGTP, which interferes with formation of receptor–cargo complexes.
Mentions: To identify transport receptors capable of recognizing the SUMO E1 subunits, we performed pull-down assays with His-CFP-Aos1 and Uba2-YFP-His immobilized on CNBr activated sepharose, and a variety of soluble import receptors (Figure 3, A and B). To control for specificity of the detected interactions, binding assays were carried out in the absence or presence of RanGTP. Inhibition of binding in the presence of RanGTP is characteristic for bona fide import receptor/cargo complexes (Rexach and Blobel, 1995; Izaurralde et al., 1997). To test whether the interactions depend on the putative NLSs identified in Aos1 and Uba2, binding was tested for both wt E1 subunits and for the import-deficient double mutants (Aos1-KR195, 196A2 and Uba2-KR623, 624A2). Figure 3A shows the analysis of Aos1 interactions: Heterodimeric importin α/β (Gorlich et al., 1995), transportin (Pollard et al., 1996), and importin 13 (Mingot et al., 2001) interacted with Aos1 in a RanGTP-sensitive manner. Importin β alone did not bind. Although importin 13 bound to wt and to mutant Aos1, transportin and importin α/β did not interact with the import-incompetent variant. Taken together, these findings pointed to both transportin and importin α/β as potentially relevant for import of Aos1 in HeLa cells; moreover, importin 13 could contribute to Aos1 import, even if it would not play a major role in HeLa cells. However, neither transportin nor importin 13 was capable of mediating import of CFP-Aos1 in vitro (Supplemental Figures 3 and 4A). Finally, we tested whether importin 13 could synergize with importin α/β to make transport more efficiently. Precedence for cooperation of import receptors comes, for example, from the finding that histone H1 can be imported by an importin β/importin 7 heterodimer (Jäkel et al., 1999). However, importin 13 impaired, rather than stimulated, CFP-Aos1 import under rate-limiting concentrations of importin α/β (Supplemental Figure 4B).

Bottom Line: Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells.Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible.These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany. f.melchior@zmbh.uni-heidelberg.de

ABSTRACT
SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

Show MeSH