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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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Inhibition of importin β prevents nuclear import of SUMO E1 holo-enzyme. Microinjection: Mixtures of CFP-Aos1/Uba2-YFP complex, TRITC-dextran, and either inhibitory anti–importin β antibody (+) or transport buffer (−) were injected into the cytoplasm of HeLa cells. After incubation for 30 min, cells were fixed and analyzed by fluorescence microscopy. Bar, 10 μm.
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Figure 7: Inhibition of importin β prevents nuclear import of SUMO E1 holo-enzyme. Microinjection: Mixtures of CFP-Aos1/Uba2-YFP complex, TRITC-dextran, and either inhibitory anti–importin β antibody (+) or transport buffer (−) were injected into the cytoplasm of HeLa cells. After incubation for 30 min, cells were fixed and analyzed by fluorescence microscopy. Bar, 10 μm.

Mentions: Finally, we wanted to test whether the importin α/β pathway is also the main import pathway for assembled E1 in intact cells. For this, we again turned to microinjection in the presence of inhibitory antibodies. Indeed, import of the assembled E1 complex (Figure 7) was efficiently blocked when anti–importin β antibodies were coinjected. This suggests that the importin α/β pathway is the major import pathway not only for single E1 subunits but also for the holo-enzyme.


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)

Inhibition of importin β prevents nuclear import of SUMO E1 holo-enzyme. Microinjection: Mixtures of CFP-Aos1/Uba2-YFP complex, TRITC-dextran, and either inhibitory anti–importin β antibody (+) or transport buffer (−) were injected into the cytoplasm of HeLa cells. After incubation for 30 min, cells were fixed and analyzed by fluorescence microscopy. Bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046061&req=5

Figure 7: Inhibition of importin β prevents nuclear import of SUMO E1 holo-enzyme. Microinjection: Mixtures of CFP-Aos1/Uba2-YFP complex, TRITC-dextran, and either inhibitory anti–importin β antibody (+) or transport buffer (−) were injected into the cytoplasm of HeLa cells. After incubation for 30 min, cells were fixed and analyzed by fluorescence microscopy. Bar, 10 μm.
Mentions: Finally, we wanted to test whether the importin α/β pathway is also the main import pathway for assembled E1 in intact cells. For this, we again turned to microinjection in the presence of inhibitory antibodies. Indeed, import of the assembled E1 complex (Figure 7) was efficiently blocked when anti–importin β antibodies were coinjected. This suggests that the importin α/β pathway is the major import pathway not only for single E1 subunits but also for the holo-enzyme.

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