Limits...
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.

Show MeSH
Aos1 and Uba2 are independently imported into HeLa cell nuclei. (A) In vitro import of CFP-Aos1 or Uba2-YFP using semipermeable HeLa cells and cytosolic extract. Nuclear accumulation of wt proteins (CFP-Aos1-wt, Uba2-wt-YFP) and of NLS mutants (CFP-Aos1-KR195, 196A2, Uba2-KR623, 624A2-YFP) was analyzed by fluorescence microscopy. +ATP, in the presence of ATP-regenerating system; –ATP, in the presence of ATP-depleting system. Bar,10 μm. (B) Microinjection of CFP-Aos1 or Uba2-YFP into the cytoplasm of HeLa cells. Wild-type proteins, Aos1-KR195, 196A2, and Uba2-KR623, 624A2 were injected together with TRITC-dextran, incubated for 30 min, fixed, and analyzed by fluorescence microscopy. Bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3046061&req=5

Figure 2: Aos1 and Uba2 are independently imported into HeLa cell nuclei. (A) In vitro import of CFP-Aos1 or Uba2-YFP using semipermeable HeLa cells and cytosolic extract. Nuclear accumulation of wt proteins (CFP-Aos1-wt, Uba2-wt-YFP) and of NLS mutants (CFP-Aos1-KR195, 196A2, Uba2-KR623, 624A2-YFP) was analyzed by fluorescence microscopy. +ATP, in the presence of ATP-regenerating system; –ATP, in the presence of ATP-depleting system. Bar,10 μm. (B) Microinjection of CFP-Aos1 or Uba2-YFP into the cytoplasm of HeLa cells. Wild-type proteins, Aos1-KR195, 196A2, and Uba2-KR623, 624A2 were injected together with TRITC-dextran, incubated for 30 min, fixed, and analyzed by fluorescence microscopy. Bar, 10 μm.

Mentions: Intracellular distribution of proteins 24 h after transfection reflects a steady-state situation that does not allow conclusions about efficiency of nuclear import. Moreover, multiple point mutations may cause folding problems that may influence import indirectly or artificially. We therefore turned next to in vitro nuclear import assays (Adam et al., 1990). For this, we generated recombinant CFP-Aos1 and Uba2-YFP wild-type (wt) and mutant variants from bacteria. Wild-type and mutant proteins behaved identically during expression and purification, which included gel filtration, excluding significant folding defects. On purification, they were analyzed for their accumulation in nuclei of semipermeable HeLa cells in the presence of cytosol and ATP. As shown in Figure 2A, wt proteins were efficiently imported in dependence of ATP, but the double mutants CFP-Aos1-KR195, 196A2 and Uba2-KR623, 624A2-YFP both failed to be imported.


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)

Aos1 and Uba2 are independently imported into HeLa cell nuclei. (A) In vitro import of CFP-Aos1 or Uba2-YFP using semipermeable HeLa cells and cytosolic extract. Nuclear accumulation of wt proteins (CFP-Aos1-wt, Uba2-wt-YFP) and of NLS mutants (CFP-Aos1-KR195, 196A2, Uba2-KR623, 624A2-YFP) was analyzed by fluorescence microscopy. +ATP, in the presence of ATP-regenerating system; –ATP, in the presence of ATP-depleting system. Bar,10 μm. (B) Microinjection of CFP-Aos1 or Uba2-YFP into the cytoplasm of HeLa cells. Wild-type proteins, Aos1-KR195, 196A2, and Uba2-KR623, 624A2 were injected together with TRITC-dextran, incubated for 30 min, 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 2: Aos1 and Uba2 are independently imported into HeLa cell nuclei. (A) In vitro import of CFP-Aos1 or Uba2-YFP using semipermeable HeLa cells and cytosolic extract. Nuclear accumulation of wt proteins (CFP-Aos1-wt, Uba2-wt-YFP) and of NLS mutants (CFP-Aos1-KR195, 196A2, Uba2-KR623, 624A2-YFP) was analyzed by fluorescence microscopy. +ATP, in the presence of ATP-regenerating system; –ATP, in the presence of ATP-depleting system. Bar,10 μm. (B) Microinjection of CFP-Aos1 or Uba2-YFP into the cytoplasm of HeLa cells. Wild-type proteins, Aos1-KR195, 196A2, and Uba2-KR623, 624A2 were injected together with TRITC-dextran, incubated for 30 min, fixed, and analyzed by fluorescence microscopy. Bar, 10 μm.
Mentions: Intracellular distribution of proteins 24 h after transfection reflects a steady-state situation that does not allow conclusions about efficiency of nuclear import. Moreover, multiple point mutations may cause folding problems that may influence import indirectly or artificially. We therefore turned next to in vitro nuclear import assays (Adam et al., 1990). For this, we generated recombinant CFP-Aos1 and Uba2-YFP wild-type (wt) and mutant variants from bacteria. Wild-type and mutant proteins behaved identically during expression and purification, which included gel filtration, excluding significant folding defects. On purification, they were analyzed for their accumulation in nuclei of semipermeable HeLa cells in the presence of cytosol and ATP. As shown in Figure 2A, wt proteins were efficiently imported in dependence of ATP, but the double mutants CFP-Aos1-KR195, 196A2 and Uba2-KR623, 624A2-YFP both failed to be imported.

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