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SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex.

Matunis MJ, Wu J, Blobel G - J. Cell Biol. (1998)

Bottom Line: SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four.Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal.This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York 10021, USA. Matunim@rockvax.rockefeller.edu

ABSTRACT
RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

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Pyruvate kinase is  targeted to the NPC by  SUMO-1 modification and  by additional determinants in  the COOH-terminal domain  of RanGAP1. Plasmids coding for the proteins illustrated in Fig. 3 A were transfected into HeLa cells, and  the transiently expressed  proteins we immunolocalized  with the anti-myc monoclonal antibody, 9E10. Representative micrographs of  the observed immunolocalizations are presented. Bar,  10 μm.
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Figure 4: Pyruvate kinase is targeted to the NPC by SUMO-1 modification and by additional determinants in the COOH-terminal domain of RanGAP1. Plasmids coding for the proteins illustrated in Fig. 3 A were transfected into HeLa cells, and the transiently expressed proteins we immunolocalized with the anti-myc monoclonal antibody, 9E10. Representative micrographs of the observed immunolocalizations are presented. Bar, 10 μm.

Mentions: To investigate the determinants specifying NPC localization further, the constructs described above were transfected into HeLa cells, and the transiently expressed proteins were localized by indirect immunofluorescence. As evident in Fig. 4 a, pyruvate kinase was localized in the cytoplasm and showed no detectable nuclear envelope staining. CΔ23, which is not a substrate for SUMO-1 modification, was also strictly cytosolic (Fig. 4 b). In contrast, NΔ419/PK was concentrated at the nuclear envelope in a pattern consistent with NPC binding and also in the nucleus (Fig. 4 c). NΔ470/PK was also localized to the nucleus but showed no evidence of NPC binding, despite the fact that it is predicted to be modified by SUMO-1 (Fig. 4 d). NΔ502/PK showed a similar intranuclear localization and no NPC staining (Fig. 4 e). These results indicate that SUMO-1 modification is required, but not sufficient, for RanGAP1 localization at the NPC. This conclusion is further supported by the finding that pyruvate kinase, expressed as a fusion protein with SUMO-1, was strictly cytosolic (Fig. 4 f). The difference in localization between NΔ419 and NΔ470 indicate that the 50 amino acid residues between 419 and 470 are also required for localization of RanGAP1 at the NPC, in addition to SUMO-1 modification.


SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex.

Matunis MJ, Wu J, Blobel G - J. Cell Biol. (1998)

Pyruvate kinase is  targeted to the NPC by  SUMO-1 modification and  by additional determinants in  the COOH-terminal domain  of RanGAP1. Plasmids coding for the proteins illustrated in Fig. 3 A were transfected into HeLa cells, and  the transiently expressed  proteins we immunolocalized  with the anti-myc monoclonal antibody, 9E10. Representative micrographs of  the observed immunolocalizations are presented. Bar,  10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140169&req=5

Figure 4: Pyruvate kinase is targeted to the NPC by SUMO-1 modification and by additional determinants in the COOH-terminal domain of RanGAP1. Plasmids coding for the proteins illustrated in Fig. 3 A were transfected into HeLa cells, and the transiently expressed proteins we immunolocalized with the anti-myc monoclonal antibody, 9E10. Representative micrographs of the observed immunolocalizations are presented. Bar, 10 μm.
Mentions: To investigate the determinants specifying NPC localization further, the constructs described above were transfected into HeLa cells, and the transiently expressed proteins were localized by indirect immunofluorescence. As evident in Fig. 4 a, pyruvate kinase was localized in the cytoplasm and showed no detectable nuclear envelope staining. CΔ23, which is not a substrate for SUMO-1 modification, was also strictly cytosolic (Fig. 4 b). In contrast, NΔ419/PK was concentrated at the nuclear envelope in a pattern consistent with NPC binding and also in the nucleus (Fig. 4 c). NΔ470/PK was also localized to the nucleus but showed no evidence of NPC binding, despite the fact that it is predicted to be modified by SUMO-1 (Fig. 4 d). NΔ502/PK showed a similar intranuclear localization and no NPC staining (Fig. 4 e). These results indicate that SUMO-1 modification is required, but not sufficient, for RanGAP1 localization at the NPC. This conclusion is further supported by the finding that pyruvate kinase, expressed as a fusion protein with SUMO-1, was strictly cytosolic (Fig. 4 f). The difference in localization between NΔ419 and NΔ470 indicate that the 50 amino acid residues between 419 and 470 are also required for localization of RanGAP1 at the NPC, in addition to SUMO-1 modification.

Bottom Line: SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four.Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal.This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York 10021, USA. Matunim@rockvax.rockefeller.edu

ABSTRACT
RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

Show MeSH