Limits...
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.

Show MeSH

Related in: MedlinePlus

SUMO-1 modification is required for localization of RanGAP1 at the  NPC. (A) HeLa cells were transfected with  plasmids expressing myc-tagged wild-type  RanGAP1 (a) or myc-tagged RanGAP1  containing a single lysine to arginine substitution at amino acid 526 (b). Subcellular  localizations were determined 36 h after  transfection by indirect immunofluorescence using the anti-myc monoclonal antibody, 9E10. (B) Cells were lysed in SDS  sample buffer 36 h after transfection and  analyzed by immunoblot analysis with the  anti-myc monoclonal antibody, 9E10.  Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140169&req=5

Figure 2: SUMO-1 modification is required for localization of RanGAP1 at the NPC. (A) HeLa cells were transfected with plasmids expressing myc-tagged wild-type RanGAP1 (a) or myc-tagged RanGAP1 containing a single lysine to arginine substitution at amino acid 526 (b). Subcellular localizations were determined 36 h after transfection by indirect immunofluorescence using the anti-myc monoclonal antibody, 9E10. (B) Cells were lysed in SDS sample buffer 36 h after transfection and analyzed by immunoblot analysis with the anti-myc monoclonal antibody, 9E10. Bar, 10 μm.

Mentions: To analyze the effects of SUMO-1 modification on RanGAP1 localization, we next transfected cells with plasmids coding for either wild-type RanGAP1 or for mutant RanGAP1 with the lysine to arginine substitution at residue 526 (K/R 526). Both proteins were designed to contain a myc epitope tag at their NH2 terminus, which was used for immunolocalization and for immunoblot analysis. Transiently expressed wild-type protein showed a pattern of localization similar to the endogenous RanGAP1; it was detected both in the cytoplasm and concentrated at the nuclear envelope (Fig. 2 A, a). Discontinuous rim staining at the equatorial plane of the nuclear envelope, and punctate staining on the surface indicated association with NPCs. In cells expressing increasing amounts of RanGAP1, NPC labeling remained constant while the cytoplasmic signal increased, indicating that NPC binding sites were saturated. In contrast to wild-type RanGAP1, the K/R 526 mutant form of RanGAP1 was confined strictly to the cytoplasm and showed no evidence of association with the nuclear envelope or NPCs even at the lowest levels of expression (Fig. 2 A, b). No signal was detected in untransfected cells (data not shown).


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)

SUMO-1 modification is required for localization of RanGAP1 at the  NPC. (A) HeLa cells were transfected with  plasmids expressing myc-tagged wild-type  RanGAP1 (a) or myc-tagged RanGAP1  containing a single lysine to arginine substitution at amino acid 526 (b). Subcellular  localizations were determined 36 h after  transfection by indirect immunofluorescence using the anti-myc monoclonal antibody, 9E10. (B) Cells were lysed in SDS  sample buffer 36 h after transfection and  analyzed by immunoblot analysis with the  anti-myc monoclonal antibody, 9E10.  Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: SUMO-1 modification is required for localization of RanGAP1 at the NPC. (A) HeLa cells were transfected with plasmids expressing myc-tagged wild-type RanGAP1 (a) or myc-tagged RanGAP1 containing a single lysine to arginine substitution at amino acid 526 (b). Subcellular localizations were determined 36 h after transfection by indirect immunofluorescence using the anti-myc monoclonal antibody, 9E10. (B) Cells were lysed in SDS sample buffer 36 h after transfection and analyzed by immunoblot analysis with the anti-myc monoclonal antibody, 9E10. Bar, 10 μm.
Mentions: To analyze the effects of SUMO-1 modification on RanGAP1 localization, we next transfected cells with plasmids coding for either wild-type RanGAP1 or for mutant RanGAP1 with the lysine to arginine substitution at residue 526 (K/R 526). Both proteins were designed to contain a myc epitope tag at their NH2 terminus, which was used for immunolocalization and for immunoblot analysis. Transiently expressed wild-type protein showed a pattern of localization similar to the endogenous RanGAP1; it was detected both in the cytoplasm and concentrated at the nuclear envelope (Fig. 2 A, a). Discontinuous rim staining at the equatorial plane of the nuclear envelope, and punctate staining on the surface indicated association with NPCs. In cells expressing increasing amounts of RanGAP1, NPC labeling remained constant while the cytoplasmic signal increased, indicating that NPC binding sites were saturated. In contrast to wild-type RanGAP1, the K/R 526 mutant form of RanGAP1 was confined strictly to the cytoplasm and showed no evidence of association with the nuclear envelope or NPCs even at the lowest levels of expression (Fig. 2 A, b). No signal was detected in untransfected cells (data not shown).

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
Related in: MedlinePlus