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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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RanGAP1 contains nine potential nuclear export signals in addition to a nuclear localization signal. (A) Schematic  representation of RanGAP1 indicating the positions of the nine  NH2-terminal nuclear export motifs and the COOH-terminal nuclear localization signal. (B) Amino acid sequence of the nuclear  localization signal in the COOH-terminal domain of RanGAP1.  (C) Amino acid sequences and alignments of the nine putative  nuclear export motifs in the NH2-terminal domain of RanGAP1  and the nuclear export motifs of Rev (Fischer et al., 1995), PKI  (Wen et al., 1995), IκB, and Rex (Fritz and Green, 1996). Hydrophobic residues comprising the core of the motif are highlighted  in dark gray, and hydrophobic residues outside of the core are  highlighted in light gray.
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Figure 7: RanGAP1 contains nine potential nuclear export signals in addition to a nuclear localization signal. (A) Schematic representation of RanGAP1 indicating the positions of the nine NH2-terminal nuclear export motifs and the COOH-terminal nuclear localization signal. (B) Amino acid sequence of the nuclear localization signal in the COOH-terminal domain of RanGAP1. (C) Amino acid sequences and alignments of the nine putative nuclear export motifs in the NH2-terminal domain of RanGAP1 and the nuclear export motifs of Rev (Fischer et al., 1995), PKI (Wen et al., 1995), IκB, and Rex (Fritz and Green, 1996). Hydrophobic residues comprising the core of the motif are highlighted in dark gray, and hydrophobic residues outside of the core are highlighted in light gray.

Mentions: The localization of the pyruvate kinase fusion proteins (Fig. 4, c–e) indicated that the COOH terminus of RanGAP1 contains a nuclear localization signal (NLS). This was further mapped using additional pyruvate kinase fusion proteins. When fused to amino acids 541–589 of RanGAP1 (NΔ540/PK), pyruvate kinase was again targeted to the nucleus (Fig. 6 a). However, when fused with amino acids 556–589 of RanGAP1 (NΔ555/PK), pyruvate kinase was detected only in the cytosol (Fig. 6 b). These results indicated that amino acids 541–589 of RanGAP1 can function as an NLS. A database search with the NLS of RanGAP1 (shown in Fig. 7 B) revealed no significant homologies with other proteins and no homologies to other known NLSs. However, the nine amino-terminal leucine-rich repeats of RanGAP1 were each found to have striking homology with the leucine-rich nuclear export sequence motif (Fig. 7 C), first described for the HIV REV protein and the cAMP-dependent protein kinase inhibitor (Fischer et al., 1995; Wen et al., 1995). The presence of potential nuclear import and export signals raises the interesting possibility that RanGAP1 may shuttle between the nucleus and the cytoplasm.


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)

RanGAP1 contains nine potential nuclear export signals in addition to a nuclear localization signal. (A) Schematic  representation of RanGAP1 indicating the positions of the nine  NH2-terminal nuclear export motifs and the COOH-terminal nuclear localization signal. (B) Amino acid sequence of the nuclear  localization signal in the COOH-terminal domain of RanGAP1.  (C) Amino acid sequences and alignments of the nine putative  nuclear export motifs in the NH2-terminal domain of RanGAP1  and the nuclear export motifs of Rev (Fischer et al., 1995), PKI  (Wen et al., 1995), IκB, and Rex (Fritz and Green, 1996). Hydrophobic residues comprising the core of the motif are highlighted  in dark gray, and hydrophobic residues outside of the core are  highlighted in light gray.
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Related In: Results  -  Collection

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Figure 7: RanGAP1 contains nine potential nuclear export signals in addition to a nuclear localization signal. (A) Schematic representation of RanGAP1 indicating the positions of the nine NH2-terminal nuclear export motifs and the COOH-terminal nuclear localization signal. (B) Amino acid sequence of the nuclear localization signal in the COOH-terminal domain of RanGAP1. (C) Amino acid sequences and alignments of the nine putative nuclear export motifs in the NH2-terminal domain of RanGAP1 and the nuclear export motifs of Rev (Fischer et al., 1995), PKI (Wen et al., 1995), IκB, and Rex (Fritz and Green, 1996). Hydrophobic residues comprising the core of the motif are highlighted in dark gray, and hydrophobic residues outside of the core are highlighted in light gray.
Mentions: The localization of the pyruvate kinase fusion proteins (Fig. 4, c–e) indicated that the COOH terminus of RanGAP1 contains a nuclear localization signal (NLS). This was further mapped using additional pyruvate kinase fusion proteins. When fused to amino acids 541–589 of RanGAP1 (NΔ540/PK), pyruvate kinase was again targeted to the nucleus (Fig. 6 a). However, when fused with amino acids 556–589 of RanGAP1 (NΔ555/PK), pyruvate kinase was detected only in the cytosol (Fig. 6 b). These results indicated that amino acids 541–589 of RanGAP1 can function as an NLS. A database search with the NLS of RanGAP1 (shown in Fig. 7 B) revealed no significant homologies with other proteins and no homologies to other known NLSs. However, the nine amino-terminal leucine-rich repeats of RanGAP1 were each found to have striking homology with the leucine-rich nuclear export sequence motif (Fig. 7 C), first described for the HIV REV protein and the cAMP-dependent protein kinase inhibitor (Fischer et al., 1995; Wen et al., 1995). The presence of potential nuclear import and export signals raises the interesting possibility that RanGAP1 may shuttle between the nucleus and the cytoplasm.

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