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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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SUMO-1–modified RanGAP1 binds to a COOH-terminal region of Nup358, between Ran-binding domains three and  four. (A) Schematic representation of Nup358. RBD, Ran-binding  domain; CycH, cyclophilin homologous domain. Long vertical  lines indicate FXFG repeats, and short vertical lines indicate FG  repeats. Fragments that were found to bind modified RanGAP1  are indicated, as well as a summary of their binding activity (+,  weak binding; ++, strong binding). Internal direct repeats in the  RanGAP1-binding domain are indicated by open boxes. (B) Full-length RanGAP1 (lane 1) and COOH-terminal regions of  RanGAP1 from amino acids 420–589 (NΔ419; lane 6) and 471– 589 (NΔ470; lane 11) were translated in vitro in the presence of  [35S]methionine. Translated proteins were assayed for binding to  the domains of Nup358 indicated in A, as described in Materials  and Methods. Molecular mass standards are indicated on the left,  and asterisks denote SUMO-1–modified proteins.
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Figure 8: SUMO-1–modified RanGAP1 binds to a COOH-terminal region of Nup358, between Ran-binding domains three and four. (A) Schematic representation of Nup358. RBD, Ran-binding domain; CycH, cyclophilin homologous domain. Long vertical lines indicate FXFG repeats, and short vertical lines indicate FG repeats. Fragments that were found to bind modified RanGAP1 are indicated, as well as a summary of their binding activity (+, weak binding; ++, strong binding). Internal direct repeats in the RanGAP1-binding domain are indicated by open boxes. (B) Full-length RanGAP1 (lane 1) and COOH-terminal regions of RanGAP1 from amino acids 420–589 (NΔ419; lane 6) and 471– 589 (NΔ470; lane 11) were translated in vitro in the presence of [35S]methionine. Translated proteins were assayed for binding to the domains of Nup358 indicated in A, as described in Materials and Methods. Molecular mass standards are indicated on the left, and asterisks denote SUMO-1–modified proteins.

Mentions: SUMO-1–modified RanGAP1 interacts with Nup358 (Mahajan et al., 1997; Saitoh et al., 1997), a nucleoporin that is a component of the cytoplasmic fibers of the NPC (Wu et al., 1995; Yokoyama et al., 1995). To characterize this interaction in more detail, we used an in vitro binding assay using bacterially expressed regions of Nup358 and radiolabeled RanGAP1 produced in rabbit reticulocyte extracts. Purified GST fusion proteins corresponding to various domains of Nup358 were bound to the wells of a microtiter plate and incubated with in vitro–translated RanGAP1. After incubation, the wells were washed, and bound proteins were eluted with SDS sample buffer. No binding of either modified or unmodified RanGAP1 was detected to regions of Nup358 corresponding to the NH2-terminal leucine-rich region, the Ran-binding domains, or the cyclophilin homologous domain (data not shown). RanGAP1 binding was, however, detected with a region of Nup358 extending from amino acid 2500 to the COOH terminus (Fig. 8 B, lane 2). Binding was specific for SUMO-1–modified RanGAP1, with no unmodified RanGAP1 being detected. The specificity of the binding was further demonstrated by the absence of interactions with GST (Fig. 8 B, lane 5). The region of Nup358 from amino acid 2500 to the COOH terminus contains two Ran-binding domains (domains three and four) separated by a 470–amino acid segment, and the cyclophilin homology domain. The 470– amino acid segment between the two Ran-binding domains contains direct repeats of ∼40 amino acids (Yokoyama et al., 1995). Because our results showed that the Ran-binding domains and cyclophilin homology domain did not interact with RanGAP1, we assayed more specifically for binding to the region separating Ran-binding domains three and four. This domain is flanked by clusters of FXFG repeats, and fragments with (amino acids 2503–2893) and without (amino acids 2550–2837) these repeats were tested in the binding assay. SUMO-1–modified RanGAP1 bound specifically to both of these fragments (Fig. 8 B, lanes 3 and 4). However, interaction with the fragment lacking FXFG repeats was of lower affinity relative to the fragment containing FXFG repeats, based on the four- to fivefold reduction in observed binding (see Fig. 9). Again, unmodified RanGAP1 did not bind to these fragments.


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–modified RanGAP1 binds to a COOH-terminal region of Nup358, between Ran-binding domains three and  four. (A) Schematic representation of Nup358. RBD, Ran-binding  domain; CycH, cyclophilin homologous domain. Long vertical  lines indicate FXFG repeats, and short vertical lines indicate FG  repeats. Fragments that were found to bind modified RanGAP1  are indicated, as well as a summary of their binding activity (+,  weak binding; ++, strong binding). Internal direct repeats in the  RanGAP1-binding domain are indicated by open boxes. (B) Full-length RanGAP1 (lane 1) and COOH-terminal regions of  RanGAP1 from amino acids 420–589 (NΔ419; lane 6) and 471– 589 (NΔ470; lane 11) were translated in vitro in the presence of  [35S]methionine. Translated proteins were assayed for binding to  the domains of Nup358 indicated in A, as described in Materials  and Methods. Molecular mass standards are indicated on the left,  and asterisks denote SUMO-1–modified proteins.
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Figure 8: SUMO-1–modified RanGAP1 binds to a COOH-terminal region of Nup358, between Ran-binding domains three and four. (A) Schematic representation of Nup358. RBD, Ran-binding domain; CycH, cyclophilin homologous domain. Long vertical lines indicate FXFG repeats, and short vertical lines indicate FG repeats. Fragments that were found to bind modified RanGAP1 are indicated, as well as a summary of their binding activity (+, weak binding; ++, strong binding). Internal direct repeats in the RanGAP1-binding domain are indicated by open boxes. (B) Full-length RanGAP1 (lane 1) and COOH-terminal regions of RanGAP1 from amino acids 420–589 (NΔ419; lane 6) and 471– 589 (NΔ470; lane 11) were translated in vitro in the presence of [35S]methionine. Translated proteins were assayed for binding to the domains of Nup358 indicated in A, as described in Materials and Methods. Molecular mass standards are indicated on the left, and asterisks denote SUMO-1–modified proteins.
Mentions: SUMO-1–modified RanGAP1 interacts with Nup358 (Mahajan et al., 1997; Saitoh et al., 1997), a nucleoporin that is a component of the cytoplasmic fibers of the NPC (Wu et al., 1995; Yokoyama et al., 1995). To characterize this interaction in more detail, we used an in vitro binding assay using bacterially expressed regions of Nup358 and radiolabeled RanGAP1 produced in rabbit reticulocyte extracts. Purified GST fusion proteins corresponding to various domains of Nup358 were bound to the wells of a microtiter plate and incubated with in vitro–translated RanGAP1. After incubation, the wells were washed, and bound proteins were eluted with SDS sample buffer. No binding of either modified or unmodified RanGAP1 was detected to regions of Nup358 corresponding to the NH2-terminal leucine-rich region, the Ran-binding domains, or the cyclophilin homologous domain (data not shown). RanGAP1 binding was, however, detected with a region of Nup358 extending from amino acid 2500 to the COOH terminus (Fig. 8 B, lane 2). Binding was specific for SUMO-1–modified RanGAP1, with no unmodified RanGAP1 being detected. The specificity of the binding was further demonstrated by the absence of interactions with GST (Fig. 8 B, lane 5). The region of Nup358 from amino acid 2500 to the COOH terminus contains two Ran-binding domains (domains three and four) separated by a 470–amino acid segment, and the cyclophilin homology domain. The 470– amino acid segment between the two Ran-binding domains contains direct repeats of ∼40 amino acids (Yokoyama et al., 1995). Because our results showed that the Ran-binding domains and cyclophilin homology domain did not interact with RanGAP1, we assayed more specifically for binding to the region separating Ran-binding domains three and four. This domain is flanked by clusters of FXFG repeats, and fragments with (amino acids 2503–2893) and without (amino acids 2550–2837) these repeats were tested in the binding assay. SUMO-1–modified RanGAP1 bound specifically to both of these fragments (Fig. 8 B, lanes 3 and 4). However, interaction with the fragment lacking FXFG repeats was of lower affinity relative to the fragment containing FXFG repeats, based on the four- to fivefold reduction in observed binding (see Fig. 9). Again, unmodified RanGAP1 did not bind to these fragments.

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