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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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Lysine 526 is essential for  SUMO-1 modification of RanGAP1 (A)  Myc-tagged wild-type RanGAP1 (lane 1)  and myc-tagged RanGAP1 containing  a single lysine to arginine substitution  at amino acid 526 (lane 2) were transcribed and translated in vitro in the  presence of [35S]methionine and analyzed by SDS-PAGE. Molecular mass  standards are indicated on the left and  an asterisk denotes SUMO-1–modified  wild-type RanGAP1.
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Figure 1: Lysine 526 is essential for SUMO-1 modification of RanGAP1 (A) Myc-tagged wild-type RanGAP1 (lane 1) and myc-tagged RanGAP1 containing a single lysine to arginine substitution at amino acid 526 (lane 2) were transcribed and translated in vitro in the presence of [35S]methionine and analyzed by SDS-PAGE. Molecular mass standards are indicated on the left and an asterisk denotes SUMO-1–modified wild-type RanGAP1.

Mentions: To identify elements of RanGAP1 required for SUMO-1 modification, we developed a rapid and simple in vitro assay using rabbit reticulocyte transcription and translation extracts. When a cDNA coding for myc-tagged RanGAP1 was transcribed and translated in an vitro extract, two products were observed, migrating with relative molecular masses of 75 and 95 kD (Fig. 1, lane 1). Both products appeared as doublets, possibly as a result of initiation of translation from an internal methionine. Their 20-kD difference suggested that the larger product represented SUMO-1–modified RanGAP1, which was confirmed by immunopurification with antibodies specific for SUMO-1 (data not shown). These results indicate that rabbit reticulocyte extracts contain all of the necessary factors for SUMO-1 modification of RanGAP1.


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)

Lysine 526 is essential for  SUMO-1 modification of RanGAP1 (A)  Myc-tagged wild-type RanGAP1 (lane 1)  and myc-tagged RanGAP1 containing  a single lysine to arginine substitution  at amino acid 526 (lane 2) were transcribed and translated in vitro in the  presence of [35S]methionine and analyzed by SDS-PAGE. Molecular mass  standards are indicated on the left and  an asterisk denotes SUMO-1–modified  wild-type RanGAP1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Lysine 526 is essential for SUMO-1 modification of RanGAP1 (A) Myc-tagged wild-type RanGAP1 (lane 1) and myc-tagged RanGAP1 containing a single lysine to arginine substitution at amino acid 526 (lane 2) were transcribed and translated in vitro in the presence of [35S]methionine and analyzed by SDS-PAGE. Molecular mass standards are indicated on the left and an asterisk denotes SUMO-1–modified wild-type RanGAP1.
Mentions: To identify elements of RanGAP1 required for SUMO-1 modification, we developed a rapid and simple in vitro assay using rabbit reticulocyte transcription and translation extracts. When a cDNA coding for myc-tagged RanGAP1 was transcribed and translated in an vitro extract, two products were observed, migrating with relative molecular masses of 75 and 95 kD (Fig. 1, lane 1). Both products appeared as doublets, possibly as a result of initiation of translation from an internal methionine. Their 20-kD difference suggested that the larger product represented SUMO-1–modified RanGAP1, which was confirmed by immunopurification with antibodies specific for SUMO-1 (data not shown). These results indicate that rabbit reticulocyte extracts contain all of the necessary factors for SUMO-1 modification of RanGAP1.

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