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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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Determinants for SUMO-1 modification reside in the COOH-terminal domain of RanGAP1. (A) Schematic representations  of RanGAP1 and pyruvate kinase fusion proteins. Asterisks indicate the presence of a myc epitope tag at the NH2 terminus of each protein. Leucine-rich repeats in RanGAP1 are indicated by dark-shaded boxes, the acidic domain by a hatched box, and the COOH-terminal domain by a light-shaded box. Pyruvate kinase is represented by very light-shaded boxes, and SUMO-1 by a black box. Results of in  vitro SUMO-1 modification and immunolocalization of the transiently expressed proteins are indicated on the right. C, cytoplasm; NE,  nuclear envelope; N, nucleus. (B) Wild-type RanGAP1 (lane 1), the COOH-terminal deletion mutant CD23 (lane 2), the pyruvate kinase fusion proteins NΔ419/PK (lane 3), NΔ470/PK (lane 4) and NΔ502/PK (lane 5), and pyruvate kinase (lane 6) were transcribed and  translated in rabbit reticulocyte extracts in the presence of [35S]methionine. Reactions were separated by SDS-PAGE and analyzed by  autoradiography. Molecular mass standards are indicated on the left and asterisks indicate SUMO-1–modified substrates.
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Figure 3: Determinants for SUMO-1 modification reside in the COOH-terminal domain of RanGAP1. (A) Schematic representations of RanGAP1 and pyruvate kinase fusion proteins. Asterisks indicate the presence of a myc epitope tag at the NH2 terminus of each protein. Leucine-rich repeats in RanGAP1 are indicated by dark-shaded boxes, the acidic domain by a hatched box, and the COOH-terminal domain by a light-shaded box. Pyruvate kinase is represented by very light-shaded boxes, and SUMO-1 by a black box. Results of in vitro SUMO-1 modification and immunolocalization of the transiently expressed proteins are indicated on the right. C, cytoplasm; NE, nuclear envelope; N, nucleus. (B) Wild-type RanGAP1 (lane 1), the COOH-terminal deletion mutant CD23 (lane 2), the pyruvate kinase fusion proteins NΔ419/PK (lane 3), NΔ470/PK (lane 4) and NΔ502/PK (lane 5), and pyruvate kinase (lane 6) were transcribed and translated in rabbit reticulocyte extracts in the presence of [35S]methionine. Reactions were separated by SDS-PAGE and analyzed by autoradiography. Molecular mass standards are indicated on the left and asterisks indicate SUMO-1–modified substrates.

Mentions: We used this in vitro assay to analyze a series of NH2- and COOH-terminal deletions of RanGAP1 and found that the COOH-terminal domain of RanGAP1 is essential for SUMO-1 modification (data not shown; also see Fig. 3 B). To identify specific amino acid residues required for SUMO-1 modification, lysines in the COOH terminus of RanGAP1 were systematically mutated to arginine, based on the assumption that SUMO-1 conjugation would occur via a lysine, similar to ubiquitination (Wilkison, 1995; Hochstrasser, 1996). Substitution of arginine for lysine residues at positions 567, 555, 532, and 530 had no effect on RanGAP1 modification in vitro (data not shown). However, when lysine 526 was mutated to arginine, RanGAP1 was no longer modified by SUMO-1 (Fig. 1, lane 2). This finding identifies lysine 526 of RanGAP1 as essential for modification and implicates this residue as the acceptor for SUMO-1 conjugation.


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)

Determinants for SUMO-1 modification reside in the COOH-terminal domain of RanGAP1. (A) Schematic representations  of RanGAP1 and pyruvate kinase fusion proteins. Asterisks indicate the presence of a myc epitope tag at the NH2 terminus of each protein. Leucine-rich repeats in RanGAP1 are indicated by dark-shaded boxes, the acidic domain by a hatched box, and the COOH-terminal domain by a light-shaded box. Pyruvate kinase is represented by very light-shaded boxes, and SUMO-1 by a black box. Results of in  vitro SUMO-1 modification and immunolocalization of the transiently expressed proteins are indicated on the right. C, cytoplasm; NE,  nuclear envelope; N, nucleus. (B) Wild-type RanGAP1 (lane 1), the COOH-terminal deletion mutant CD23 (lane 2), the pyruvate kinase fusion proteins NΔ419/PK (lane 3), NΔ470/PK (lane 4) and NΔ502/PK (lane 5), and pyruvate kinase (lane 6) were transcribed and  translated in rabbit reticulocyte extracts in the presence of [35S]methionine. Reactions were separated by SDS-PAGE and analyzed by  autoradiography. Molecular mass standards are indicated on the left and asterisks indicate SUMO-1–modified substrates.
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Related In: Results  -  Collection

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Figure 3: Determinants for SUMO-1 modification reside in the COOH-terminal domain of RanGAP1. (A) Schematic representations of RanGAP1 and pyruvate kinase fusion proteins. Asterisks indicate the presence of a myc epitope tag at the NH2 terminus of each protein. Leucine-rich repeats in RanGAP1 are indicated by dark-shaded boxes, the acidic domain by a hatched box, and the COOH-terminal domain by a light-shaded box. Pyruvate kinase is represented by very light-shaded boxes, and SUMO-1 by a black box. Results of in vitro SUMO-1 modification and immunolocalization of the transiently expressed proteins are indicated on the right. C, cytoplasm; NE, nuclear envelope; N, nucleus. (B) Wild-type RanGAP1 (lane 1), the COOH-terminal deletion mutant CD23 (lane 2), the pyruvate kinase fusion proteins NΔ419/PK (lane 3), NΔ470/PK (lane 4) and NΔ502/PK (lane 5), and pyruvate kinase (lane 6) were transcribed and translated in rabbit reticulocyte extracts in the presence of [35S]methionine. Reactions were separated by SDS-PAGE and analyzed by autoradiography. Molecular mass standards are indicated on the left and asterisks indicate SUMO-1–modified substrates.
Mentions: We used this in vitro assay to analyze a series of NH2- and COOH-terminal deletions of RanGAP1 and found that the COOH-terminal domain of RanGAP1 is essential for SUMO-1 modification (data not shown; also see Fig. 3 B). To identify specific amino acid residues required for SUMO-1 modification, lysines in the COOH terminus of RanGAP1 were systematically mutated to arginine, based on the assumption that SUMO-1 conjugation would occur via a lysine, similar to ubiquitination (Wilkison, 1995; Hochstrasser, 1996). Substitution of arginine for lysine residues at positions 567, 555, 532, and 530 had no effect on RanGAP1 modification in vitro (data not shown). However, when lysine 526 was mutated to arginine, RanGAP1 was no longer modified by SUMO-1 (Fig. 1, lane 2). This finding identifies lysine 526 of RanGAP1 as essential for modification and implicates this residue as the acceptor for SUMO-1 conjugation.

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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