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
The COOH-terminal domain of RanGAP1 contains a  nuclear localization signal. The pyruvate kinase fusion proteins  NΔ540/PK (a) and NΔ555/PK (b) were transiently expressed in  HeLa cells and localized with the anti-myc monoclonal antibody,  9E10.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140169&req=5

Figure 6: The COOH-terminal domain of RanGAP1 contains a nuclear localization signal. The pyruvate kinase fusion proteins NΔ540/PK (a) and NΔ555/PK (b) were transiently expressed in HeLa cells and localized with the anti-myc monoclonal antibody, 9E10.

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)

The COOH-terminal domain of RanGAP1 contains a  nuclear localization signal. The pyruvate kinase fusion proteins  NΔ540/PK (a) and NΔ555/PK (b) were transiently expressed in  HeLa cells and localized with the anti-myc monoclonal antibody,  9E10.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: The COOH-terminal domain of RanGAP1 contains a nuclear localization signal. The pyruvate kinase fusion proteins NΔ540/PK (a) and NΔ555/PK (b) were transiently expressed in HeLa cells and localized with the anti-myc monoclonal antibody, 9E10.
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