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Subcellular localization of SUN2 is regulated by lamin A and Rab5.

Liang Y, Chiu PH, Yip KY, Chan SY - PLoS ONE (2011)

Bottom Line: We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2.Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process.These findings support a role of SUN2 in endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong SAR, China.

ABSTRACT
SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2. SUN2 was redistributed to endosomes upon overexpression of Rab5, but remained on the nuclear envelope when the SUN domain was deleted. To explore the physiological function of SUN2 in vesicle trafficking and endocytosis, we demonstrated the colocalization of endogenous SUN2 and Rab5. Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process. These findings support a role of SUN2 in endocytosis.

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Rab5 and the GTPase-deficient mutant, Rab5Q79L, lead to SUN2 redistribution to endosomes.(A) Left: SUN2 coimmunoprecipitated with Rab5, using antibodies against c-myc and Rab5, in the protein lysate from HeLa cells cotransfected with pcDNA3-HA-SUN2-myc and pEGFP-Rab5. Middle: No binding between SUN2 and EGFP was observed in cells overexpressing HA-SUN2-myc and EGFP. Right: The antibody against c-myc could not recognize any bands in HeLa cells transfected with pEGFP-Rab5 only. Molecular weight is shown in kDa. (B) Immunofluorescence staining reveals that endogenous SUN2 partially colocalized with transfected EGFP-Rab5 in endosomes (arrows). (C) In the cell expressing pEGFP-Rab5Q79L, SUN2 was relocated to the enlarged endosomes from the NE (arrow), but not in Rab5Q79L-negative cells (arrowheads). (D) In Rab5S34N-positive cells, SUN2 remained on the NE (arrow). Scale bar, 10 µm. (E) Antibodies against HA and Rab5 were used as precipitating/immunoblotting antibodies in HeLa cells cotransfected with plasmids abbreviated on the top. HA-SUN2 coimmunoprecipitated with wild type or mutant forms of EGFP-Rab5. In the control shown on the right, the antibody against HA did not immunoprecipitate Rab5 in HeLa cells transfected with pEGFP-Rab5 only. IB, immunoblotting; IP, immunoprecipitation.
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pone-0020507-g003: Rab5 and the GTPase-deficient mutant, Rab5Q79L, lead to SUN2 redistribution to endosomes.(A) Left: SUN2 coimmunoprecipitated with Rab5, using antibodies against c-myc and Rab5, in the protein lysate from HeLa cells cotransfected with pcDNA3-HA-SUN2-myc and pEGFP-Rab5. Middle: No binding between SUN2 and EGFP was observed in cells overexpressing HA-SUN2-myc and EGFP. Right: The antibody against c-myc could not recognize any bands in HeLa cells transfected with pEGFP-Rab5 only. Molecular weight is shown in kDa. (B) Immunofluorescence staining reveals that endogenous SUN2 partially colocalized with transfected EGFP-Rab5 in endosomes (arrows). (C) In the cell expressing pEGFP-Rab5Q79L, SUN2 was relocated to the enlarged endosomes from the NE (arrow), but not in Rab5Q79L-negative cells (arrowheads). (D) In Rab5S34N-positive cells, SUN2 remained on the NE (arrow). Scale bar, 10 µm. (E) Antibodies against HA and Rab5 were used as precipitating/immunoblotting antibodies in HeLa cells cotransfected with plasmids abbreviated on the top. HA-SUN2 coimmunoprecipitated with wild type or mutant forms of EGFP-Rab5. In the control shown on the right, the antibody against HA did not immunoprecipitate Rab5 in HeLa cells transfected with pEGFP-Rab5 only. IB, immunoblotting; IP, immunoprecipitation.

Mentions: Unlike most other INM proteins exhibiting an exclusive NE localization, we observed punctate SUN2 staining in addition to the NE staining in a small proportion of HeLa cells, which resembled cytoplasmic vesicles. This staining pattern can also be found in other reports but is essentially ignored [11], [12], [25]. A previous study has shown that truncated SUN2 localizes in Rab5-positive vesicles in cotransfected HeLa cells. Furthermore, endogenous SUN2 is found in the membrane fraction after removal of nuclei [16]. By immunoprecipitation, we confirmed the interaction between full length SUN2 and Rab5 in HeLa cells overexpressing SUN2 and EGFP-Rab5 (Fig. 3A). Rab5 is a key regulator of early endocytosis. To further analyze the involvement of endogenous SUN2 in endocytosis, we used HeLa cells transiently transfected with EGFP-Rab5 and its two mutants. Rab5Q79L is a GTPase-deficient mutant which stimulates endosome fusion, whereas Rab5S34N is a dominant negative mutant preferentially GDP-bound [26]. Colocalization of endogenous SUN2 with Rab5 in endosomes was detected in cells overexpressing Rab5 (Fig. 3B). In cells expressing Rab5Q79L, SUN2 expression overlapped with Rab5Q79L in enlarged early endosomes (Fig. 3C, arrow). In contrast, nontransfected HeLa cells maintained a uniform expression of SUN2 on the NE (Fig. 3C, arrowheads). SUN2 did not colocalize with Rab5S34N but was found on the NE and some punctate structures in the nucleus (Fig. 3D, arrow). However, all the Rab5 mutant forms could interact with SUN2 (Fig. 3E). Close proximity between Rab5 and SUN2 (<10 Å) was confirmed by FRET. Using wildtype Rab5 and its mutant Rab5Q79L, the results confirm direct interaction between SUN2 and the GTP-bound form of Rab5 (Fig. 4).


Subcellular localization of SUN2 is regulated by lamin A and Rab5.

Liang Y, Chiu PH, Yip KY, Chan SY - PLoS ONE (2011)

Rab5 and the GTPase-deficient mutant, Rab5Q79L, lead to SUN2 redistribution to endosomes.(A) Left: SUN2 coimmunoprecipitated with Rab5, using antibodies against c-myc and Rab5, in the protein lysate from HeLa cells cotransfected with pcDNA3-HA-SUN2-myc and pEGFP-Rab5. Middle: No binding between SUN2 and EGFP was observed in cells overexpressing HA-SUN2-myc and EGFP. Right: The antibody against c-myc could not recognize any bands in HeLa cells transfected with pEGFP-Rab5 only. Molecular weight is shown in kDa. (B) Immunofluorescence staining reveals that endogenous SUN2 partially colocalized with transfected EGFP-Rab5 in endosomes (arrows). (C) In the cell expressing pEGFP-Rab5Q79L, SUN2 was relocated to the enlarged endosomes from the NE (arrow), but not in Rab5Q79L-negative cells (arrowheads). (D) In Rab5S34N-positive cells, SUN2 remained on the NE (arrow). Scale bar, 10 µm. (E) Antibodies against HA and Rab5 were used as precipitating/immunoblotting antibodies in HeLa cells cotransfected with plasmids abbreviated on the top. HA-SUN2 coimmunoprecipitated with wild type or mutant forms of EGFP-Rab5. In the control shown on the right, the antibody against HA did not immunoprecipitate Rab5 in HeLa cells transfected with pEGFP-Rab5 only. IB, immunoblotting; IP, immunoprecipitation.
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pone-0020507-g003: Rab5 and the GTPase-deficient mutant, Rab5Q79L, lead to SUN2 redistribution to endosomes.(A) Left: SUN2 coimmunoprecipitated with Rab5, using antibodies against c-myc and Rab5, in the protein lysate from HeLa cells cotransfected with pcDNA3-HA-SUN2-myc and pEGFP-Rab5. Middle: No binding between SUN2 and EGFP was observed in cells overexpressing HA-SUN2-myc and EGFP. Right: The antibody against c-myc could not recognize any bands in HeLa cells transfected with pEGFP-Rab5 only. Molecular weight is shown in kDa. (B) Immunofluorescence staining reveals that endogenous SUN2 partially colocalized with transfected EGFP-Rab5 in endosomes (arrows). (C) In the cell expressing pEGFP-Rab5Q79L, SUN2 was relocated to the enlarged endosomes from the NE (arrow), but not in Rab5Q79L-negative cells (arrowheads). (D) In Rab5S34N-positive cells, SUN2 remained on the NE (arrow). Scale bar, 10 µm. (E) Antibodies against HA and Rab5 were used as precipitating/immunoblotting antibodies in HeLa cells cotransfected with plasmids abbreviated on the top. HA-SUN2 coimmunoprecipitated with wild type or mutant forms of EGFP-Rab5. In the control shown on the right, the antibody against HA did not immunoprecipitate Rab5 in HeLa cells transfected with pEGFP-Rab5 only. IB, immunoblotting; IP, immunoprecipitation.
Mentions: Unlike most other INM proteins exhibiting an exclusive NE localization, we observed punctate SUN2 staining in addition to the NE staining in a small proportion of HeLa cells, which resembled cytoplasmic vesicles. This staining pattern can also be found in other reports but is essentially ignored [11], [12], [25]. A previous study has shown that truncated SUN2 localizes in Rab5-positive vesicles in cotransfected HeLa cells. Furthermore, endogenous SUN2 is found in the membrane fraction after removal of nuclei [16]. By immunoprecipitation, we confirmed the interaction between full length SUN2 and Rab5 in HeLa cells overexpressing SUN2 and EGFP-Rab5 (Fig. 3A). Rab5 is a key regulator of early endocytosis. To further analyze the involvement of endogenous SUN2 in endocytosis, we used HeLa cells transiently transfected with EGFP-Rab5 and its two mutants. Rab5Q79L is a GTPase-deficient mutant which stimulates endosome fusion, whereas Rab5S34N is a dominant negative mutant preferentially GDP-bound [26]. Colocalization of endogenous SUN2 with Rab5 in endosomes was detected in cells overexpressing Rab5 (Fig. 3B). In cells expressing Rab5Q79L, SUN2 expression overlapped with Rab5Q79L in enlarged early endosomes (Fig. 3C, arrow). In contrast, nontransfected HeLa cells maintained a uniform expression of SUN2 on the NE (Fig. 3C, arrowheads). SUN2 did not colocalize with Rab5S34N but was found on the NE and some punctate structures in the nucleus (Fig. 3D, arrow). However, all the Rab5 mutant forms could interact with SUN2 (Fig. 3E). Close proximity between Rab5 and SUN2 (<10 Å) was confirmed by FRET. Using wildtype Rab5 and its mutant Rab5Q79L, the results confirm direct interaction between SUN2 and the GTP-bound form of Rab5 (Fig. 4).

Bottom Line: We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2.Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process.These findings support a role of SUN2 in endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong SAR, China.

ABSTRACT
SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2. SUN2 was redistributed to endosomes upon overexpression of Rab5, but remained on the nuclear envelope when the SUN domain was deleted. To explore the physiological function of SUN2 in vesicle trafficking and endocytosis, we demonstrated the colocalization of endogenous SUN2 and Rab5. Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process. These findings support a role of SUN2 in endocytosis.

Show MeSH
Related in: MedlinePlus