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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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SUN domain is required for the translocation of SUN2 from the NE to the cytoplasm upon Rab5 overexpression.(A) HeLa cells transfected with pcDNA3-HA-SUN2-myc (SUN2) or pcDNA3-HA-ΔSUN (ΔSUN) were analyzed by the antibody against HA. ΔSUN is identified at ∼60 kDa; IB, immunoblotting. (B) Immunofluoresence staining using the antibody against HA shows that both full length HA-SUN2 and HA-ΔSUN localized on the NE of transfected HeLa cells. Scale bar, 10 µm. (C) HA-ΔSUN detected by anti-HA localized on the NE in HeLa cells transfected with either pEGFP-Rab5 or pEGFP-Rab5Q79L. Scale bar, 20 µm. (D) Antibodies against HA and Rab5 were used as precipitating (IP) / IB anibodies in cotransfected HeLa cells. HA-ΔSUN coimmunoprecipitated with EGFP-Rab5 and its mutant forms.
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pone-0020507-g005: SUN domain is required for the translocation of SUN2 from the NE to the cytoplasm upon Rab5 overexpression.(A) HeLa cells transfected with pcDNA3-HA-SUN2-myc (SUN2) or pcDNA3-HA-ΔSUN (ΔSUN) were analyzed by the antibody against HA. ΔSUN is identified at ∼60 kDa; IB, immunoblotting. (B) Immunofluoresence staining using the antibody against HA shows that both full length HA-SUN2 and HA-ΔSUN localized on the NE of transfected HeLa cells. Scale bar, 10 µm. (C) HA-ΔSUN detected by anti-HA localized on the NE in HeLa cells transfected with either pEGFP-Rab5 or pEGFP-Rab5Q79L. Scale bar, 20 µm. (D) Antibodies against HA and Rab5 were used as precipitating (IP) / IB anibodies in cotransfected HeLa cells. HA-ΔSUN coimmunoprecipitated with EGFP-Rab5 and its mutant forms.

Mentions: To investigate the importance of the SUN domain, a SUN2 construct with SUN domain deletion as shown in Figure 1A was generated. The anti-HA antibody efficiently identified this truncated protein, termed ΔSUN at its calculated size of ∼60 kDa (Fig. 5A). Like the full length protein, ΔSUN localized mainly to the NE, together with some cytoplasmic vesicles (Fig. 5B). In contrast to the endogenous SUN2, ΔSUN was concentrated at the NE after transfection with either Rab5 or its positive mutant (Fig. 5C). The result of coimmunoprecipitation showed ΔSUN interacted with Rab5 and its two mutants in vitro (Fig. 5D). This suggests that the SUN domain is not essential for the binding between SUN2 and Rab5, but it is required for the translocation of SUN2 to the cyotplasmic vesicles.


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

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

SUN domain is required for the translocation of SUN2 from the NE to the cytoplasm upon Rab5 overexpression.(A) HeLa cells transfected with pcDNA3-HA-SUN2-myc (SUN2) or pcDNA3-HA-ΔSUN (ΔSUN) were analyzed by the antibody against HA. ΔSUN is identified at ∼60 kDa; IB, immunoblotting. (B) Immunofluoresence staining using the antibody against HA shows that both full length HA-SUN2 and HA-ΔSUN localized on the NE of transfected HeLa cells. Scale bar, 10 µm. (C) HA-ΔSUN detected by anti-HA localized on the NE in HeLa cells transfected with either pEGFP-Rab5 or pEGFP-Rab5Q79L. Scale bar, 20 µm. (D) Antibodies against HA and Rab5 were used as precipitating (IP) / IB anibodies in cotransfected HeLa cells. HA-ΔSUN coimmunoprecipitated with EGFP-Rab5 and its mutant forms.
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Related In: Results  -  Collection

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pone-0020507-g005: SUN domain is required for the translocation of SUN2 from the NE to the cytoplasm upon Rab5 overexpression.(A) HeLa cells transfected with pcDNA3-HA-SUN2-myc (SUN2) or pcDNA3-HA-ΔSUN (ΔSUN) were analyzed by the antibody against HA. ΔSUN is identified at ∼60 kDa; IB, immunoblotting. (B) Immunofluoresence staining using the antibody against HA shows that both full length HA-SUN2 and HA-ΔSUN localized on the NE of transfected HeLa cells. Scale bar, 10 µm. (C) HA-ΔSUN detected by anti-HA localized on the NE in HeLa cells transfected with either pEGFP-Rab5 or pEGFP-Rab5Q79L. Scale bar, 20 µm. (D) Antibodies against HA and Rab5 were used as precipitating (IP) / IB anibodies in cotransfected HeLa cells. HA-ΔSUN coimmunoprecipitated with EGFP-Rab5 and its mutant forms.
Mentions: To investigate the importance of the SUN domain, a SUN2 construct with SUN domain deletion as shown in Figure 1A was generated. The anti-HA antibody efficiently identified this truncated protein, termed ΔSUN at its calculated size of ∼60 kDa (Fig. 5A). Like the full length protein, ΔSUN localized mainly to the NE, together with some cytoplasmic vesicles (Fig. 5B). In contrast to the endogenous SUN2, ΔSUN was concentrated at the NE after transfection with either Rab5 or its positive mutant (Fig. 5C). The result of coimmunoprecipitation showed ΔSUN interacted with Rab5 and its two mutants in vitro (Fig. 5D). This suggests that the SUN domain is not essential for the binding between SUN2 and Rab5, but it is required for the translocation of SUN2 to the cyotplasmic vesicles.

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