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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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Expression constructs and antibodies for SUN2.(A) Schematic diagram of recombinant SUN2. Full length SUN2 contains a transmembrane domain (TM) and a conserved SUN domain. Each domain is labeled according to human UNC-84 homolog B (NP_056189) numbering. The N-terminal 1–50 amino acid sequence of SUN2 is also shown. The two methionine residues corresponding to the two potential translation initiation sites are highlighted in black. The position of the peptide used to generate the anti-SUN2 antibody is indicated as Ab. Regions shown in light gray are the locations of two epitopes, hemagglutinin A (HA) and myc. The construct lacking the SUN domain is labeled as ΔSUN. The construct lacking most of the lumen region C-terminal to TM is labeled as SUN2ΔL. (B) Total protein extracts (50 µg) from non-transfected HeLa cells (NT) or cells transfected with pcDNA3-HA-SUN2-myc (T) were analyzed by antibodies against HA, c-myc, SUN2 and its pre-immune serum. A dominant band at ∼85 kDa is found in all T lanes except immunoblotting with the pre-immune serum. Antibodies against c-myc and SUN2 also recognize an additional minor band at 80 kDa. Endogenous SUN2 as shown in the SUN2-NT lane is ∼85 kDa. IB, immunoblotting.
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pone-0020507-g001: Expression constructs and antibodies for SUN2.(A) Schematic diagram of recombinant SUN2. Full length SUN2 contains a transmembrane domain (TM) and a conserved SUN domain. Each domain is labeled according to human UNC-84 homolog B (NP_056189) numbering. The N-terminal 1–50 amino acid sequence of SUN2 is also shown. The two methionine residues corresponding to the two potential translation initiation sites are highlighted in black. The position of the peptide used to generate the anti-SUN2 antibody is indicated as Ab. Regions shown in light gray are the locations of two epitopes, hemagglutinin A (HA) and myc. The construct lacking the SUN domain is labeled as ΔSUN. The construct lacking most of the lumen region C-terminal to TM is labeled as SUN2ΔL. (B) Total protein extracts (50 µg) from non-transfected HeLa cells (NT) or cells transfected with pcDNA3-HA-SUN2-myc (T) were analyzed by antibodies against HA, c-myc, SUN2 and its pre-immune serum. A dominant band at ∼85 kDa is found in all T lanes except immunoblotting with the pre-immune serum. Antibodies against c-myc and SUN2 also recognize an additional minor band at 80 kDa. Endogenous SUN2 as shown in the SUN2-NT lane is ∼85 kDa. IB, immunoblotting.

Mentions: To detect endogenous SUN2 expression, we raised an antiserum against SUN2 and examined its specificity after affinity purification. HeLa cells were transfected with a plasmid expressing HA and myc-tagged full length SUN2 (Fig. 1A). Lysates of both transfected and nontransfected cells were analyzed with antibodies against HA, c-myc and SUN2. As shown in Figure 1B, all the three antibodies, but not the pre-immune serum, recognized the same band at ∼85 kDa. This indicates that SUN2 antibody specifically recognized SUN2. In the transfected cells, a less prominent band at 80 kDa was also detected with antibodies against c-myc and SUN2. Possible explanations include alternative splicing, alternative initiation at the second methionine codon (in the +50 position) (Fig. 1A) or incomplete posttranslational modifications.


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

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

Expression constructs and antibodies for SUN2.(A) Schematic diagram of recombinant SUN2. Full length SUN2 contains a transmembrane domain (TM) and a conserved SUN domain. Each domain is labeled according to human UNC-84 homolog B (NP_056189) numbering. The N-terminal 1–50 amino acid sequence of SUN2 is also shown. The two methionine residues corresponding to the two potential translation initiation sites are highlighted in black. The position of the peptide used to generate the anti-SUN2 antibody is indicated as Ab. Regions shown in light gray are the locations of two epitopes, hemagglutinin A (HA) and myc. The construct lacking the SUN domain is labeled as ΔSUN. The construct lacking most of the lumen region C-terminal to TM is labeled as SUN2ΔL. (B) Total protein extracts (50 µg) from non-transfected HeLa cells (NT) or cells transfected with pcDNA3-HA-SUN2-myc (T) were analyzed by antibodies against HA, c-myc, SUN2 and its pre-immune serum. A dominant band at ∼85 kDa is found in all T lanes except immunoblotting with the pre-immune serum. Antibodies against c-myc and SUN2 also recognize an additional minor band at 80 kDa. Endogenous SUN2 as shown in the SUN2-NT lane is ∼85 kDa. IB, immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105078&req=5

pone-0020507-g001: Expression constructs and antibodies for SUN2.(A) Schematic diagram of recombinant SUN2. Full length SUN2 contains a transmembrane domain (TM) and a conserved SUN domain. Each domain is labeled according to human UNC-84 homolog B (NP_056189) numbering. The N-terminal 1–50 amino acid sequence of SUN2 is also shown. The two methionine residues corresponding to the two potential translation initiation sites are highlighted in black. The position of the peptide used to generate the anti-SUN2 antibody is indicated as Ab. Regions shown in light gray are the locations of two epitopes, hemagglutinin A (HA) and myc. The construct lacking the SUN domain is labeled as ΔSUN. The construct lacking most of the lumen region C-terminal to TM is labeled as SUN2ΔL. (B) Total protein extracts (50 µg) from non-transfected HeLa cells (NT) or cells transfected with pcDNA3-HA-SUN2-myc (T) were analyzed by antibodies against HA, c-myc, SUN2 and its pre-immune serum. A dominant band at ∼85 kDa is found in all T lanes except immunoblotting with the pre-immune serum. Antibodies against c-myc and SUN2 also recognize an additional minor band at 80 kDa. Endogenous SUN2 as shown in the SUN2-NT lane is ∼85 kDa. IB, immunoblotting.
Mentions: To detect endogenous SUN2 expression, we raised an antiserum against SUN2 and examined its specificity after affinity purification. HeLa cells were transfected with a plasmid expressing HA and myc-tagged full length SUN2 (Fig. 1A). Lysates of both transfected and nontransfected cells were analyzed with antibodies against HA, c-myc and SUN2. As shown in Figure 1B, all the three antibodies, but not the pre-immune serum, recognized the same band at ∼85 kDa. This indicates that SUN2 antibody specifically recognized SUN2. In the transfected cells, a less prominent band at 80 kDa was also detected with antibodies against c-myc and SUN2. Possible explanations include alternative splicing, alternative initiation at the second methionine codon (in the +50 position) (Fig. 1A) or incomplete posttranslational modifications.

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