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SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.

Cabasso O, Pekar O, Horowitz M - PLoS ONE (2015)

Bottom Line: We show, both in-vitro and in cell culture, that EHD3 undergoes SUMOylation.Non-SUMOylated EHD3 delays transferrin recycling from the ERC to the cell surface.Our findings indicate that SUMOylation of EHD3 is involved in tubulation of the ERC membranes, which is important for efficient recycling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, Israel.

ABSTRACT
Endocytosis defines the entry of molecules or macromolecules through the plasma membrane as well as membrane trafficking in the cell. It depends on a large number of proteins that undergo protein-protein and protein-phospholipid interactions. EH Domain containing (EHDs) proteins formulate a family, whose members participate in different stages of endocytosis. Of the four mammalian EHDs (EHD1-EHD4) EHD1 and EHD3 control traffic to the endocytic recycling compartment (ERC) and from the ERC to the plasma membrane, while EHD2 modulates internalization. Recently, we have shown that EHD2 undergoes SUMOylation, which facilitates its exit from the nucleus, where it serves as a co-repressor. In the present study, we tested whether EHD3 undergoes SUMOylation and what is its role in endocytic recycling. We show, both in-vitro and in cell culture, that EHD3 undergoes SUMOylation. Localization of EHD3 to the tubular structures of the ERC depends on its SUMOylation on lysines 315 and 511. Absence of SUMOylation of EHD3 has no effect on its dimerization, an important factor in membrane localization of EHD3, but has a dominant negative effect on its appearance in tubular ERC structures. Non-SUMOylated EHD3 delays transferrin recycling from the ERC to the cell surface. Our findings indicate that SUMOylation of EHD3 is involved in tubulation of the ERC membranes, which is important for efficient recycling.

No MeSH data available.


Related in: MedlinePlus

The effect of EHD3 SUMOylation on EHD1 localization.A. Lysates of HEK293T cells, transiently cotransfected with GFP-EHD1 and either wt or one of the SUMOylation mutants of EHD3, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates and 5% of cell lysates were subjected to SDS-PAGE and the corresponding blots were interacted with anti-myc and anti-GFP antibodies. IP: immunoprecipitation; WB: western blot. B. COS-7 cells were transiently cotransfected with plasmids expressing GFP-EHD1 together with either myc-EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315+511)R]. Twenty-four hours later cells were fixed with 4% paraformaldehyde and visualized (left panel). Right panels depict enlarged regions of the cells. Scale bars represent 10 μm.
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pone.0134053.g006: The effect of EHD3 SUMOylation on EHD1 localization.A. Lysates of HEK293T cells, transiently cotransfected with GFP-EHD1 and either wt or one of the SUMOylation mutants of EHD3, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates and 5% of cell lysates were subjected to SDS-PAGE and the corresponding blots were interacted with anti-myc and anti-GFP antibodies. IP: immunoprecipitation; WB: western blot. B. COS-7 cells were transiently cotransfected with plasmids expressing GFP-EHD1 together with either myc-EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315+511)R]. Twenty-four hours later cells were fixed with 4% paraformaldehyde and visualized (left panel). Right panels depict enlarged regions of the cells. Scale bars represent 10 μm.

Mentions: EHD1, another EHD family member, interacts with EHD3 [20] through their EH-NPF motifs [21]. Also, EHD3 was shown to regulate tubular localization of EHD1 [20]. Therefore, it was interesting to test whether SUMOylation of EHD3 plays a role in this interaction and whether it affects EHD1 localization. Results of coimmunoprecipitation analysis of lysates from HEK293 cells, transfected with plasmids expressing the four variants of EHD3 (Fig 6A), showed that interaction between EHD1 and EHD3 does not depend on EHD3 SUMOylation. However, EHD1 lost its tubular localization in cells expressing EHD3K(315+511)R variant and concentrated in the perinuclear area (Fig 6B). It localized both to very short tubules and vesicular structures in cells expressing EHD3K511R (Fig 6B). However, in cells expressing either EHD3K315R or wt EHD3 variants, EHD1 localized to long tubules. These results imply that SUMOylation of EHD3 is involved in the formation of tubular ERC and therefore, affects both EHD3 and EHD1 localization to the peripheral tubular recycling endosomes".


SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.

Cabasso O, Pekar O, Horowitz M - PLoS ONE (2015)

The effect of EHD3 SUMOylation on EHD1 localization.A. Lysates of HEK293T cells, transiently cotransfected with GFP-EHD1 and either wt or one of the SUMOylation mutants of EHD3, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates and 5% of cell lysates were subjected to SDS-PAGE and the corresponding blots were interacted with anti-myc and anti-GFP antibodies. IP: immunoprecipitation; WB: western blot. B. COS-7 cells were transiently cotransfected with plasmids expressing GFP-EHD1 together with either myc-EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315+511)R]. Twenty-four hours later cells were fixed with 4% paraformaldehyde and visualized (left panel). Right panels depict enlarged regions of the cells. Scale bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4520680&req=5

pone.0134053.g006: The effect of EHD3 SUMOylation on EHD1 localization.A. Lysates of HEK293T cells, transiently cotransfected with GFP-EHD1 and either wt or one of the SUMOylation mutants of EHD3, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates and 5% of cell lysates were subjected to SDS-PAGE and the corresponding blots were interacted with anti-myc and anti-GFP antibodies. IP: immunoprecipitation; WB: western blot. B. COS-7 cells were transiently cotransfected with plasmids expressing GFP-EHD1 together with either myc-EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315+511)R]. Twenty-four hours later cells were fixed with 4% paraformaldehyde and visualized (left panel). Right panels depict enlarged regions of the cells. Scale bars represent 10 μm.
Mentions: EHD1, another EHD family member, interacts with EHD3 [20] through their EH-NPF motifs [21]. Also, EHD3 was shown to regulate tubular localization of EHD1 [20]. Therefore, it was interesting to test whether SUMOylation of EHD3 plays a role in this interaction and whether it affects EHD1 localization. Results of coimmunoprecipitation analysis of lysates from HEK293 cells, transfected with plasmids expressing the four variants of EHD3 (Fig 6A), showed that interaction between EHD1 and EHD3 does not depend on EHD3 SUMOylation. However, EHD1 lost its tubular localization in cells expressing EHD3K(315+511)R variant and concentrated in the perinuclear area (Fig 6B). It localized both to very short tubules and vesicular structures in cells expressing EHD3K511R (Fig 6B). However, in cells expressing either EHD3K315R or wt EHD3 variants, EHD1 localized to long tubules. These results imply that SUMOylation of EHD3 is involved in the formation of tubular ERC and therefore, affects both EHD3 and EHD1 localization to the peripheral tubular recycling endosomes".

Bottom Line: We show, both in-vitro and in cell culture, that EHD3 undergoes SUMOylation.Non-SUMOylated EHD3 delays transferrin recycling from the ERC to the cell surface.Our findings indicate that SUMOylation of EHD3 is involved in tubulation of the ERC membranes, which is important for efficient recycling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, Israel.

ABSTRACT
Endocytosis defines the entry of molecules or macromolecules through the plasma membrane as well as membrane trafficking in the cell. It depends on a large number of proteins that undergo protein-protein and protein-phospholipid interactions. EH Domain containing (EHDs) proteins formulate a family, whose members participate in different stages of endocytosis. Of the four mammalian EHDs (EHD1-EHD4) EHD1 and EHD3 control traffic to the endocytic recycling compartment (ERC) and from the ERC to the plasma membrane, while EHD2 modulates internalization. Recently, we have shown that EHD2 undergoes SUMOylation, which facilitates its exit from the nucleus, where it serves as a co-repressor. In the present study, we tested whether EHD3 undergoes SUMOylation and what is its role in endocytic recycling. We show, both in-vitro and in cell culture, that EHD3 undergoes SUMOylation. Localization of EHD3 to the tubular structures of the ERC depends on its SUMOylation on lysines 315 and 511. Absence of SUMOylation of EHD3 has no effect on its dimerization, an important factor in membrane localization of EHD3, but has a dominant negative effect on its appearance in tubular ERC structures. Non-SUMOylated EHD3 delays transferrin recycling from the ERC to the cell surface. Our findings indicate that SUMOylation of EHD3 is involved in tubulation of the ERC membranes, which is important for efficient recycling.

No MeSH data available.


Related in: MedlinePlus