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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 SUMOylation on dimerization of EHD3.A. Lysates of HEK293T cells, transiently transfected with different combinations of EHD3 variants, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates and 5% of the 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 myc-EHD3 together with either GFP-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.g005: The effect of SUMOylation on dimerization of EHD3.A. Lysates of HEK293T cells, transiently transfected with different combinations of EHD3 variants, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates and 5% of the 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 myc-EHD3 together with either GFP-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: EHD2 form dimers, which allow their membrane binding. Further oligomerization of EHD2 allows membrane tubulation (bending) [16]. Membrane tubulation has been shown recently for EHD3 as well [19]. Since we suggest a role for SUMOylation in regulating localization of EHD3 to recycling endosomal tubules, we tested whether it plays a role in dimerization of EHD3. To this aim, coimmunoprecipitation was performed on lysates prepared from HEK293T cells, transfected with plasmids expressing different tagged wt or mutant variants of EHD3. The results depicted in Fig 5A indicated that any variant of EHD3 (wt, single mutant or double mutant) was present in the same complex with either wt or any of the EHD3 SUMOylation mutants. These results strongly suggest that SUMOylation does not affect dimerization. We next tested whether SUMOylation affects localization of EHD3 dimers to the tubular structures. We did so by immunostaining COS cells, transiently transfected with myc-EHD3 and all GFP-EHD3 SUMOylation variants, with the corresponding antibodies. The results strongly indicated that myc-EHD3 colocalized with all GFP-EHD3 SUMOylation variants and were consistent with the immunoprecipitation results (Fig 5B). In cells expressing myc-EHD3 together with either GFP-EHD3 or GFP-EHD3K315R colocalization appeared mainly to the tubular structures. In contrast, myc-EHD3 poorly localized to the tubular structures when it was expressed together with GFP-EHD3K511R and hardly did so in tandem with GFP-EHD3K(315+511)R. Taken together, these results indicate that SUMOylation is not necessary for EHD3 dimerization. However localization of EHD3 (most probably as oligomers) to tubular recycling endosomes depends on SUMOylation of both monomers.


SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.

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

The effect of SUMOylation on dimerization of EHD3.A. Lysates of HEK293T cells, transiently transfected with different combinations of EHD3 variants, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates and 5% of the 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 myc-EHD3 together with either GFP-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

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

pone.0134053.g005: The effect of SUMOylation on dimerization of EHD3.A. Lysates of HEK293T cells, transiently transfected with different combinations of EHD3 variants, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates and 5% of the 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 myc-EHD3 together with either GFP-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: EHD2 form dimers, which allow their membrane binding. Further oligomerization of EHD2 allows membrane tubulation (bending) [16]. Membrane tubulation has been shown recently for EHD3 as well [19]. Since we suggest a role for SUMOylation in regulating localization of EHD3 to recycling endosomal tubules, we tested whether it plays a role in dimerization of EHD3. To this aim, coimmunoprecipitation was performed on lysates prepared from HEK293T cells, transfected with plasmids expressing different tagged wt or mutant variants of EHD3. The results depicted in Fig 5A indicated that any variant of EHD3 (wt, single mutant or double mutant) was present in the same complex with either wt or any of the EHD3 SUMOylation mutants. These results strongly suggest that SUMOylation does not affect dimerization. We next tested whether SUMOylation affects localization of EHD3 dimers to the tubular structures. We did so by immunostaining COS cells, transiently transfected with myc-EHD3 and all GFP-EHD3 SUMOylation variants, with the corresponding antibodies. The results strongly indicated that myc-EHD3 colocalized with all GFP-EHD3 SUMOylation variants and were consistent with the immunoprecipitation results (Fig 5B). In cells expressing myc-EHD3 together with either GFP-EHD3 or GFP-EHD3K315R colocalization appeared mainly to the tubular structures. In contrast, myc-EHD3 poorly localized to the tubular structures when it was expressed together with GFP-EHD3K511R and hardly did so in tandem with GFP-EHD3K(315+511)R. Taken together, these results indicate that SUMOylation is not necessary for EHD3 dimerization. However localization of EHD3 (most probably as oligomers) to tubular recycling endosomes depends on SUMOylation of both monomers.

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