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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

EHD3 undergoes SUMOylation.A. Lysates of HEK293T cells, transiently transfected with myc–EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315R+511)R] and HA–SUMO, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA (to visualize SUMOylation) and anti-myc (to visualize the EHD3 variant) antibodies. In parallel, 5% of the lysates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA antibody (in order to assess transfection with SUMO1) and anti-myc antibody (to follow presence of transfected EHD3 variant).B. Two micrograms of bacterially purified EHD3 or its SUMOylation mutants (EHD3K315A, EHD3K511A, EHD3K315A/K511A) were incubated with human SUMO1 as detailed in Materials and Methods. The reaction products were subjected to SDS-PAGE and the corresponding blot was incubated with anti-His antibody. IP: immunoprecipitation; WB: western blot.
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pone.0134053.g004: EHD3 undergoes SUMOylation.A. Lysates of HEK293T cells, transiently transfected with myc–EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315R+511)R] and HA–SUMO, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA (to visualize SUMOylation) and anti-myc (to visualize the EHD3 variant) antibodies. In parallel, 5% of the lysates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA antibody (in order to assess transfection with SUMO1) and anti-myc antibody (to follow presence of transfected EHD3 variant).B. Two micrograms of bacterially purified EHD3 or its SUMOylation mutants (EHD3K315A, EHD3K511A, EHD3K315A/K511A) were incubated with human SUMO1 as detailed in Materials and Methods. The reaction products were subjected to SDS-PAGE and the corresponding blot was incubated with anti-His antibody. IP: immunoprecipitation; WB: western blot.

Mentions: Aiming at showing that EHD3 undergoes SUMOylation, we tested association of SUMO with EHD3 in cell lysates, prepared from COS cells, transfected with HA-SUMO and different variants of myc-tagged EHD3 expressing plasmids. The results indicated that either wt EHD3 or the EHD3K315R mutant coimmunoprecipitated with HA-SUMO almost at the same level and created a typical ladder of increasing protein masses. However, the Lys511 mutant form of EHD3 presented a significant decrease in its association with SUMO in comparison to wt EHD3. Moreover, the EHD3 double mutant showed almost no association with SUMO protein (Fig 4A). To confirm the ability of EHD3 to undergo SUMOylation, in-vitro SUMOylation experiments were performed using bacterially expressed proteins. In-vitro sumoylation of wt and K315A EHD3 variants resulted in the appearance of several high molecular weight forms of EHD3 (Fig 4B). Mutation in Lys511 led to a significant decrease in the appearance of modified forms of EHD3, while double mutation in both Lys315 and Lys511 resulted in an almost complete disappearance of the SUMOylated forms of EHD3 in comparison to wt EHD3 or the single K315AEHD3 mutant (Fig 4B), confirming SUMOylation of EHD3 on Lys315 and Lys511.


SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.

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

EHD3 undergoes SUMOylation.A. Lysates of HEK293T cells, transiently transfected with myc–EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315R+511)R] and HA–SUMO, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA (to visualize SUMOylation) and anti-myc (to visualize the EHD3 variant) antibodies. In parallel, 5% of the lysates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA antibody (in order to assess transfection with SUMO1) and anti-myc antibody (to follow presence of transfected EHD3 variant).B. Two micrograms of bacterially purified EHD3 or its SUMOylation mutants (EHD3K315A, EHD3K511A, EHD3K315A/K511A) were incubated with human SUMO1 as detailed in Materials and Methods. The reaction products were subjected to SDS-PAGE and the corresponding blot was incubated with anti-His antibody. IP: immunoprecipitation; WB: western blot.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520680&req=5

pone.0134053.g004: EHD3 undergoes SUMOylation.A. Lysates of HEK293T cells, transiently transfected with myc–EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315R+511)R] and HA–SUMO, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA (to visualize SUMOylation) and anti-myc (to visualize the EHD3 variant) antibodies. In parallel, 5% of the lysates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA antibody (in order to assess transfection with SUMO1) and anti-myc antibody (to follow presence of transfected EHD3 variant).B. Two micrograms of bacterially purified EHD3 or its SUMOylation mutants (EHD3K315A, EHD3K511A, EHD3K315A/K511A) were incubated with human SUMO1 as detailed in Materials and Methods. The reaction products were subjected to SDS-PAGE and the corresponding blot was incubated with anti-His antibody. IP: immunoprecipitation; WB: western blot.
Mentions: Aiming at showing that EHD3 undergoes SUMOylation, we tested association of SUMO with EHD3 in cell lysates, prepared from COS cells, transfected with HA-SUMO and different variants of myc-tagged EHD3 expressing plasmids. The results indicated that either wt EHD3 or the EHD3K315R mutant coimmunoprecipitated with HA-SUMO almost at the same level and created a typical ladder of increasing protein masses. However, the Lys511 mutant form of EHD3 presented a significant decrease in its association with SUMO in comparison to wt EHD3. Moreover, the EHD3 double mutant showed almost no association with SUMO protein (Fig 4A). To confirm the ability of EHD3 to undergo SUMOylation, in-vitro SUMOylation experiments were performed using bacterially expressed proteins. In-vitro sumoylation of wt and K315A EHD3 variants resulted in the appearance of several high molecular weight forms of EHD3 (Fig 4B). Mutation in Lys511 led to a significant decrease in the appearance of modified forms of EHD3, while double mutation in both Lys315 and Lys511 resulted in an almost complete disappearance of the SUMOylated forms of EHD3 in comparison to wt EHD3 or the single K315AEHD3 mutant (Fig 4B), confirming SUMOylation of EHD3 on Lys315 and Lys511.

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