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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

SUMOylation affects ERC localization of EHD3A. COS-7 cells were transiently transfected with plasmids expressing GFP-EHD3 or its SUMOylation mutants. Twenty-four hours later cells were fixed with 4% paraformaldehyde, permeabilized and incubated with anti-Rab11 antibody. Detection was performed with rhodamine conjugated goat anti-rabbit antibodies. The results were visualized using a confocal microscopy (left panel). Right panels depict enlarged regions of the cells. Scale bars represent 10 μm. B. Quantification of signal intensity obtained from the perinuclear area of the ERC (the signal was measured from non-tubular structures or tubules, which are less than 2 μm in length). About 40 cells were analyzed for each type of protein. The level of signal in the wt sample was considered 100%. C. The percent increase in perinuclear signal, calculated from the mean values.
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pone.0134053.g002: SUMOylation affects ERC localization of EHD3A. COS-7 cells were transiently transfected with plasmids expressing GFP-EHD3 or its SUMOylation mutants. Twenty-four hours later cells were fixed with 4% paraformaldehyde, permeabilized and incubated with anti-Rab11 antibody. Detection was performed with rhodamine conjugated goat anti-rabbit antibodies. The results were visualized using a confocal microscopy (left panel). Right panels depict enlarged regions of the cells. Scale bars represent 10 μm. B. Quantification of signal intensity obtained from the perinuclear area of the ERC (the signal was measured from non-tubular structures or tubules, which are less than 2 μm in length). About 40 cells were analyzed for each type of protein. The level of signal in the wt sample was considered 100%. C. The percent increase in perinuclear signal, calculated from the mean values.

Mentions: Since we have shown in the past that EHD3 localizes to endocytic recycling tubules [20], we tested whether the SUMOylation variants of EHD3 localize to Rab11-positive ERC structures [42]. All EHD3 variants colocalized with Rab11 (Fig 2). WT and K315R variants colocalized with Rab11 in typical tubular structures (of size longer than 2 μm), while EHD3K511R colocalized in shorter tubules (less than 2 μm in length). However, the double mutant lost almost completely its tubular localization and concentrated in the perinuclear area of the ERC (defined as closest to the nucleus area, marked by colocalization with Rab11) (Fig 2A). Quantification of perinuclear, non-tubular, ERC staining (Fig 2B and 2C) showed a 2.2 fold increase in the perinuclear signal of the double EHD3 mutant compared to wt EHD3 while EHD3K511R variant presented 1.85 fold increase, indicating the important role of Lys511 in the localization of EHD3 to the tubular structures. Although there was only a slight increase in the signal of perinuclear, non-tubular GFP-EHD3K315R compared to wt EHD3, it is likely that Lys315 has a minor contribution to the phenotype of the double mutant variant. We also tested the colocalization of EHD3 and its SUMOylation mutants with a known marker for recycling ERC tubules, Rab11-FIP2 [21]. All EHD3 variants colocalized with transfected Rab11-FIP2 (Fig 3). WT and K315R variants colocalized with Rab11-FIP2 in typical tubular structures, while EHD3K511R colocalized with this ERC marker in shorter tubules (less than 2 μm in length). However, the double mutant of EHD3 and transfected Rab11-FIP2 lost almost completely their tubular localization and both concentrated in the perinuclear area of the ERC (Fig 3A). These results are in agreement with published data, which showed that EHD3 modulates localization of Rab11-FIP2 [21]. Quantification of tubular ERC staining (Fig 3B and 3C) revealed almost complete loss of double mutant of EHD3 from the ERC tubular structures. EHDK511R presented 4 fold decrease in the tubular ERC signal, while EHD3K315R showed 1.2 fold decrease, reinforcing our observation that EHD3 SUMOylation on both sites has a synergistic effect. These data imply that SUMOylation of EHD3 is involved in regulation of its localization to the peripheral tubular recycling endosomes and disruption of this posttranslational modification results in accumulation of EHD3 in the perinuclear, non-tubular fraction of the ERC.


SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.

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

SUMOylation affects ERC localization of EHD3A. COS-7 cells were transiently transfected with plasmids expressing GFP-EHD3 or its SUMOylation mutants. Twenty-four hours later cells were fixed with 4% paraformaldehyde, permeabilized and incubated with anti-Rab11 antibody. Detection was performed with rhodamine conjugated goat anti-rabbit antibodies. The results were visualized using a confocal microscopy (left panel). Right panels depict enlarged regions of the cells. Scale bars represent 10 μm. B. Quantification of signal intensity obtained from the perinuclear area of the ERC (the signal was measured from non-tubular structures or tubules, which are less than 2 μm in length). About 40 cells were analyzed for each type of protein. The level of signal in the wt sample was considered 100%. C. The percent increase in perinuclear signal, calculated from the mean values.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520680&req=5

pone.0134053.g002: SUMOylation affects ERC localization of EHD3A. COS-7 cells were transiently transfected with plasmids expressing GFP-EHD3 or its SUMOylation mutants. Twenty-four hours later cells were fixed with 4% paraformaldehyde, permeabilized and incubated with anti-Rab11 antibody. Detection was performed with rhodamine conjugated goat anti-rabbit antibodies. The results were visualized using a confocal microscopy (left panel). Right panels depict enlarged regions of the cells. Scale bars represent 10 μm. B. Quantification of signal intensity obtained from the perinuclear area of the ERC (the signal was measured from non-tubular structures or tubules, which are less than 2 μm in length). About 40 cells were analyzed for each type of protein. The level of signal in the wt sample was considered 100%. C. The percent increase in perinuclear signal, calculated from the mean values.
Mentions: Since we have shown in the past that EHD3 localizes to endocytic recycling tubules [20], we tested whether the SUMOylation variants of EHD3 localize to Rab11-positive ERC structures [42]. All EHD3 variants colocalized with Rab11 (Fig 2). WT and K315R variants colocalized with Rab11 in typical tubular structures (of size longer than 2 μm), while EHD3K511R colocalized in shorter tubules (less than 2 μm in length). However, the double mutant lost almost completely its tubular localization and concentrated in the perinuclear area of the ERC (defined as closest to the nucleus area, marked by colocalization with Rab11) (Fig 2A). Quantification of perinuclear, non-tubular, ERC staining (Fig 2B and 2C) showed a 2.2 fold increase in the perinuclear signal of the double EHD3 mutant compared to wt EHD3 while EHD3K511R variant presented 1.85 fold increase, indicating the important role of Lys511 in the localization of EHD3 to the tubular structures. Although there was only a slight increase in the signal of perinuclear, non-tubular GFP-EHD3K315R compared to wt EHD3, it is likely that Lys315 has a minor contribution to the phenotype of the double mutant variant. We also tested the colocalization of EHD3 and its SUMOylation mutants with a known marker for recycling ERC tubules, Rab11-FIP2 [21]. All EHD3 variants colocalized with transfected Rab11-FIP2 (Fig 3). WT and K315R variants colocalized with Rab11-FIP2 in typical tubular structures, while EHD3K511R colocalized with this ERC marker in shorter tubules (less than 2 μm in length). However, the double mutant of EHD3 and transfected Rab11-FIP2 lost almost completely their tubular localization and both concentrated in the perinuclear area of the ERC (Fig 3A). These results are in agreement with published data, which showed that EHD3 modulates localization of Rab11-FIP2 [21]. Quantification of tubular ERC staining (Fig 3B and 3C) revealed almost complete loss of double mutant of EHD3 from the ERC tubular structures. EHDK511R presented 4 fold decrease in the tubular ERC signal, while EHD3K315R showed 1.2 fold decrease, reinforcing our observation that EHD3 SUMOylation on both sites has a synergistic effect. These data imply that SUMOylation of EHD3 is involved in regulation of its localization to the peripheral tubular recycling endosomes and disruption of this posttranslational modification results in accumulation of EHD3 in the perinuclear, non-tubular fraction of the ERC.

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