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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

Localization of predicted SUMOylation mutants of EHD3.A. Multiple alignment of potential SUMOylation sites in different EHD homologs and their scores (given by SUMOplot™ Analysis Program). The positions of consensus SUMOylation sites are underlined. Position numbers are relevant to human EHD3. B. COS-7 cells were transiently transfected with wt GFP-EHD3 or its SUMOylation mutants: GFP-EHD3K315R, GFP-EHD3K511R, GFP-EHD3K(315+511)R. Twenty-four hours later cells were fixed with 4% paraformaldehyde and visualized using confocal microscopy. Right panels depict enlarged regions of the cells and show tubular structures of EHD3. Scale bars represent 10 μm. C. Quantification of signal intensity obtained from tubular structures (%) longer than 2 μm in length of either wt or its SUMOylation mutants. The level of signal in the wt sample was considered 100%. ***P<0.0001. Eighty to 100 cells were analyzed for each type of EHD3 variant. D. Shown is the percent reduction in tubular structure signal, calculated from the mean values.
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pone.0134053.g001: Localization of predicted SUMOylation mutants of EHD3.A. Multiple alignment of potential SUMOylation sites in different EHD homologs and their scores (given by SUMOplot™ Analysis Program). The positions of consensus SUMOylation sites are underlined. Position numbers are relevant to human EHD3. B. COS-7 cells were transiently transfected with wt GFP-EHD3 or its SUMOylation mutants: GFP-EHD3K315R, GFP-EHD3K511R, GFP-EHD3K(315+511)R. Twenty-four hours later cells were fixed with 4% paraformaldehyde and visualized using confocal microscopy. Right panels depict enlarged regions of the cells and show tubular structures of EHD3. Scale bars represent 10 μm. C. Quantification of signal intensity obtained from tubular structures (%) longer than 2 μm in length of either wt or its SUMOylation mutants. The level of signal in the wt sample was considered 100%. ***P<0.0001. Eighty to 100 cells were analyzed for each type of EHD3 variant. D. Shown is the percent reduction in tubular structure signal, calculated from the mean values.

Mentions: All mammalian EHDs have one or two putative SUMOylation sites with scores over 0.90 (Fig 1A). We have shown in the past that SUMOylation of EHD2 is important for its exit from the nucleus [30]. Here, we extended our study to test whether EHD3 undergoes SUMOylation and what function it serves, taking into consideration that EHD3 controls trafficking from the early endosomes to the ERC [21] and from the ERC to the plasma membrane [20, 22]. Thus, we created three variants of EHD3, altered at the predicted SUMOylated Lys315 and Lys511 (EHD3K315R and EHD3K511R, respectively), and the double mutant [EHD3K(315+511)R] (Fig 1A), and tested their cellular localization in COS transfected cells. As shown in Fig 1B and 1C wt EHD3 and EHD3K315R variants were localized to the tubular structures, with a slight reduction in the amount of GFP-EHD3K315R stained tubules compared to wt EHD3. A significant decrease in the number of EHD3 stained tubular structures was observed for the EHD3K511R variant, while the double mutant [EHD3K(315+511)R] lost its tubular localization and was vesicular. These results suggested that SUMOylation of EHD3 on both Lys315 and Lys511 is essential for the localization of ehd3 to the tubular structures and that the effect of two sites is synergistic (Fig 1D). Similar results were observed in COS cells, transfected with the different EHD3 SUMOylated variants, in which the predicted SUMOylated lysines were mutated to alanines (S1 Fig). These findings confirmed that EHD3 SUMOylation on Lys315 and Lys511 is important for its tubular localization and that this effect is synergistic. The results also confirmed that the change of either lysine to alanine (ie: charge change) or lysine to arginine (ie: no change in charge) has the same physiological effect, similarly to what we have shown for EHD2 SUMOylation[30].


SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.

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

Localization of predicted SUMOylation mutants of EHD3.A. Multiple alignment of potential SUMOylation sites in different EHD homologs and their scores (given by SUMOplot™ Analysis Program). The positions of consensus SUMOylation sites are underlined. Position numbers are relevant to human EHD3. B. COS-7 cells were transiently transfected with wt GFP-EHD3 or its SUMOylation mutants: GFP-EHD3K315R, GFP-EHD3K511R, GFP-EHD3K(315+511)R. Twenty-four hours later cells were fixed with 4% paraformaldehyde and visualized using confocal microscopy. Right panels depict enlarged regions of the cells and show tubular structures of EHD3. Scale bars represent 10 μm. C. Quantification of signal intensity obtained from tubular structures (%) longer than 2 μm in length of either wt or its SUMOylation mutants. The level of signal in the wt sample was considered 100%. ***P<0.0001. Eighty to 100 cells were analyzed for each type of EHD3 variant. D. Shown is the percent reduction in tubular structure 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.g001: Localization of predicted SUMOylation mutants of EHD3.A. Multiple alignment of potential SUMOylation sites in different EHD homologs and their scores (given by SUMOplot™ Analysis Program). The positions of consensus SUMOylation sites are underlined. Position numbers are relevant to human EHD3. B. COS-7 cells were transiently transfected with wt GFP-EHD3 or its SUMOylation mutants: GFP-EHD3K315R, GFP-EHD3K511R, GFP-EHD3K(315+511)R. Twenty-four hours later cells were fixed with 4% paraformaldehyde and visualized using confocal microscopy. Right panels depict enlarged regions of the cells and show tubular structures of EHD3. Scale bars represent 10 μm. C. Quantification of signal intensity obtained from tubular structures (%) longer than 2 μm in length of either wt or its SUMOylation mutants. The level of signal in the wt sample was considered 100%. ***P<0.0001. Eighty to 100 cells were analyzed for each type of EHD3 variant. D. Shown is the percent reduction in tubular structure signal, calculated from the mean values.
Mentions: All mammalian EHDs have one or two putative SUMOylation sites with scores over 0.90 (Fig 1A). We have shown in the past that SUMOylation of EHD2 is important for its exit from the nucleus [30]. Here, we extended our study to test whether EHD3 undergoes SUMOylation and what function it serves, taking into consideration that EHD3 controls trafficking from the early endosomes to the ERC [21] and from the ERC to the plasma membrane [20, 22]. Thus, we created three variants of EHD3, altered at the predicted SUMOylated Lys315 and Lys511 (EHD3K315R and EHD3K511R, respectively), and the double mutant [EHD3K(315+511)R] (Fig 1A), and tested their cellular localization in COS transfected cells. As shown in Fig 1B and 1C wt EHD3 and EHD3K315R variants were localized to the tubular structures, with a slight reduction in the amount of GFP-EHD3K315R stained tubules compared to wt EHD3. A significant decrease in the number of EHD3 stained tubular structures was observed for the EHD3K511R variant, while the double mutant [EHD3K(315+511)R] lost its tubular localization and was vesicular. These results suggested that SUMOylation of EHD3 on both Lys315 and Lys511 is essential for the localization of ehd3 to the tubular structures and that the effect of two sites is synergistic (Fig 1D). Similar results were observed in COS cells, transfected with the different EHD3 SUMOylated variants, in which the predicted SUMOylated lysines were mutated to alanines (S1 Fig). These findings confirmed that EHD3 SUMOylation on Lys315 and Lys511 is important for its tubular localization and that this effect is synergistic. The results also confirmed that the change of either lysine to alanine (ie: charge change) or lysine to arginine (ie: no change in charge) has the same physiological effect, similarly to what we have shown for EHD2 SUMOylation[30].

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