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Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.

Bache KG, Brech A, Mehlum A, Stenmark H - J. Cell Biol. (2003)

Bottom Line: Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes.Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes.We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

ABSTRACT
Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.

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Overexpression of Hrs prevents the localization of Tsg101 to late endosomes. HeLa cells were transfected (D–L) or not (A–C) with Hrs for 48 h and permeabilized before fixation. The cells were labeled with anti-Tsg101 (A, D, and G), anti-EEA1 (B and H), or anti-LAMP-1 (I). Yellow (C, F, and J) or purple (K) color in merged images indicates colocalization. An interference contrast image of the Hrs-transfected cell (G–K) is shown in L (colors are inverted in this panel). Bar, 5 μm.
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fig7: Overexpression of Hrs prevents the localization of Tsg101 to late endosomes. HeLa cells were transfected (D–L) or not (A–C) with Hrs for 48 h and permeabilized before fixation. The cells were labeled with anti-Tsg101 (A, D, and G), anti-EEA1 (B and H), or anti-LAMP-1 (I). Yellow (C, F, and J) or purple (K) color in merged images indicates colocalization. An interference contrast image of the Hrs-transfected cell (G–K) is shown in L (colors are inverted in this panel). Bar, 5 μm.

Mentions: The finding that Hrs is required for recruitment of Tsg101 to endosomes prompted us to ask whether an increased expression of Hrs would cause an increased amount of Tsg101 on early endosomes. Therefore, we compared the intracellular localization of endogenous Tsg101 in untransfected cells and cells that overexpressed Hrs. Confocal immunofluorescence microscopy showed that there was little colocalization between Tsg101 and EEA1 in untransfected cells (Fig. 7, A–C), in agreement with the finding that Tsg101 colocalizes extensively with LAMP-1 (Fig. 5, A–C). In contrast, Hrs overexpression led to increased colocalization between Hrs and Tsg101 (Fig. 7, D–F), and caused a strong redistribution of Tsg101 to early endosomes, which were now found clustered in the perinuclear region (Fig. 7, G and H). Interestingly, although Tsg101 colocalized extensively with LAMP-1 in control cells (Fig. 5, A–C), Tsg101 was almost absent from LAMP-1–positive late endocytic structures in the Hrs-transfected cells (Fig. 7, G, I, and K), even though these were frequently located close to early endosomes. Instead, it colocalized strongly with EEA1 (Fig. 7, G, H, and J), which was the opposite of what was seen in untransfected cells (compare with Fig. 7, A–C). This suggests that Hrs overexpression retains Tsg101 on early endosomes and prevents its association with late endosomes and lysosomes, which is consistent with the idea that Hrs mediates recruitment of Tsg101 to early endosomes.


Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.

Bache KG, Brech A, Mehlum A, Stenmark H - J. Cell Biol. (2003)

Overexpression of Hrs prevents the localization of Tsg101 to late endosomes. HeLa cells were transfected (D–L) or not (A–C) with Hrs for 48 h and permeabilized before fixation. The cells were labeled with anti-Tsg101 (A, D, and G), anti-EEA1 (B and H), or anti-LAMP-1 (I). Yellow (C, F, and J) or purple (K) color in merged images indicates colocalization. An interference contrast image of the Hrs-transfected cell (G–K) is shown in L (colors are inverted in this panel). Bar, 5 μm.
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Related In: Results  -  Collection

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fig7: Overexpression of Hrs prevents the localization of Tsg101 to late endosomes. HeLa cells were transfected (D–L) or not (A–C) with Hrs for 48 h and permeabilized before fixation. The cells were labeled with anti-Tsg101 (A, D, and G), anti-EEA1 (B and H), or anti-LAMP-1 (I). Yellow (C, F, and J) or purple (K) color in merged images indicates colocalization. An interference contrast image of the Hrs-transfected cell (G–K) is shown in L (colors are inverted in this panel). Bar, 5 μm.
Mentions: The finding that Hrs is required for recruitment of Tsg101 to endosomes prompted us to ask whether an increased expression of Hrs would cause an increased amount of Tsg101 on early endosomes. Therefore, we compared the intracellular localization of endogenous Tsg101 in untransfected cells and cells that overexpressed Hrs. Confocal immunofluorescence microscopy showed that there was little colocalization between Tsg101 and EEA1 in untransfected cells (Fig. 7, A–C), in agreement with the finding that Tsg101 colocalizes extensively with LAMP-1 (Fig. 5, A–C). In contrast, Hrs overexpression led to increased colocalization between Hrs and Tsg101 (Fig. 7, D–F), and caused a strong redistribution of Tsg101 to early endosomes, which were now found clustered in the perinuclear region (Fig. 7, G and H). Interestingly, although Tsg101 colocalized extensively with LAMP-1 in control cells (Fig. 5, A–C), Tsg101 was almost absent from LAMP-1–positive late endocytic structures in the Hrs-transfected cells (Fig. 7, G, I, and K), even though these were frequently located close to early endosomes. Instead, it colocalized strongly with EEA1 (Fig. 7, G, H, and J), which was the opposite of what was seen in untransfected cells (compare with Fig. 7, A–C). This suggests that Hrs overexpression retains Tsg101 on early endosomes and prevents its association with late endosomes and lysosomes, which is consistent with the idea that Hrs mediates recruitment of Tsg101 to early endosomes.

Bottom Line: Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes.Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes.We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

ABSTRACT
Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.

Show MeSH
Related in: MedlinePlus