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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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Tsg101 and Hrs colocalize on LBPA-containing endosomes. HeLa cells were permeabilized before fixation and labeled with anti-Hrs (C) and anti-LBPA (B) primary and secondary antibodies before staining with Zenon®-labeled anti-Tsg101 (A) as described in Materials and methods. Colocalization between LBPA and Tsg101 is shown in yellow (D), between LBPA and Hrs in turquoise (E), and between all three molecules in white (F). Examples of profiles positive for all three molecules are indicated by arrows. Bar, 5 μm.
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fig6: Tsg101 and Hrs colocalize on LBPA-containing endosomes. HeLa cells were permeabilized before fixation and labeled with anti-Hrs (C) and anti-LBPA (B) primary and secondary antibodies before staining with Zenon®-labeled anti-Tsg101 (A) as described in Materials and methods. Colocalization between LBPA and Tsg101 is shown in yellow (D), between LBPA and Hrs in turquoise (E), and between all three molecules in white (F). Examples of profiles positive for all three molecules are indicated by arrows. Bar, 5 μm.

Mentions: Assuming that Tsg101 is recruited to endosomes by Hrs but is also found on late, LAMP-1–positive structures, we would expect a significant pool of Tsg101 to be located on intermediates between early and late endosomes. Therefore, we triple labeled HeLa cells for confocal microscopy with antibodies against Tsg101, Hrs, and LBPA, a well-characterized marker for multivesicular late endosomes (Kobayashi et al., 1998). We found extensive colocalization between Tsg101 and LBPA (Fig. 6, A, B, and D). In agreement with Fig. 5, some of the endosomes were positive for both Hrs and Tsg101, and most of these structures also contained LBPA (Fig. 6 A, C, and F). This suggests that Hrs and Tsg101 colocalize on a subset of LBPA-positive structures. Because Hrs colocalizes poorly with the late endosome/lysosome marker LAMP-1 (Fig. 5), we assume that these Hrs-containing LBPA-positive structures represent intermediates between early and late endosomes that are positive for LBPA but negative for LAMP1. In agreement with this idea, previous electron microscopy studies have shown that Hrs is present on the limiting membrane of MVBs (Tsujimoto et al., 1999; Sachse et al., 2002), and we observed LPBA associated with intraluminal vesicles of MVBs (unpublished data).


Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.

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

Tsg101 and Hrs colocalize on LBPA-containing endosomes. HeLa cells were permeabilized before fixation and labeled with anti-Hrs (C) and anti-LBPA (B) primary and secondary antibodies before staining with Zenon®-labeled anti-Tsg101 (A) as described in Materials and methods. Colocalization between LBPA and Tsg101 is shown in yellow (D), between LBPA and Hrs in turquoise (E), and between all three molecules in white (F). Examples of profiles positive for all three molecules are indicated by arrows. Bar, 5 μm.
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Related In: Results  -  Collection

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fig6: Tsg101 and Hrs colocalize on LBPA-containing endosomes. HeLa cells were permeabilized before fixation and labeled with anti-Hrs (C) and anti-LBPA (B) primary and secondary antibodies before staining with Zenon®-labeled anti-Tsg101 (A) as described in Materials and methods. Colocalization between LBPA and Tsg101 is shown in yellow (D), between LBPA and Hrs in turquoise (E), and between all three molecules in white (F). Examples of profiles positive for all three molecules are indicated by arrows. Bar, 5 μm.
Mentions: Assuming that Tsg101 is recruited to endosomes by Hrs but is also found on late, LAMP-1–positive structures, we would expect a significant pool of Tsg101 to be located on intermediates between early and late endosomes. Therefore, we triple labeled HeLa cells for confocal microscopy with antibodies against Tsg101, Hrs, and LBPA, a well-characterized marker for multivesicular late endosomes (Kobayashi et al., 1998). We found extensive colocalization between Tsg101 and LBPA (Fig. 6, A, B, and D). In agreement with Fig. 5, some of the endosomes were positive for both Hrs and Tsg101, and most of these structures also contained LBPA (Fig. 6 A, C, and F). This suggests that Hrs and Tsg101 colocalize on a subset of LBPA-positive structures. Because Hrs colocalizes poorly with the late endosome/lysosome marker LAMP-1 (Fig. 5), we assume that these Hrs-containing LBPA-positive structures represent intermediates between early and late endosomes that are positive for LBPA but negative for LAMP1. In agreement with this idea, previous electron microscopy studies have shown that Hrs is present on the limiting membrane of MVBs (Tsujimoto et al., 1999; Sachse et al., 2002), and we observed LPBA associated with intraluminal vesicles of MVBs (unpublished data).

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