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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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Hrs depletion affects the morphology of late endosomes and lysosomes. HeLa cells treated with control double-stranded RNA (A and B) or siRNA against Hrs (C and D) were prepared for electron microscopy or immunofluorescence microscopy as described in Materials and methods. Late endosomes and lysosomes were visualized by 7 nm internalized BSA gold (A, arrow) or staining with antibodies against LAMP-2 followed by 15 nm protein A–gold (A–D, arrowheads). The boxed area in C is shown magnified in D. Note the different sizes of bars. Immunofluorescence images were obtained by labeling with antibodies against LAMP-1 (E and F). Bar, (E and F) 5 μm. E shows a control cell, whereas F shows an siRNA-treated cell.
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fig4: Hrs depletion affects the morphology of late endosomes and lysosomes. HeLa cells treated with control double-stranded RNA (A and B) or siRNA against Hrs (C and D) were prepared for electron microscopy or immunofluorescence microscopy as described in Materials and methods. Late endosomes and lysosomes were visualized by 7 nm internalized BSA gold (A, arrow) or staining with antibodies against LAMP-2 followed by 15 nm protein A–gold (A–D, arrowheads). The boxed area in C is shown magnified in D. Note the different sizes of bars. Immunofluorescence images were obtained by labeling with antibodies against LAMP-1 (E and F). Bar, (E and F) 5 μm. E shows a control cell, whereas F shows an siRNA-treated cell.

Mentions: Because MVBs fuse with late endosomes and lysosomes (Gruenberg, 2001; Katzmann et al., 2002), we next asked whether Hrs depletion would affect the morphology of these organelles. First, we studied this by confocal microscopy using an antibody against the late endosome/lysosome marker LAMP-1. In cells treated with a control RNA molecule, numerous LAMP-1–positive structures were found in the perinuclear region, as expected (Fig. 4 E). However, in cells treated with siRNA against Hrs, the LAMP-1–positive structures were found concentrated close to the nucleus (Fig. 4 F). Next, to study this morphological change by electron microscopy, we labeled lysosomes by performing a 3-h pulse/overnight chase incubation with BSA gold. In control cells, we observed LAMP-2 staining on gold-containing electron dense lysosomes with a membranous content (Fig. 4 A), and more occasionally on electron lucent vesicles largely devoid of endocytic tracers and with no intraluminal membranes (Fig. 4 B). In siRNA-treated cells, we observed similar LAMP-2–positive structures, but they often appeared clustered and were greatly enlarged (up to threefold increase in diameter; Fig. 4, C and D). These results indicate that Hrs is required both for MVB formation (Fig. 3) and for normal morphology of late endosomes/lysosomes.


Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.

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

Hrs depletion affects the morphology of late endosomes and lysosomes. HeLa cells treated with control double-stranded RNA (A and B) or siRNA against Hrs (C and D) were prepared for electron microscopy or immunofluorescence microscopy as described in Materials and methods. Late endosomes and lysosomes were visualized by 7 nm internalized BSA gold (A, arrow) or staining with antibodies against LAMP-2 followed by 15 nm protein A–gold (A–D, arrowheads). The boxed area in C is shown magnified in D. Note the different sizes of bars. Immunofluorescence images were obtained by labeling with antibodies against LAMP-1 (E and F). Bar, (E and F) 5 μm. E shows a control cell, whereas F shows an siRNA-treated cell.
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Related In: Results  -  Collection

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fig4: Hrs depletion affects the morphology of late endosomes and lysosomes. HeLa cells treated with control double-stranded RNA (A and B) or siRNA against Hrs (C and D) were prepared for electron microscopy or immunofluorescence microscopy as described in Materials and methods. Late endosomes and lysosomes were visualized by 7 nm internalized BSA gold (A, arrow) or staining with antibodies against LAMP-2 followed by 15 nm protein A–gold (A–D, arrowheads). The boxed area in C is shown magnified in D. Note the different sizes of bars. Immunofluorescence images were obtained by labeling with antibodies against LAMP-1 (E and F). Bar, (E and F) 5 μm. E shows a control cell, whereas F shows an siRNA-treated cell.
Mentions: Because MVBs fuse with late endosomes and lysosomes (Gruenberg, 2001; Katzmann et al., 2002), we next asked whether Hrs depletion would affect the morphology of these organelles. First, we studied this by confocal microscopy using an antibody against the late endosome/lysosome marker LAMP-1. In cells treated with a control RNA molecule, numerous LAMP-1–positive structures were found in the perinuclear region, as expected (Fig. 4 E). However, in cells treated with siRNA against Hrs, the LAMP-1–positive structures were found concentrated close to the nucleus (Fig. 4 F). Next, to study this morphological change by electron microscopy, we labeled lysosomes by performing a 3-h pulse/overnight chase incubation with BSA gold. In control cells, we observed LAMP-2 staining on gold-containing electron dense lysosomes with a membranous content (Fig. 4 A), and more occasionally on electron lucent vesicles largely devoid of endocytic tracers and with no intraluminal membranes (Fig. 4 B). In siRNA-treated cells, we observed similar LAMP-2–positive structures, but they often appeared clustered and were greatly enlarged (up to threefold increase in diameter; Fig. 4, C and D). These results indicate that Hrs is required both for MVB formation (Fig. 3) and for normal morphology of late endosomes/lysosomes.

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