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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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Related in: MedlinePlus

Tsg101 has a broader endosomal distribution than Hrs. HeLa cells grown on coverslips were permeabilized before fixation and double labeled with anti-Tsg101 and anti–LAMP-1 (A–C), anti-Tsg101 and Hrs (D–F), or anti-Hrs and anti–LAMP-1 (G–I). Yellow color in the merged images (C, F, and I) indicates colocalization. Bar, 5 μm.
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fig5: Tsg101 has a broader endosomal distribution than Hrs. HeLa cells grown on coverslips were permeabilized before fixation and double labeled with anti-Tsg101 and anti–LAMP-1 (A–C), anti-Tsg101 and Hrs (D–F), or anti-Hrs and anti–LAMP-1 (G–I). Yellow color in the merged images (C, F, and I) indicates colocalization. Bar, 5 μm.

Mentions: Both Hrs and Tsg101 are known to localize to endosomes (Komada et al., 1997; Bishop and Woodman, 2001), but their intracellular localizations have not been compared directly. Because Hrs is required for the endosomal recruitment of Tsg101, and because both these proteins are involved in endosomal protein sorting and MVB formation, we expected Hrs and Tsg101 to colocalize. Electron microscopy showed that overexpression of Tsg101, even at moderate levels, causes formation of aggresomes (unpublished data). To study the relative localization of Tsg101 and Hrs, we, therefore, stained HeLa cells with antibodies that detect the two endogenous proteins by immunofluorescence microscopy (Fig. 5). As expected, there was a significant overlap in the distribution of Hrs and Tsg101 (Fig. 5, D–F). However, whereas Hrs partially localized to membranes positive for the early endosome marker EEA1 (Urbé et al., 2000; Raiborg et al., 2001b) and was largely absent from LAMP-1–positive late endocytic structures (Fig. 5, G–I), Tsg101 was also found on LAMP-1–positive structures (Fig. 5, A–C). These results suggest that even though Tsg101 requires Hrs for its initial recruitment to endosomes, it stays associated with endosomes much longer than Hrs during endosomal maturation or trafficking.


Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.

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

Tsg101 has a broader endosomal distribution than Hrs. HeLa cells grown on coverslips were permeabilized before fixation and double labeled with anti-Tsg101 and anti–LAMP-1 (A–C), anti-Tsg101 and Hrs (D–F), or anti-Hrs and anti–LAMP-1 (G–I). Yellow color in the merged images (C, F, and I) indicates colocalization. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172703&req=5

fig5: Tsg101 has a broader endosomal distribution than Hrs. HeLa cells grown on coverslips were permeabilized before fixation and double labeled with anti-Tsg101 and anti–LAMP-1 (A–C), anti-Tsg101 and Hrs (D–F), or anti-Hrs and anti–LAMP-1 (G–I). Yellow color in the merged images (C, F, and I) indicates colocalization. Bar, 5 μm.
Mentions: Both Hrs and Tsg101 are known to localize to endosomes (Komada et al., 1997; Bishop and Woodman, 2001), but their intracellular localizations have not been compared directly. Because Hrs is required for the endosomal recruitment of Tsg101, and because both these proteins are involved in endosomal protein sorting and MVB formation, we expected Hrs and Tsg101 to colocalize. Electron microscopy showed that overexpression of Tsg101, even at moderate levels, causes formation of aggresomes (unpublished data). To study the relative localization of Tsg101 and Hrs, we, therefore, stained HeLa cells with antibodies that detect the two endogenous proteins by immunofluorescence microscopy (Fig. 5). As expected, there was a significant overlap in the distribution of Hrs and Tsg101 (Fig. 5, D–F). However, whereas Hrs partially localized to membranes positive for the early endosome marker EEA1 (Urbé et al., 2000; Raiborg et al., 2001b) and was largely absent from LAMP-1–positive late endocytic structures (Fig. 5, G–I), Tsg101 was also found on LAMP-1–positive structures (Fig. 5, A–C). These results suggest that even though Tsg101 requires Hrs for its initial recruitment to endosomes, it stays associated with endosomes much longer than Hrs during endosomal maturation or trafficking.

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