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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 is required for MVB formation. HeLa cells treated with control RNA or siRNA against Hrs were incubated with 5 mg/ml HRP for 15 min and processed for electron microscopy. In control cells, we observed early endosomes of varying sizes (A) and MVBs (B). (C) In siRNA-treated cells we also observed early endosomes, but significantly less MVBs. (A–C) Arrowheads indicate HRP-positive structures. (D) To quantify the effect of siRNA on MVB formation, we estimated the number of MVBs per cell section. We included only MVBs with an appearance as seen in B in the estimation and omitted early endosomal structures as seen in A and C. MVBs were counted and expressed as the mean number of MVBs per cell section. Three separate experiments were performed and 20 cells were counted for each condition. Statistical significance was estimated with the t test. Error bars denote SEM.
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fig3: Hrs is required for MVB formation. HeLa cells treated with control RNA or siRNA against Hrs were incubated with 5 mg/ml HRP for 15 min and processed for electron microscopy. In control cells, we observed early endosomes of varying sizes (A) and MVBs (B). (C) In siRNA-treated cells we also observed early endosomes, but significantly less MVBs. (A–C) Arrowheads indicate HRP-positive structures. (D) To quantify the effect of siRNA on MVB formation, we estimated the number of MVBs per cell section. We included only MVBs with an appearance as seen in B in the estimation and omitted early endosomal structures as seen in A and C. MVBs were counted and expressed as the mean number of MVBs per cell section. Three separate experiments were performed and 20 cells were counted for each condition. Statistical significance was estimated with the t test. Error bars denote SEM.

Mentions: Yeast cells devoid of the Hrs homologue Vps27p have vacuoles that lack intraluminal vesicles (even when vacuolar hydrolase activity is inhibited), and they contain a multilamellar late endosome, the class E compartment (Odorizzi et al., 1998). Similarly, Garland cells and synaptic boutons from hrs mutant Drosophila larvae have reduced numbers of MVBs (Lloyd et al., 2002). Because the endocytic pathway in mammalian cells is better characterized at the morphological level than that of yeast and flies, we found it important to study the ultrastructure of endosomes in HeLa cells depleted of Hrs with siRNA. To label endocytic compartments, the cells were allowed to endocytose HRP for 15 min before fixation and electron microscopy. Whereas early endosomes (Fig. 3 A) and MVBs (Fig. 3 B) could be readily detected in untreated cells, siRNA-treated cells (Fig. 3 C) had ∼50% less MVBs (see quantification in Fig. 3 D). These results indicate that mammalian Hrs, similar to its yeast and fly counterparts, is required for MVB formation.


Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.

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

Hrs is required for MVB formation. HeLa cells treated with control RNA or siRNA against Hrs were incubated with 5 mg/ml HRP for 15 min and processed for electron microscopy. In control cells, we observed early endosomes of varying sizes (A) and MVBs (B). (C) In siRNA-treated cells we also observed early endosomes, but significantly less MVBs. (A–C) Arrowheads indicate HRP-positive structures. (D) To quantify the effect of siRNA on MVB formation, we estimated the number of MVBs per cell section. We included only MVBs with an appearance as seen in B in the estimation and omitted early endosomal structures as seen in A and C. MVBs were counted and expressed as the mean number of MVBs per cell section. Three separate experiments were performed and 20 cells were counted for each condition. Statistical significance was estimated with the t test. Error bars denote SEM.
© Copyright Policy
Related In: Results  -  Collection

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fig3: Hrs is required for MVB formation. HeLa cells treated with control RNA or siRNA against Hrs were incubated with 5 mg/ml HRP for 15 min and processed for electron microscopy. In control cells, we observed early endosomes of varying sizes (A) and MVBs (B). (C) In siRNA-treated cells we also observed early endosomes, but significantly less MVBs. (A–C) Arrowheads indicate HRP-positive structures. (D) To quantify the effect of siRNA on MVB formation, we estimated the number of MVBs per cell section. We included only MVBs with an appearance as seen in B in the estimation and omitted early endosomal structures as seen in A and C. MVBs were counted and expressed as the mean number of MVBs per cell section. Three separate experiments were performed and 20 cells were counted for each condition. Statistical significance was estimated with the t test. Error bars denote SEM.
Mentions: Yeast cells devoid of the Hrs homologue Vps27p have vacuoles that lack intraluminal vesicles (even when vacuolar hydrolase activity is inhibited), and they contain a multilamellar late endosome, the class E compartment (Odorizzi et al., 1998). Similarly, Garland cells and synaptic boutons from hrs mutant Drosophila larvae have reduced numbers of MVBs (Lloyd et al., 2002). Because the endocytic pathway in mammalian cells is better characterized at the morphological level than that of yeast and flies, we found it important to study the ultrastructure of endosomes in HeLa cells depleted of Hrs with siRNA. To label endocytic compartments, the cells were allowed to endocytose HRP for 15 min before fixation and electron microscopy. Whereas early endosomes (Fig. 3 A) and MVBs (Fig. 3 B) could be readily detected in untreated cells, siRNA-treated cells (Fig. 3 C) had ∼50% less MVBs (see quantification in Fig. 3 D). These results indicate that mammalian Hrs, similar to its yeast and fly counterparts, is required for MVB formation.

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