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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 the efficient association of Tsg101 with membranes. HeLa cells treated with control RNA (−) or with siRNA against Hrs (+) were fractionated into membrane and cytosolic fractions as described in Materials and methods. Hrs left in the cytosol and on membranes after siRNA treatment, as described in Materials and methods, was analyzed by SDS-PAGE (A and B, top lanes), and the corresponding levels of Tsg101 and hVps28 were shown by sequential blotting of the same membrane with anti-Tsg101 (A) or anti-hVps28 (B). The loaded amount of membrane fraction was sixfold higher than that of the cytosol fraction in A, and twofold higher in B. To visualize transferred proteins, the blots were stained with Ponceau S (bottom panels) before detection of Hrs and Tsg101 (A), and Hrs and hVps28 (B). The relative intensities of the bands from membrane fractions of control and siRNA-treated cells were quantified using ImageQuant 5.0 (C), and are presented as the average of three experiments. Error bars denote SEM.
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fig2: Hrs is required for the efficient association of Tsg101 with membranes. HeLa cells treated with control RNA (−) or with siRNA against Hrs (+) were fractionated into membrane and cytosolic fractions as described in Materials and methods. Hrs left in the cytosol and on membranes after siRNA treatment, as described in Materials and methods, was analyzed by SDS-PAGE (A and B, top lanes), and the corresponding levels of Tsg101 and hVps28 were shown by sequential blotting of the same membrane with anti-Tsg101 (A) or anti-hVps28 (B). The loaded amount of membrane fraction was sixfold higher than that of the cytosol fraction in A, and twofold higher in B. To visualize transferred proteins, the blots were stained with Ponceau S (bottom panels) before detection of Hrs and Tsg101 (A), and Hrs and hVps28 (B). The relative intensities of the bands from membrane fractions of control and siRNA-treated cells were quantified using ImageQuant 5.0 (C), and are presented as the average of three experiments. Error bars denote SEM.

Mentions: Because Hrs binds to the endosome-specific lipid phosphatidylinositol 3-phosphate (Gaullier et al., 1998; Gillooly et al., 2000), this protein is likely to interact directly with the endosome membrane. The finding that Hrs interacts with Tsg101, therefore, raised the possibility that Hrs may function to recruit ESCRT-I to endosome membranes. Should this be the case, then a reduction in the cellular level of Hrs might lead to a decrease in membrane-associated ESCRT-I subunits. To investigate this possibility, we used a specific small interfering RNA (siRNA) molecule for the knockdown of Hrs expression (Elbashir et al., 2002; Bache et al., 2003). As shown in Fig. 2 (A and B), HeLa cells incubated with the siRNA duplex showed a strong decrease in Hrs expression (as assessed with Western blotting with antibodies specific for Hrs) when compared with cells that had been incubated in the presence of a control RNA. Importantly, Western blotting with anti-Tsg101 antibodies showed that there was a significant reduction of membrane-associated Tsg101 levels in cells treated with siRNA to Hrs. We also investigated whether Hrs is required for membrane localization of the other known human ESCRT-I subunit, hVps28, which does not bind directly to Hrs. Like Tsg101, the amount of hVps28 in the membrane fraction was reduced by ∼50% in cells treated with siRNA against Hrs (Fig. 2, B and C). In the case of both Tsg101 and hVps28, the decrease in membrane-associated proteins was less than that of Hrs in Hrs-depleted cells. We do not know the reason for this, but one explanation could be that ESCRT-I is also associated with endosomes via other molecules than Hrs (see Discussion). In any case, these results indicate that Hrs is required for efficient membrane targeting of ESCRT-I.


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 the efficient association of Tsg101 with membranes. HeLa cells treated with control RNA (−) or with siRNA against Hrs (+) were fractionated into membrane and cytosolic fractions as described in Materials and methods. Hrs left in the cytosol and on membranes after siRNA treatment, as described in Materials and methods, was analyzed by SDS-PAGE (A and B, top lanes), and the corresponding levels of Tsg101 and hVps28 were shown by sequential blotting of the same membrane with anti-Tsg101 (A) or anti-hVps28 (B). The loaded amount of membrane fraction was sixfold higher than that of the cytosol fraction in A, and twofold higher in B. To visualize transferred proteins, the blots were stained with Ponceau S (bottom panels) before detection of Hrs and Tsg101 (A), and Hrs and hVps28 (B). The relative intensities of the bands from membrane fractions of control and siRNA-treated cells were quantified using ImageQuant 5.0 (C), and are presented as the average of three experiments. Error bars denote SEM.
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Related In: Results  -  Collection

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fig2: Hrs is required for the efficient association of Tsg101 with membranes. HeLa cells treated with control RNA (−) or with siRNA against Hrs (+) were fractionated into membrane and cytosolic fractions as described in Materials and methods. Hrs left in the cytosol and on membranes after siRNA treatment, as described in Materials and methods, was analyzed by SDS-PAGE (A and B, top lanes), and the corresponding levels of Tsg101 and hVps28 were shown by sequential blotting of the same membrane with anti-Tsg101 (A) or anti-hVps28 (B). The loaded amount of membrane fraction was sixfold higher than that of the cytosol fraction in A, and twofold higher in B. To visualize transferred proteins, the blots were stained with Ponceau S (bottom panels) before detection of Hrs and Tsg101 (A), and Hrs and hVps28 (B). The relative intensities of the bands from membrane fractions of control and siRNA-treated cells were quantified using ImageQuant 5.0 (C), and are presented as the average of three experiments. Error bars denote SEM.
Mentions: Because Hrs binds to the endosome-specific lipid phosphatidylinositol 3-phosphate (Gaullier et al., 1998; Gillooly et al., 2000), this protein is likely to interact directly with the endosome membrane. The finding that Hrs interacts with Tsg101, therefore, raised the possibility that Hrs may function to recruit ESCRT-I to endosome membranes. Should this be the case, then a reduction in the cellular level of Hrs might lead to a decrease in membrane-associated ESCRT-I subunits. To investigate this possibility, we used a specific small interfering RNA (siRNA) molecule for the knockdown of Hrs expression (Elbashir et al., 2002; Bache et al., 2003). As shown in Fig. 2 (A and B), HeLa cells incubated with the siRNA duplex showed a strong decrease in Hrs expression (as assessed with Western blotting with antibodies specific for Hrs) when compared with cells that had been incubated in the presence of a control RNA. Importantly, Western blotting with anti-Tsg101 antibodies showed that there was a significant reduction of membrane-associated Tsg101 levels in cells treated with siRNA to Hrs. We also investigated whether Hrs is required for membrane localization of the other known human ESCRT-I subunit, hVps28, which does not bind directly to Hrs. Like Tsg101, the amount of hVps28 in the membrane fraction was reduced by ∼50% in cells treated with siRNA against Hrs (Fig. 2, B and C). In the case of both Tsg101 and hVps28, the decrease in membrane-associated proteins was less than that of Hrs in Hrs-depleted cells. We do not know the reason for this, but one explanation could be that ESCRT-I is also associated with endosomes via other molecules than Hrs (see Discussion). In any case, these results indicate that Hrs is required for efficient membrane targeting of ESCRT-I.

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