Limits...
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.

Show MeSH

Related in: MedlinePlus

Hrs interacts with Tsg101. (A) A schematic representation of Hrs. The VHS, FYVE, coiled-coil (CC), and clathrin-binding domain (CBD) are indicated. The ubiquitin interacting motif (UIM), PSAP sequence, and proline- and proline/glutamine-rich regions are also indicated. (B) Interaction between Hrs and Tsg101 in the yeast two-hybrid system. The indicated Hrs constructs were used as bait and Tsg101 as prey. The values indicate β-galactosidase activities in arbitrary units. Neither of the bait constructs showed any significant reporter activation (<1 U) in the absence of a prey construct. All determinations were done in triplicate. Error bars denote SEM. (C) Interaction between Hrs and Tsg101 in vitro. GST alone or fused to Hrs287–573 or Hrs287–573P351A were immobilized on glutathione-Sepharose beads and incubated with in vitro–translated 35S-labeled Tsg101 for 1 h at 4°C. The beads were washed and analyzed by SDS-PAGE and fluorography. The amounts bound in lanes 1 and 7 correspond to 2–3% of the input amount. The doublet band is presumably due to translational initiation downstream of the initiator ATG.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172703&req=5

fig1: Hrs interacts with Tsg101. (A) A schematic representation of Hrs. The VHS, FYVE, coiled-coil (CC), and clathrin-binding domain (CBD) are indicated. The ubiquitin interacting motif (UIM), PSAP sequence, and proline- and proline/glutamine-rich regions are also indicated. (B) Interaction between Hrs and Tsg101 in the yeast two-hybrid system. The indicated Hrs constructs were used as bait and Tsg101 as prey. The values indicate β-galactosidase activities in arbitrary units. Neither of the bait constructs showed any significant reporter activation (<1 U) in the absence of a prey construct. All determinations were done in triplicate. Error bars denote SEM. (C) Interaction between Hrs and Tsg101 in vitro. GST alone or fused to Hrs287–573 or Hrs287–573P351A were immobilized on glutathione-Sepharose beads and incubated with in vitro–translated 35S-labeled Tsg101 for 1 h at 4°C. The beads were washed and analyzed by SDS-PAGE and fluorography. The amounts bound in lanes 1 and 7 correspond to 2–3% of the input amount. The doublet band is presumably due to translational initiation downstream of the initiator ATG.

Mentions: The fact that Hrs contains a PSAP sequence (residues 348–351, Fig. 1 A), similar to that found in Tsg101-binding viral proteins (Carter, 2002; Pornillos et al., 2002c), prompted us to ask whether Tsg101 may interact with Hrs. First, we investigated this possibility using the yeast two-hybrid system. Full-length or truncated Hrs constructs were prepared as baits and Tsg101 as prey, and β-galactosidase reporter activity was measured in reporter yeast cells. The results indicated that Hrs does interact with Tsg101 (Fig. 1 B). Although the NH2-terminal part of Hrs, containing the VHS and FYVE domains and ubiquitin-interacting motif (UIM), showed no interaction with Tsg101, the remaining part, containing the proline-rich, coiled-coil, and clathrin binding domains, bound strongly. To study if the PSAP sequence is involved in the interaction, we mutated the final proline residue in this sequence to alanine. Interestingly, this P351A mutation markedly reduced the interaction, but did not abolish it completely. Consistent with this, we also detected a weak interaction between the coiled-coil region of Hrs and Tsg101, and a Hrs construct containing only the proline-rich and coiled-coil domains of (Hrs287–573) showed a strong interaction with Tsg101 (Fig. 1 B). In the companion paper, Pornillos et al. (2003)(in this issue) show that Hrs truncated just after the coiled-coiled domain (at residue 559) binds poorly to Tsg101. Because Hrs287–573, which binds Tsg101 strongly, contains a few additional residues after the coiled-coiled domain, it appears that these residues are important for Tsg101 binding, possibly because they allow correct folding of the coiled-coil region. Together, these results suggest that Tsg101 interacts with several interfaces of Hrs, including the PSAP sequence and the coiled-coil region. We also cannot rule out the possibility that the latter regions join to form a single Tsg101-interacting interface.


Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.

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

Hrs interacts with Tsg101. (A) A schematic representation of Hrs. The VHS, FYVE, coiled-coil (CC), and clathrin-binding domain (CBD) are indicated. The ubiquitin interacting motif (UIM), PSAP sequence, and proline- and proline/glutamine-rich regions are also indicated. (B) Interaction between Hrs and Tsg101 in the yeast two-hybrid system. The indicated Hrs constructs were used as bait and Tsg101 as prey. The values indicate β-galactosidase activities in arbitrary units. Neither of the bait constructs showed any significant reporter activation (<1 U) in the absence of a prey construct. All determinations were done in triplicate. Error bars denote SEM. (C) Interaction between Hrs and Tsg101 in vitro. GST alone or fused to Hrs287–573 or Hrs287–573P351A were immobilized on glutathione-Sepharose beads and incubated with in vitro–translated 35S-labeled Tsg101 for 1 h at 4°C. The beads were washed and analyzed by SDS-PAGE and fluorography. The amounts bound in lanes 1 and 7 correspond to 2–3% of the input amount. The doublet band is presumably due to translational initiation downstream of the initiator ATG.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Hrs interacts with Tsg101. (A) A schematic representation of Hrs. The VHS, FYVE, coiled-coil (CC), and clathrin-binding domain (CBD) are indicated. The ubiquitin interacting motif (UIM), PSAP sequence, and proline- and proline/glutamine-rich regions are also indicated. (B) Interaction between Hrs and Tsg101 in the yeast two-hybrid system. The indicated Hrs constructs were used as bait and Tsg101 as prey. The values indicate β-galactosidase activities in arbitrary units. Neither of the bait constructs showed any significant reporter activation (<1 U) in the absence of a prey construct. All determinations were done in triplicate. Error bars denote SEM. (C) Interaction between Hrs and Tsg101 in vitro. GST alone or fused to Hrs287–573 or Hrs287–573P351A were immobilized on glutathione-Sepharose beads and incubated with in vitro–translated 35S-labeled Tsg101 for 1 h at 4°C. The beads were washed and analyzed by SDS-PAGE and fluorography. The amounts bound in lanes 1 and 7 correspond to 2–3% of the input amount. The doublet band is presumably due to translational initiation downstream of the initiator ATG.
Mentions: The fact that Hrs contains a PSAP sequence (residues 348–351, Fig. 1 A), similar to that found in Tsg101-binding viral proteins (Carter, 2002; Pornillos et al., 2002c), prompted us to ask whether Tsg101 may interact with Hrs. First, we investigated this possibility using the yeast two-hybrid system. Full-length or truncated Hrs constructs were prepared as baits and Tsg101 as prey, and β-galactosidase reporter activity was measured in reporter yeast cells. The results indicated that Hrs does interact with Tsg101 (Fig. 1 B). Although the NH2-terminal part of Hrs, containing the VHS and FYVE domains and ubiquitin-interacting motif (UIM), showed no interaction with Tsg101, the remaining part, containing the proline-rich, coiled-coil, and clathrin binding domains, bound strongly. To study if the PSAP sequence is involved in the interaction, we mutated the final proline residue in this sequence to alanine. Interestingly, this P351A mutation markedly reduced the interaction, but did not abolish it completely. Consistent with this, we also detected a weak interaction between the coiled-coil region of Hrs and Tsg101, and a Hrs construct containing only the proline-rich and coiled-coil domains of (Hrs287–573) showed a strong interaction with Tsg101 (Fig. 1 B). In the companion paper, Pornillos et al. (2003)(in this issue) show that Hrs truncated just after the coiled-coiled domain (at residue 559) binds poorly to Tsg101. Because Hrs287–573, which binds Tsg101 strongly, contains a few additional residues after the coiled-coiled domain, it appears that these residues are important for Tsg101 binding, possibly because they allow correct folding of the coiled-coil region. Together, these results suggest that Tsg101 interacts with several interfaces of Hrs, including the PSAP sequence and the coiled-coil region. We also cannot rule out the possibility that the latter regions join to form a single Tsg101-interacting interface.

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