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Vps27 recruits ESCRT machinery to endosomes during MVB sorting.

Katzmann DJ, Stefan CJ, Babst M, Emr SD - J. Cell Biol. (2003)

Bottom Line: A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23.We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin.Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, and Howard Hughes Medical Institute, University of California, San Diego School of Medicine, La Jolla, CA 92093-0688, USA.

ABSTRACT
Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23. We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.

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The steady state localization of Vps27 on PI(3)P-containing endosomal membranes is dependent on Vps34 activity and the FYVE domain in Vps27. (A) Schematic representations of Vps27 and its mammalian orthologue, Hrs. The VHS (light green), FYVE (blue), and UIM (yellow) domains are indicated. The proline/glutamine-rich region (P/Q rich), and clathrin-binding motifs (CB) are indicated in Vps27 and Hrs. The proline-rich (PRD) and coiled-coil domain (coil) in Hrs are also indicated. The asterisks indicate the relative positions of candidate PTAP-like motifs found in Vps27 (PSDP447–450, PSDP524–527 and PTVP581–584) and Hrs (PSAP348–351, PSGP583–586 and PSMP620–623). (B) Wild-type cells (SEY6210) coexpressing GFP-Vps27 and DsRed-FYVEEEA1 fusions were observed by fluorescent and Nomarski microscopy using a DeltaVision deconvolution system. (C) Wild-type (top) or vps34Δ cells (PHY102; middle) coexpressing GFP-Vps27 and DsRed-FYVEEEA1 fusions or wild-type cells coexpressing GFP-Vps27ΔFYVE and DsRed-FYVEEEA1 fusions (bottom) were observed by fluorescent and Nomarski microscopy.
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fig2: The steady state localization of Vps27 on PI(3)P-containing endosomal membranes is dependent on Vps34 activity and the FYVE domain in Vps27. (A) Schematic representations of Vps27 and its mammalian orthologue, Hrs. The VHS (light green), FYVE (blue), and UIM (yellow) domains are indicated. The proline/glutamine-rich region (P/Q rich), and clathrin-binding motifs (CB) are indicated in Vps27 and Hrs. The proline-rich (PRD) and coiled-coil domain (coil) in Hrs are also indicated. The asterisks indicate the relative positions of candidate PTAP-like motifs found in Vps27 (PSDP447–450, PSDP524–527 and PTVP581–584) and Hrs (PSAP348–351, PSGP583–586 and PSMP620–623). (B) Wild-type cells (SEY6210) coexpressing GFP-Vps27 and DsRed-FYVEEEA1 fusions were observed by fluorescent and Nomarski microscopy using a DeltaVision deconvolution system. (C) Wild-type (top) or vps34Δ cells (PHY102; middle) coexpressing GFP-Vps27 and DsRed-FYVEEEA1 fusions or wild-type cells coexpressing GFP-Vps27ΔFYVE and DsRed-FYVEEEA1 fusions (bottom) were observed by fluorescent and Nomarski microscopy.

Mentions: To examine the subcellular localization of Vps27 in vivo, we constructed a GFP-Vps27 chimera and expressed this in cells. We chose an approach that employed intact, viable cells because we have found that PI(3)P and other phosphoinositide species were rapidly degraded upon cell lysis (unpublished data). This GFP-Vps27 fusion protein was functional, as addressed by its ability to complement the sorting defects for the vacuolar hydrolysis carboxypeptidase Y and carboxypepidase S (CPS) observed in vps27Δ cells (unpublished data). In wild-type cells, the fusion protein localized to intracellular punctate structures (Figs. 2 B, left; Fig. 2 C, top left). Next, we examined whether these punctate structures represented endosomal membranes. To do so, we fused DsRed to the FYVE domain from mammalian EEA1 as a reporter for PI(3)P-enriched membranes and expressed this fusion protein in cells together with the GFP-Vps27 chimera. The FYVE domain of EEA1 has previously been shown to bind specifically to PI(3)P in vitro and direct endosomal localization in vivo (Burd and Emr, 1998; Gaullier et al., 1998). In wild-type cells expressing DsRed-FYVE, red fluorescence was observed on punctate structures adjacent to the vacuole, weakly on the vacuole limiting membrane, and in some cases within the vacuole itself, consistent with previous studies that used GFP-FYVEEEA1 and GST-FYVEEEA1 fusions (Burd and Emr, 1998; Gillooly et al., 2000; Fig. 2 B, middle; Fig. 2 C, top middle). In cells coexpressing GFP-Vps27 and DsRed-FYVE significant colocalization was observed between GFP-Vps27 and DsRed-FYVE (Fig. 2 B, right), which is consistent with Vps27 being recruited to PI(3)P-containing endosomal membranes.


Vps27 recruits ESCRT machinery to endosomes during MVB sorting.

Katzmann DJ, Stefan CJ, Babst M, Emr SD - J. Cell Biol. (2003)

The steady state localization of Vps27 on PI(3)P-containing endosomal membranes is dependent on Vps34 activity and the FYVE domain in Vps27. (A) Schematic representations of Vps27 and its mammalian orthologue, Hrs. The VHS (light green), FYVE (blue), and UIM (yellow) domains are indicated. The proline/glutamine-rich region (P/Q rich), and clathrin-binding motifs (CB) are indicated in Vps27 and Hrs. The proline-rich (PRD) and coiled-coil domain (coil) in Hrs are also indicated. The asterisks indicate the relative positions of candidate PTAP-like motifs found in Vps27 (PSDP447–450, PSDP524–527 and PTVP581–584) and Hrs (PSAP348–351, PSGP583–586 and PSMP620–623). (B) Wild-type cells (SEY6210) coexpressing GFP-Vps27 and DsRed-FYVEEEA1 fusions were observed by fluorescent and Nomarski microscopy using a DeltaVision deconvolution system. (C) Wild-type (top) or vps34Δ cells (PHY102; middle) coexpressing GFP-Vps27 and DsRed-FYVEEEA1 fusions or wild-type cells coexpressing GFP-Vps27ΔFYVE and DsRed-FYVEEEA1 fusions (bottom) were observed by fluorescent and Nomarski microscopy.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172707&req=5

fig2: The steady state localization of Vps27 on PI(3)P-containing endosomal membranes is dependent on Vps34 activity and the FYVE domain in Vps27. (A) Schematic representations of Vps27 and its mammalian orthologue, Hrs. The VHS (light green), FYVE (blue), and UIM (yellow) domains are indicated. The proline/glutamine-rich region (P/Q rich), and clathrin-binding motifs (CB) are indicated in Vps27 and Hrs. The proline-rich (PRD) and coiled-coil domain (coil) in Hrs are also indicated. The asterisks indicate the relative positions of candidate PTAP-like motifs found in Vps27 (PSDP447–450, PSDP524–527 and PTVP581–584) and Hrs (PSAP348–351, PSGP583–586 and PSMP620–623). (B) Wild-type cells (SEY6210) coexpressing GFP-Vps27 and DsRed-FYVEEEA1 fusions were observed by fluorescent and Nomarski microscopy using a DeltaVision deconvolution system. (C) Wild-type (top) or vps34Δ cells (PHY102; middle) coexpressing GFP-Vps27 and DsRed-FYVEEEA1 fusions or wild-type cells coexpressing GFP-Vps27ΔFYVE and DsRed-FYVEEEA1 fusions (bottom) were observed by fluorescent and Nomarski microscopy.
Mentions: To examine the subcellular localization of Vps27 in vivo, we constructed a GFP-Vps27 chimera and expressed this in cells. We chose an approach that employed intact, viable cells because we have found that PI(3)P and other phosphoinositide species were rapidly degraded upon cell lysis (unpublished data). This GFP-Vps27 fusion protein was functional, as addressed by its ability to complement the sorting defects for the vacuolar hydrolysis carboxypeptidase Y and carboxypepidase S (CPS) observed in vps27Δ cells (unpublished data). In wild-type cells, the fusion protein localized to intracellular punctate structures (Figs. 2 B, left; Fig. 2 C, top left). Next, we examined whether these punctate structures represented endosomal membranes. To do so, we fused DsRed to the FYVE domain from mammalian EEA1 as a reporter for PI(3)P-enriched membranes and expressed this fusion protein in cells together with the GFP-Vps27 chimera. The FYVE domain of EEA1 has previously been shown to bind specifically to PI(3)P in vitro and direct endosomal localization in vivo (Burd and Emr, 1998; Gaullier et al., 1998). In wild-type cells expressing DsRed-FYVE, red fluorescence was observed on punctate structures adjacent to the vacuole, weakly on the vacuole limiting membrane, and in some cases within the vacuole itself, consistent with previous studies that used GFP-FYVEEEA1 and GST-FYVEEEA1 fusions (Burd and Emr, 1998; Gillooly et al., 2000; Fig. 2 B, middle; Fig. 2 C, top middle). In cells coexpressing GFP-Vps27 and DsRed-FYVE significant colocalization was observed between GFP-Vps27 and DsRed-FYVE (Fig. 2 B, right), which is consistent with Vps27 being recruited to PI(3)P-containing endosomal membranes.

Bottom Line: A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23.We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin.Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, and Howard Hughes Medical Institute, University of California, San Diego School of Medicine, La Jolla, CA 92093-0688, USA.

ABSTRACT
Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23. We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.

Show MeSH