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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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Mutant forms of Vps27 defective in the efficient recruitment of ESCRT-I to endosomes display MVB sorting defects. Localization of GFP-CPS (encoded by pGO45) in vps27Δ cells (MBY21) expressing either wild-type or the indicated mutant forms of Vps27 was observed by fluorescence and Normarski microscopy. Schematic representations of wild-type and mutant forms of Vps27 are shown. Truncations in the proline/glutamine-rich region (P/Q rich) and substitutions in the PTVP peptide motif are indicated. Asterisks indicate the presence of candidate PTAP-like motifs (PSDP447–450, PSDP524–527, and PTVP581–584) in the various forms of Vps27.
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fig6: Mutant forms of Vps27 defective in the efficient recruitment of ESCRT-I to endosomes display MVB sorting defects. Localization of GFP-CPS (encoded by pGO45) in vps27Δ cells (MBY21) expressing either wild-type or the indicated mutant forms of Vps27 was observed by fluorescence and Normarski microscopy. Schematic representations of wild-type and mutant forms of Vps27 are shown. Truncations in the proline/glutamine-rich region (P/Q rich) and substitutions in the PTVP peptide motif are indicated. Asterisks indicate the presence of candidate PTAP-like motifs (PSDP447–450, PSDP524–527, and PTVP581–584) in the various forms of Vps27.

Mentions: Next, we expressed mutant forms of Vps27 in vps27Δ cells and examined MVB sorting of a GFP-CPS fusion reporter (Odorizzi et al., 1998). As expected, in cells expressing wild-type Vps27, GFP-CPS was delivered to the lumen of the vacuole, resulting in GFP fluorescence localized to the vacuole lumen (Fig. 6). As shown previously, GFP-CPS was mislocalized to the outer, limiting membrane of the vacuole and aberrant endosomal structures in cells lacking Vps27 (Odorizzi et al., 2000; Shih et al., 2002; Fig. 6). Consistent with a defect in ESCRT-I recruitment, GFP-CPS was missorted to the limiting membrane of the vacuole and accumulated on prevacuolar endosomes in cells expressing Vps27Δ524–622 alone (Fig. 6). Interestingly, truncation of Vps27 at residue 581 (Vps27Δ581–622) conferred a defect in MVB sorting of GFP-CPS similar to that found in vps27Δ cells (Fig. 6), likely due to impaired endosomal recruitment of Vps23-GFP as observed in vps27Δ581–622 mutant cells (Fig. 5). Likewise, in cells expressing Vps27P581G, T582S, GFP-CPS was missorted to the outer, limiting membrane of the vacuole (Fig. 6), in further support of a role for this conserved PTVP motif in Vps27/ESCRT-I association. We also followed the localization of another MVB reporter, 7NBD-labeled phosphatidylcholine (NBD-PC), in vps27 mutant cells. In pep4Δ cells expressing wild-type Vps27, NBD-PC was efficiently sorted into the MVB pathway and subsequently transported to the vacuole lumen as previously shown (Bilodeau et al., 2002). However, pep4Δ vps27Δ524–622 and pep4Δ vps27Δ581–622 mutant cells were impaired in the transport of NBD-PC to the vacuole lumen. Instead, NBD-PC accumulated in aberrant prevacuolar compartments (unpublished data), suggesting that Vps27/ESCRT-I association was necessary for the formation of MVB vesicles. Altogether, our results have identified a region in the COOH-terminal domain of Vps27 necessary for efficient recruitment of ESCRT-I to endosomes and implicated the PTVP motif in this process.


Vps27 recruits ESCRT machinery to endosomes during MVB sorting.

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

Mutant forms of Vps27 defective in the efficient recruitment of ESCRT-I to endosomes display MVB sorting defects. Localization of GFP-CPS (encoded by pGO45) in vps27Δ cells (MBY21) expressing either wild-type or the indicated mutant forms of Vps27 was observed by fluorescence and Normarski microscopy. Schematic representations of wild-type and mutant forms of Vps27 are shown. Truncations in the proline/glutamine-rich region (P/Q rich) and substitutions in the PTVP peptide motif are indicated. Asterisks indicate the presence of candidate PTAP-like motifs (PSDP447–450, PSDP524–527, and PTVP581–584) in the various forms of Vps27.
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Related In: Results  -  Collection

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fig6: Mutant forms of Vps27 defective in the efficient recruitment of ESCRT-I to endosomes display MVB sorting defects. Localization of GFP-CPS (encoded by pGO45) in vps27Δ cells (MBY21) expressing either wild-type or the indicated mutant forms of Vps27 was observed by fluorescence and Normarski microscopy. Schematic representations of wild-type and mutant forms of Vps27 are shown. Truncations in the proline/glutamine-rich region (P/Q rich) and substitutions in the PTVP peptide motif are indicated. Asterisks indicate the presence of candidate PTAP-like motifs (PSDP447–450, PSDP524–527, and PTVP581–584) in the various forms of Vps27.
Mentions: Next, we expressed mutant forms of Vps27 in vps27Δ cells and examined MVB sorting of a GFP-CPS fusion reporter (Odorizzi et al., 1998). As expected, in cells expressing wild-type Vps27, GFP-CPS was delivered to the lumen of the vacuole, resulting in GFP fluorescence localized to the vacuole lumen (Fig. 6). As shown previously, GFP-CPS was mislocalized to the outer, limiting membrane of the vacuole and aberrant endosomal structures in cells lacking Vps27 (Odorizzi et al., 2000; Shih et al., 2002; Fig. 6). Consistent with a defect in ESCRT-I recruitment, GFP-CPS was missorted to the limiting membrane of the vacuole and accumulated on prevacuolar endosomes in cells expressing Vps27Δ524–622 alone (Fig. 6). Interestingly, truncation of Vps27 at residue 581 (Vps27Δ581–622) conferred a defect in MVB sorting of GFP-CPS similar to that found in vps27Δ cells (Fig. 6), likely due to impaired endosomal recruitment of Vps23-GFP as observed in vps27Δ581–622 mutant cells (Fig. 5). Likewise, in cells expressing Vps27P581G, T582S, GFP-CPS was missorted to the outer, limiting membrane of the vacuole (Fig. 6), in further support of a role for this conserved PTVP motif in Vps27/ESCRT-I association. We also followed the localization of another MVB reporter, 7NBD-labeled phosphatidylcholine (NBD-PC), in vps27 mutant cells. In pep4Δ cells expressing wild-type Vps27, NBD-PC was efficiently sorted into the MVB pathway and subsequently transported to the vacuole lumen as previously shown (Bilodeau et al., 2002). However, pep4Δ vps27Δ524–622 and pep4Δ vps27Δ581–622 mutant cells were impaired in the transport of NBD-PC to the vacuole lumen. Instead, NBD-PC accumulated in aberrant prevacuolar compartments (unpublished data), suggesting that Vps27/ESCRT-I association was necessary for the formation of MVB vesicles. Altogether, our results have identified a region in the COOH-terminal domain of Vps27 necessary for efficient recruitment of ESCRT-I to endosomes and implicated the PTVP motif in this process.

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