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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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ESCRT-I and Vps27 bind Ub independently of one another in vitro. (A) Extracts were prepared from MBY52 cells expressing the indicated VPS27 alleles and incubated with immobilized GST or Ub-GST (Ub-GST). ESCRT-I binding was visualized by Western blotting with anti-Vps23 antisera. (B) Extracts were prepared from DKY61 cells expressing the indicated VPS23 alleles and incubated with immobilized GST or Ub-GST. Vps27-HA binding was visualized by Western blotting with anti-HA antisera. (C) Cellular membranes were isolated from DKY61, solubilized and protein A–Vps27 was purified under native conditions and bound material was probed by Western blotting with anti-Vps27 or anti-CPS antisera.
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fig1: ESCRT-I and Vps27 bind Ub independently of one another in vitro. (A) Extracts were prepared from MBY52 cells expressing the indicated VPS27 alleles and incubated with immobilized GST or Ub-GST (Ub-GST). ESCRT-I binding was visualized by Western blotting with anti-Vps23 antisera. (B) Extracts were prepared from DKY61 cells expressing the indicated VPS23 alleles and incubated with immobilized GST or Ub-GST. Vps27-HA binding was visualized by Western blotting with anti-HA antisera. (C) Cellular membranes were isolated from DKY61, solubilized and protein A–Vps27 was purified under native conditions and bound material was probed by Western blotting with anti-Vps27 or anti-CPS antisera.

Mentions: Among the class E Vps proteins, both Vps27 and the Vps23-containing ESCRT-I complex have been shown to bind Ub and play a critical role in the function of the MVB sorting pathway (Katzmann et al., 2001; Bilodeau et al., 2002; Shih et al., 2002). To address the functional relationship between these Ub-interacting proteins in the context of the MVB sorting reaction, we initially analyzed the ability of each of these class E Vps proteins to bind Ub independently. This was accomplished by performing protein–protein interaction studies using either GST or Ub fused to GST as described previously (Katzmann et al., 2001; Shih et al., 2002). To address a requirement for Vps27 in ESCRT-I binding to Ub, immobilized GST or Ub-GST was incubated with cellular extracts prepared from wild-type cells, vps27Δ cells, or cells expressing a mutant form of Vps27 bearing substitutions in the Ub-interacting motifs (UIM domains; Vps27S313D, S270D), previously shown to display dramatically reduced binding affinity for Ub in vitro (Shih et al., 2002). Subsequent to incubation with cell extracts, beads were washed and bound material was analyzed by Western blotting with anti-Vps23 antisera. As expected, the ESCRT-I subunit Vps23 bound to Ub-GST, but not GST, when lysate was prepared from wild-type cells (Fig. 1 A). Vps27 was not essential for this interaction, but appears to contribute to this binding, as deletion of VPS27 resulted in a small decrease (less than twofold) in the amount of bound ESCRT-I, whereas the vps27S313D, S270D UIM mutation caused no obvious defect in binding. This indicated that ESCRT-I binding to Ub did not require the Ub-binding function of Vps27, although Vps27 may contribute to the stability of this interaction.


Vps27 recruits ESCRT machinery to endosomes during MVB sorting.

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

ESCRT-I and Vps27 bind Ub independently of one another in vitro. (A) Extracts were prepared from MBY52 cells expressing the indicated VPS27 alleles and incubated with immobilized GST or Ub-GST (Ub-GST). ESCRT-I binding was visualized by Western blotting with anti-Vps23 antisera. (B) Extracts were prepared from DKY61 cells expressing the indicated VPS23 alleles and incubated with immobilized GST or Ub-GST. Vps27-HA binding was visualized by Western blotting with anti-HA antisera. (C) Cellular membranes were isolated from DKY61, solubilized and protein A–Vps27 was purified under native conditions and bound material was probed by Western blotting with anti-Vps27 or anti-CPS antisera.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172707&req=5

fig1: ESCRT-I and Vps27 bind Ub independently of one another in vitro. (A) Extracts were prepared from MBY52 cells expressing the indicated VPS27 alleles and incubated with immobilized GST or Ub-GST (Ub-GST). ESCRT-I binding was visualized by Western blotting with anti-Vps23 antisera. (B) Extracts were prepared from DKY61 cells expressing the indicated VPS23 alleles and incubated with immobilized GST or Ub-GST. Vps27-HA binding was visualized by Western blotting with anti-HA antisera. (C) Cellular membranes were isolated from DKY61, solubilized and protein A–Vps27 was purified under native conditions and bound material was probed by Western blotting with anti-Vps27 or anti-CPS antisera.
Mentions: Among the class E Vps proteins, both Vps27 and the Vps23-containing ESCRT-I complex have been shown to bind Ub and play a critical role in the function of the MVB sorting pathway (Katzmann et al., 2001; Bilodeau et al., 2002; Shih et al., 2002). To address the functional relationship between these Ub-interacting proteins in the context of the MVB sorting reaction, we initially analyzed the ability of each of these class E Vps proteins to bind Ub independently. This was accomplished by performing protein–protein interaction studies using either GST or Ub fused to GST as described previously (Katzmann et al., 2001; Shih et al., 2002). To address a requirement for Vps27 in ESCRT-I binding to Ub, immobilized GST or Ub-GST was incubated with cellular extracts prepared from wild-type cells, vps27Δ cells, or cells expressing a mutant form of Vps27 bearing substitutions in the Ub-interacting motifs (UIM domains; Vps27S313D, S270D), previously shown to display dramatically reduced binding affinity for Ub in vitro (Shih et al., 2002). Subsequent to incubation with cell extracts, beads were washed and bound material was analyzed by Western blotting with anti-Vps23 antisera. As expected, the ESCRT-I subunit Vps23 bound to Ub-GST, but not GST, when lysate was prepared from wild-type cells (Fig. 1 A). Vps27 was not essential for this interaction, but appears to contribute to this binding, as deletion of VPS27 resulted in a small decrease (less than twofold) in the amount of bound ESCRT-I, whereas the vps27S313D, S270D UIM mutation caused no obvious defect in binding. This indicated that ESCRT-I binding to Ub did not require the Ub-binding function of Vps27, although Vps27 may contribute to the stability of this interaction.

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