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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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Vps27 and ESCRT-I interact on membranes. (A) Wild-type cells (TVY614) expressing Vps23–protein A and Vps27-HA were lysed and separated into low speed membrane pellet (M) and soluble fractions (S). Both fractions were adjusted to 0.5% LDAO, cleared, and Vps23–protein A was purified under native conditions. Isolated material was probed with anti-HA antibody to detect Vps27-HA. Input lanes show 20% of the total Vps27-HA present in the lysates. (B) MBY52 cells expressing protein A alone (A), protein A–Vps27 (Vps27), protein A–Vps27S313D, S270D (uim), or protein A–Vps27DVHS (vhsΔ) were lysed. Low speed membrane fractions were generated and solubilized with 0.5% LDAO and protein A–Vps27 was purified under native conditions. Bound material was Western blotted using antisera as indicated.
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fig4: Vps27 and ESCRT-I interact on membranes. (A) Wild-type cells (TVY614) expressing Vps23–protein A and Vps27-HA were lysed and separated into low speed membrane pellet (M) and soluble fractions (S). Both fractions were adjusted to 0.5% LDAO, cleared, and Vps23–protein A was purified under native conditions. Isolated material was probed with anti-HA antibody to detect Vps27-HA. Input lanes show 20% of the total Vps27-HA present in the lysates. (B) MBY52 cells expressing protein A alone (A), protein A–Vps27 (Vps27), protein A–Vps27S313D, S270D (uim), or protein A–Vps27DVHS (vhsΔ) were lysed. Low speed membrane fractions were generated and solubilized with 0.5% LDAO and protein A–Vps27 was purified under native conditions. Bound material was Western blotted using antisera as indicated.

Mentions: To clarify the role of Vps27 in the recruitment of ESCRT-I to endosomal membranes, we examined whether Vps27 and ESCRT-I associate on membranes. Wild-type cells expressing functional Vps23–protein A (Babst et al., 2000) and Vps27-HA (Shih et al., 2002) fusion proteins were fractionated into soluble and membrane fractions. After solubilizing the membrane fraction, Vps23–protein A was isolated from both cell fractions under native conditions. Bound material was probed for the presence of Vps27-HA. The majority of Vps27 in cell lysates was present in the soluble fraction, likely due to PI(3)P hydrolysis upon cell lysis (Fig. 4 A, Input). Regardless, the vast majority of Vps27-HA copurified with Vps23–protein A in the membrane fraction, but not the soluble fraction (Fig. 4 A). This indicated that Vps27 and ESCRT-I specifically associate on membranes—consistent with the previous finding that soluble ESCRT-I did not contain Vps27 (Katzmann et al., 2001)—and suggested that this interaction did not occur after lysis.


Vps27 recruits ESCRT machinery to endosomes during MVB sorting.

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

Vps27 and ESCRT-I interact on membranes. (A) Wild-type cells (TVY614) expressing Vps23–protein A and Vps27-HA were lysed and separated into low speed membrane pellet (M) and soluble fractions (S). Both fractions were adjusted to 0.5% LDAO, cleared, and Vps23–protein A was purified under native conditions. Isolated material was probed with anti-HA antibody to detect Vps27-HA. Input lanes show 20% of the total Vps27-HA present in the lysates. (B) MBY52 cells expressing protein A alone (A), protein A–Vps27 (Vps27), protein A–Vps27S313D, S270D (uim), or protein A–Vps27DVHS (vhsΔ) were lysed. Low speed membrane fractions were generated and solubilized with 0.5% LDAO and protein A–Vps27 was purified under native conditions. Bound material was Western blotted using antisera as indicated.
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Related In: Results  -  Collection

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fig4: Vps27 and ESCRT-I interact on membranes. (A) Wild-type cells (TVY614) expressing Vps23–protein A and Vps27-HA were lysed and separated into low speed membrane pellet (M) and soluble fractions (S). Both fractions were adjusted to 0.5% LDAO, cleared, and Vps23–protein A was purified under native conditions. Isolated material was probed with anti-HA antibody to detect Vps27-HA. Input lanes show 20% of the total Vps27-HA present in the lysates. (B) MBY52 cells expressing protein A alone (A), protein A–Vps27 (Vps27), protein A–Vps27S313D, S270D (uim), or protein A–Vps27DVHS (vhsΔ) were lysed. Low speed membrane fractions were generated and solubilized with 0.5% LDAO and protein A–Vps27 was purified under native conditions. Bound material was Western blotted using antisera as indicated.
Mentions: To clarify the role of Vps27 in the recruitment of ESCRT-I to endosomal membranes, we examined whether Vps27 and ESCRT-I associate on membranes. Wild-type cells expressing functional Vps23–protein A (Babst et al., 2000) and Vps27-HA (Shih et al., 2002) fusion proteins were fractionated into soluble and membrane fractions. After solubilizing the membrane fraction, Vps23–protein A was isolated from both cell fractions under native conditions. Bound material was probed for the presence of Vps27-HA. The majority of Vps27 in cell lysates was present in the soluble fraction, likely due to PI(3)P hydrolysis upon cell lysis (Fig. 4 A, Input). Regardless, the vast majority of Vps27-HA copurified with Vps23–protein A in the membrane fraction, but not the soluble fraction (Fig. 4 A). This indicated that Vps27 and ESCRT-I specifically associate on membranes—consistent with the previous finding that soluble ESCRT-I did not contain Vps27 (Katzmann et al., 2001)—and suggested that this interaction did not occur after lysis.

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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