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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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Localization of ESCRT-I to endosomes is dependent on Vps34 activity and function of the PI(3)P-binding protein, Vps27. (A) Localization of Vps23-GFP in wild-type (DKY54), vps34Δ (DKY82), vps27Δ (DKY78), vps4Δ (DKY55), or vps4Δ vps27Δ (DKY79) living cells by fluorescence and Normarski microscopy. (B) ESCRT-I is not essential for the recruitment of Vps27 to membranes. Wild-type (top) or vps23Δ cells (EEY6–2; bottom) expressing GFP-Vps27 were observed by fluorescence and Nomarski microscopy. Bar: (A and B) 5 μM.
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fig3: Localization of ESCRT-I to endosomes is dependent on Vps34 activity and function of the PI(3)P-binding protein, Vps27. (A) Localization of Vps23-GFP in wild-type (DKY54), vps34Δ (DKY82), vps27Δ (DKY78), vps4Δ (DKY55), or vps4Δ vps27Δ (DKY79) living cells by fluorescence and Normarski microscopy. (B) ESCRT-I is not essential for the recruitment of Vps27 to membranes. Wild-type (top) or vps23Δ cells (EEY6–2; bottom) expressing GFP-Vps27 were observed by fluorescence and Nomarski microscopy. Bar: (A and B) 5 μM.

Mentions: Next, we addressed the mechanism by which ESCRT-I is recruited to its site of action, as there are clearly numerous membrane sites within the cell that contain ubiquitinated proteins (e.g., endoplasmic reticulum, Golgi compartments, plasma membrane, and endosomes), indicating this cannot be the sole mechanism for its localization. To examine the subcellular localization of ESCRT-I, we used a previously described chromosomally integrated gene fusion between Vps23 and GFP (Katzmann et al., 2001). As expected, in wild-type cells, Vps23-GFP was observed on intracellular punctate structures as well as in the cytoplasm (Fig. 3 A). As efficient Vps27 localization to endosomal membranes required the production of PI(3)P, we examined whether PI(3)P was also required for the recruitment of ESCRT-I. In vps34Δ cells, Vps23-GFP exhibited largely a cytoplasmic distribution with almost no intracellular puncta (Fig. 3 A). One interpretation of this result would be that the PI(3)P effector Vps27 is required for bridging the interaction between ESCRT-I and PI(3)P. To test this idea, Vps23-GFP localization was analyzed in vps27Δ cells. Vps23-GFP redistributed to a diffuse cytoplasmic pattern in vps27Δ cells (Fig. 3 A).


Vps27 recruits ESCRT machinery to endosomes during MVB sorting.

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

Localization of ESCRT-I to endosomes is dependent on Vps34 activity and function of the PI(3)P-binding protein, Vps27. (A) Localization of Vps23-GFP in wild-type (DKY54), vps34Δ (DKY82), vps27Δ (DKY78), vps4Δ (DKY55), or vps4Δ vps27Δ (DKY79) living cells by fluorescence and Normarski microscopy. (B) ESCRT-I is not essential for the recruitment of Vps27 to membranes. Wild-type (top) or vps23Δ cells (EEY6–2; bottom) expressing GFP-Vps27 were observed by fluorescence and Nomarski microscopy. Bar: (A and B) 5 μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Localization of ESCRT-I to endosomes is dependent on Vps34 activity and function of the PI(3)P-binding protein, Vps27. (A) Localization of Vps23-GFP in wild-type (DKY54), vps34Δ (DKY82), vps27Δ (DKY78), vps4Δ (DKY55), or vps4Δ vps27Δ (DKY79) living cells by fluorescence and Normarski microscopy. (B) ESCRT-I is not essential for the recruitment of Vps27 to membranes. Wild-type (top) or vps23Δ cells (EEY6–2; bottom) expressing GFP-Vps27 were observed by fluorescence and Nomarski microscopy. Bar: (A and B) 5 μM.
Mentions: Next, we addressed the mechanism by which ESCRT-I is recruited to its site of action, as there are clearly numerous membrane sites within the cell that contain ubiquitinated proteins (e.g., endoplasmic reticulum, Golgi compartments, plasma membrane, and endosomes), indicating this cannot be the sole mechanism for its localization. To examine the subcellular localization of ESCRT-I, we used a previously described chromosomally integrated gene fusion between Vps23 and GFP (Katzmann et al., 2001). As expected, in wild-type cells, Vps23-GFP was observed on intracellular punctate structures as well as in the cytoplasm (Fig. 3 A). As efficient Vps27 localization to endosomal membranes required the production of PI(3)P, we examined whether PI(3)P was also required for the recruitment of ESCRT-I. In vps34Δ cells, Vps23-GFP exhibited largely a cytoplasmic distribution with almost no intracellular puncta (Fig. 3 A). One interpretation of this result would be that the PI(3)P effector Vps27 is required for bridging the interaction between ESCRT-I and PI(3)P. To test this idea, Vps23-GFP localization was analyzed in vps27Δ cells. Vps23-GFP redistributed to a diffuse cytoplasmic pattern in vps27Δ cells (Fig. 3 A).

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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