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PI3P signaling regulates receptor sorting but not transport in the endosomal pathway.

Petiot A, Faure J, Stenmark H, Gruenberg J - J. Cell Biol. (2003)

Bottom Line: We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes.Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting.They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211-Geneva-4, Switzerland.

ABSTRACT
While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

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GFP-2xFYVE does not inhibit bulk transport to late endosomes in vivo. (A) As in Fig. 1 A, a pulse of rhodamine dextran was endocytosed with or without chase in cells expressing a double FYVE domain associated to GFP (GFP-2xFYVE). Cells were analyzed as described in the legend to Fig. 1 A. (B) Cells expressing or not expressing GFP-2xFYVE were incubated overnight with 40 μg/ml mouse IgGs with or without 50 μg/ml leupeptin. Endocytosed IgGs were revealed using anti-mouse antibodies and cells were analyzed by double channel fluorescence microscopy. The total number of labeled vesicles (containing IgGs in red) was counted for each condition in control cells or cells expressing GFP-2xFYVE. Vesicles in ≥20 cells were counted for each condition in three different experiments. Numbers were similar whether GFP-2xFYVE was present or not, and are expressed as a percentage of the total number of vesicles in leupeptin-treated cells, to facilitate direct comparison. Bar, 2.5 μm.
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fig2: GFP-2xFYVE does not inhibit bulk transport to late endosomes in vivo. (A) As in Fig. 1 A, a pulse of rhodamine dextran was endocytosed with or without chase in cells expressing a double FYVE domain associated to GFP (GFP-2xFYVE). Cells were analyzed as described in the legend to Fig. 1 A. (B) Cells expressing or not expressing GFP-2xFYVE were incubated overnight with 40 μg/ml mouse IgGs with or without 50 μg/ml leupeptin. Endocytosed IgGs were revealed using anti-mouse antibodies and cells were analyzed by double channel fluorescence microscopy. The total number of labeled vesicles (containing IgGs in red) was counted for each condition in control cells or cells expressing GFP-2xFYVE. Vesicles in ≥20 cells were counted for each condition in three different experiments. Numbers were similar whether GFP-2xFYVE was present or not, and are expressed as a percentage of the total number of vesicles in leupeptin-treated cells, to facilitate direct comparison. Bar, 2.5 μm.

Mentions: When transport from early to late endosomes is inhibited, HRP is regurgitated into the medium and fails to accumulate intracellularly (Clague et al., 1994; Gu et al., 1997; Mayran et al., 2003). In contrast, when PI3 kinase was inhibited with wortmannin, although HRP internalization was decreased, accumulation was not affected (not shown), suggesting that transport from early to late endosomes does not depend on PI3P signaling. Indeed, PI3 kinase inhibition did not affect transport of dextran to late endosomes containing LBPA (Fig. 1 D, and see quantification in Fig. 3 C) or Lamp1 (see Fig. 3 A). As a control, we confirmed that wortmannin did however lead to the release of EEA1 from early endosomes (Fig. 1 D), and reduced the PI3P content of early endosomal fractions (not shown). To interfere more specifically with PI3P-dependent functions, we used a double FYVE PI3P-binding domain (2xFYVE) that binds PI3P with high specificity (not shown) and inhibits early endosome fusion in vitro (see Fig. 4 B), as expected (Gillooly et al., 2000). When linked to GFP, 2xFYVE colocalized with dextran internalized for 10 min and EEA1 on early endosomes (Fig. 2 A). In agreement with the lack of effect of wortmannin, GFP-2xFYVE did not affect dextran transport to late endosomes (Fig. 2 A, and quantification in Fig. 3 C). Similarly, endocytosed mouse IgGs, a fluid phase marker, were transported to lysosomes and degraded whether or not GFP-2xFYVE was expressed, whereas IgGs accumulated intracellularly when lysosomal degradation was blocked with leupeptin (Fig. 2 B). We thus concluded that PI3P signaling is not involved in bulk endosomal membrane transport.


PI3P signaling regulates receptor sorting but not transport in the endosomal pathway.

Petiot A, Faure J, Stenmark H, Gruenberg J - J. Cell Biol. (2003)

GFP-2xFYVE does not inhibit bulk transport to late endosomes in vivo. (A) As in Fig. 1 A, a pulse of rhodamine dextran was endocytosed with or without chase in cells expressing a double FYVE domain associated to GFP (GFP-2xFYVE). Cells were analyzed as described in the legend to Fig. 1 A. (B) Cells expressing or not expressing GFP-2xFYVE were incubated overnight with 40 μg/ml mouse IgGs with or without 50 μg/ml leupeptin. Endocytosed IgGs were revealed using anti-mouse antibodies and cells were analyzed by double channel fluorescence microscopy. The total number of labeled vesicles (containing IgGs in red) was counted for each condition in control cells or cells expressing GFP-2xFYVE. Vesicles in ≥20 cells were counted for each condition in three different experiments. Numbers were similar whether GFP-2xFYVE was present or not, and are expressed as a percentage of the total number of vesicles in leupeptin-treated cells, to facilitate direct comparison. Bar, 2.5 μm.
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Related In: Results  -  Collection

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fig2: GFP-2xFYVE does not inhibit bulk transport to late endosomes in vivo. (A) As in Fig. 1 A, a pulse of rhodamine dextran was endocytosed with or without chase in cells expressing a double FYVE domain associated to GFP (GFP-2xFYVE). Cells were analyzed as described in the legend to Fig. 1 A. (B) Cells expressing or not expressing GFP-2xFYVE were incubated overnight with 40 μg/ml mouse IgGs with or without 50 μg/ml leupeptin. Endocytosed IgGs were revealed using anti-mouse antibodies and cells were analyzed by double channel fluorescence microscopy. The total number of labeled vesicles (containing IgGs in red) was counted for each condition in control cells or cells expressing GFP-2xFYVE. Vesicles in ≥20 cells were counted for each condition in three different experiments. Numbers were similar whether GFP-2xFYVE was present or not, and are expressed as a percentage of the total number of vesicles in leupeptin-treated cells, to facilitate direct comparison. Bar, 2.5 μm.
Mentions: When transport from early to late endosomes is inhibited, HRP is regurgitated into the medium and fails to accumulate intracellularly (Clague et al., 1994; Gu et al., 1997; Mayran et al., 2003). In contrast, when PI3 kinase was inhibited with wortmannin, although HRP internalization was decreased, accumulation was not affected (not shown), suggesting that transport from early to late endosomes does not depend on PI3P signaling. Indeed, PI3 kinase inhibition did not affect transport of dextran to late endosomes containing LBPA (Fig. 1 D, and see quantification in Fig. 3 C) or Lamp1 (see Fig. 3 A). As a control, we confirmed that wortmannin did however lead to the release of EEA1 from early endosomes (Fig. 1 D), and reduced the PI3P content of early endosomal fractions (not shown). To interfere more specifically with PI3P-dependent functions, we used a double FYVE PI3P-binding domain (2xFYVE) that binds PI3P with high specificity (not shown) and inhibits early endosome fusion in vitro (see Fig. 4 B), as expected (Gillooly et al., 2000). When linked to GFP, 2xFYVE colocalized with dextran internalized for 10 min and EEA1 on early endosomes (Fig. 2 A). In agreement with the lack of effect of wortmannin, GFP-2xFYVE did not affect dextran transport to late endosomes (Fig. 2 A, and quantification in Fig. 3 C). Similarly, endocytosed mouse IgGs, a fluid phase marker, were transported to lysosomes and degraded whether or not GFP-2xFYVE was expressed, whereas IgGs accumulated intracellularly when lysosomal degradation was blocked with leupeptin (Fig. 2 B). We thus concluded that PI3P signaling is not involved in bulk endosomal membrane transport.

Bottom Line: We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes.Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting.They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211-Geneva-4, Switzerland.

ABSTRACT
While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

Show MeSH
Related in: MedlinePlus