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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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PI3P signaling is not necessary for ECV biogenesis. (A) The different transport steps that were studied in vitro are shown with reference to the corresponding panels: homotypic early endosome (EE) fusion (B), ECV biogenesis (C), and ECV fusion with late endosomes (LE) (D). ECVs lose the capacity to fuse with EEs in vitro (E) and acquire the capacity to fuse with LEs (D). (B–F) The homotypic fusion of early endosomes (B), the formation of ECVs (C), and the fusion of ECVs formed in vitro with late (D) or early (E) endosomes were measured in vitro in the absence (control) or presence of 100 nM wortmannin, 3 μg/100 μg endosomal protein GST-2xFYVE or C215S mutant, and ATP, as indicated. Note the relatively high efficiency of ECV fusion with late (D) but not early (E) endosomes, when compared with the efficiency of ECV formation (C). (F) Early endosomes were incubated with or without GST-2xFYVE and analyzed by Western blotting using antibodies against the indicated proteins.
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fig4: PI3P signaling is not necessary for ECV biogenesis. (A) The different transport steps that were studied in vitro are shown with reference to the corresponding panels: homotypic early endosome (EE) fusion (B), ECV biogenesis (C), and ECV fusion with late endosomes (LE) (D). ECVs lose the capacity to fuse with EEs in vitro (E) and acquire the capacity to fuse with LEs (D). (B–F) The homotypic fusion of early endosomes (B), the formation of ECVs (C), and the fusion of ECVs formed in vitro with late (D) or early (E) endosomes were measured in vitro in the absence (control) or presence of 100 nM wortmannin, 3 μg/100 μg endosomal protein GST-2xFYVE or C215S mutant, and ATP, as indicated. Note the relatively high efficiency of ECV fusion with late (D) but not early (E) endosomes, when compared with the efficiency of ECV formation (C). (F) Early endosomes were incubated with or without GST-2xFYVE and analyzed by Western blotting using antibodies against the indicated proteins.

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)

PI3P signaling is not necessary for ECV biogenesis. (A) The different transport steps that were studied in vitro are shown with reference to the corresponding panels: homotypic early endosome (EE) fusion (B), ECV biogenesis (C), and ECV fusion with late endosomes (LE) (D). ECVs lose the capacity to fuse with EEs in vitro (E) and acquire the capacity to fuse with LEs (D). (B–F) The homotypic fusion of early endosomes (B), the formation of ECVs (C), and the fusion of ECVs formed in vitro with late (D) or early (E) endosomes were measured in vitro in the absence (control) or presence of 100 nM wortmannin, 3 μg/100 μg endosomal protein GST-2xFYVE or C215S mutant, and ATP, as indicated. Note the relatively high efficiency of ECV fusion with late (D) but not early (E) endosomes, when compared with the efficiency of ECV formation (C). (F) Early endosomes were incubated with or without GST-2xFYVE and analyzed by Western blotting using antibodies against the indicated proteins.
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Related In: Results  -  Collection

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fig4: PI3P signaling is not necessary for ECV biogenesis. (A) The different transport steps that were studied in vitro are shown with reference to the corresponding panels: homotypic early endosome (EE) fusion (B), ECV biogenesis (C), and ECV fusion with late endosomes (LE) (D). ECVs lose the capacity to fuse with EEs in vitro (E) and acquire the capacity to fuse with LEs (D). (B–F) The homotypic fusion of early endosomes (B), the formation of ECVs (C), and the fusion of ECVs formed in vitro with late (D) or early (E) endosomes were measured in vitro in the absence (control) or presence of 100 nM wortmannin, 3 μg/100 μg endosomal protein GST-2xFYVE or C215S mutant, and ATP, as indicated. Note the relatively high efficiency of ECV fusion with late (D) but not early (E) endosomes, when compared with the efficiency of ECV formation (C). (F) Early endosomes were incubated with or without GST-2xFYVE and analyzed by Western blotting using antibodies against the indicated proteins.
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