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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 regulates EGFR sorting in early endosomes. (A) Cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A) were incubated for 10 min at 37°C with rhodamine-dextran, followed by a 90-min chase with 100 nM wortmannin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (B) Cells transfected with GFP-2xFYVE were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C followed (chase) or not (pulse) by a 90-min chase in the presence of leupeptin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (C) Cells expressing GFP-2xFYVE (FYVE), or treated (wort) or not treated (control: ctrl) with wortmannin were labeled with endocytosed EGF-biotin/streptavidin-phycoerythrin (as in B) or dextran (as in A), except that the chase time period was 45 min, and then processed for immunofluorescence using antibodies against the early endosomal (EE) marker EEA1 or the late endosomal (LE) marker LBPA, as indicated. For each condition, the total number of vesicles labeled with EGF or dextran was counted from ≥15 cells in three different experiments (expressed as 100%), as well as the percentage of these vesicles that also contained the EE (EEA1 in control cells; FYVE in FYVE-expressing cells) or the LE endosomal marker LBPA (except with wortmannin, since the drug releases EEA1). (D) After EGF pretreatment (as in Fig. 1 A), cells overexpressing EGFR-GFP were incubated for 10 min at 37°C followed (chase) or not (pulse) by a 35-min chase with wortmannin (wort) and processed for immunofluorescence. (E) The number of vesicles in D that contained both GFP-EGFR and TfR was counted, and is expressed as a percentage of the total number of GFP-EGFR–positive vesicles (C). Bars: (A and B) 2.5 μm; (D) 5 μm.
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fig3: PI3P signaling regulates EGFR sorting in early endosomes. (A) Cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A) were incubated for 10 min at 37°C with rhodamine-dextran, followed by a 90-min chase with 100 nM wortmannin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (B) Cells transfected with GFP-2xFYVE were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C followed (chase) or not (pulse) by a 90-min chase in the presence of leupeptin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (C) Cells expressing GFP-2xFYVE (FYVE), or treated (wort) or not treated (control: ctrl) with wortmannin were labeled with endocytosed EGF-biotin/streptavidin-phycoerythrin (as in B) or dextran (as in A), except that the chase time period was 45 min, and then processed for immunofluorescence using antibodies against the early endosomal (EE) marker EEA1 or the late endosomal (LE) marker LBPA, as indicated. For each condition, the total number of vesicles labeled with EGF or dextran was counted from ≥15 cells in three different experiments (expressed as 100%), as well as the percentage of these vesicles that also contained the EE (EEA1 in control cells; FYVE in FYVE-expressing cells) or the LE endosomal marker LBPA (except with wortmannin, since the drug releases EEA1). (D) After EGF pretreatment (as in Fig. 1 A), cells overexpressing EGFR-GFP were incubated for 10 min at 37°C followed (chase) or not (pulse) by a 35-min chase with wortmannin (wort) and processed for immunofluorescence. (E) The number of vesicles in D that contained both GFP-EGFR and TfR was counted, and is expressed as a percentage of the total number of GFP-EGFR–positive vesicles (C). Bars: (A and B) 2.5 μm; (D) 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)

PI3P signaling regulates EGFR sorting in early endosomes. (A) Cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A) were incubated for 10 min at 37°C with rhodamine-dextran, followed by a 90-min chase with 100 nM wortmannin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (B) Cells transfected with GFP-2xFYVE were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C followed (chase) or not (pulse) by a 90-min chase in the presence of leupeptin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (C) Cells expressing GFP-2xFYVE (FYVE), or treated (wort) or not treated (control: ctrl) with wortmannin were labeled with endocytosed EGF-biotin/streptavidin-phycoerythrin (as in B) or dextran (as in A), except that the chase time period was 45 min, and then processed for immunofluorescence using antibodies against the early endosomal (EE) marker EEA1 or the late endosomal (LE) marker LBPA, as indicated. For each condition, the total number of vesicles labeled with EGF or dextran was counted from ≥15 cells in three different experiments (expressed as 100%), as well as the percentage of these vesicles that also contained the EE (EEA1 in control cells; FYVE in FYVE-expressing cells) or the LE endosomal marker LBPA (except with wortmannin, since the drug releases EEA1). (D) After EGF pretreatment (as in Fig. 1 A), cells overexpressing EGFR-GFP were incubated for 10 min at 37°C followed (chase) or not (pulse) by a 35-min chase with wortmannin (wort) and processed for immunofluorescence. (E) The number of vesicles in D that contained both GFP-EGFR and TfR was counted, and is expressed as a percentage of the total number of GFP-EGFR–positive vesicles (C). Bars: (A and B) 2.5 μm; (D) 5 μm.
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fig3: PI3P signaling regulates EGFR sorting in early endosomes. (A) Cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A) were incubated for 10 min at 37°C with rhodamine-dextran, followed by a 90-min chase with 100 nM wortmannin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (B) Cells transfected with GFP-2xFYVE were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C followed (chase) or not (pulse) by a 90-min chase in the presence of leupeptin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (C) Cells expressing GFP-2xFYVE (FYVE), or treated (wort) or not treated (control: ctrl) with wortmannin were labeled with endocytosed EGF-biotin/streptavidin-phycoerythrin (as in B) or dextran (as in A), except that the chase time period was 45 min, and then processed for immunofluorescence using antibodies against the early endosomal (EE) marker EEA1 or the late endosomal (LE) marker LBPA, as indicated. For each condition, the total number of vesicles labeled with EGF or dextran was counted from ≥15 cells in three different experiments (expressed as 100%), as well as the percentage of these vesicles that also contained the EE (EEA1 in control cells; FYVE in FYVE-expressing cells) or the LE endosomal marker LBPA (except with wortmannin, since the drug releases EEA1). (D) After EGF pretreatment (as in Fig. 1 A), cells overexpressing EGFR-GFP were incubated for 10 min at 37°C followed (chase) or not (pulse) by a 35-min chase with wortmannin (wort) and processed for immunofluorescence. (E) The number of vesicles in D that contained both GFP-EGFR and TfR was counted, and is expressed as a percentage of the total number of GFP-EGFR–positive vesicles (C). Bars: (A and B) 2.5 μm; (D) 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