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Phosphatidylinositol 3,4,5-trisphosphate localization in recycling endosomes is necessary for AP-1B-dependent sorting in polarized epithelial cells.

Fields IC, King SM, Shteyn E, Kang RS, Fölsch H - Mol. Biol. Cell (2009)

Bottom Line: We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)].Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane.In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell-specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits micro1A or micro1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of micro1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

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A patch of three amino acid residues in μ1B is necessary for AP-1B localization. (A) Schematic diagram of the C-terminus of μ1B and model of the C-terminus of μ1B threaded according to the atomic structure of μ2 using Swiss-Pdb Viewer (DeepView) software (www.expasy.org/spdbv; Guex and Peitsch, 1997). Residues highlighted in yellow are involved in cargo binding, and residues highlighted in green are residues important for membrane recruitment of AP-1B. Amino acid residues of the putative membrane-recruitment patch show regions of high similarity across species for μ1B and dissimilarity in other μ subunits. In this region, basic residues are shown in blue and acidic residues in red. Site-directed mutagenesis was used to replace residues in μ1B with corresponding residues from μ1A (underlined residues). (B) MDCK cells were seeded on collagen/fibronectin-coated coverslips and transiently transfected with cDNAs encoding μ1B-HA (top) or μ1Bloca-HA (bottom) and human TfnR. (C) LLC- PK1::μ1B-HA (top) and LLC-PK1::μ1Bloca-HA (bottom) cell lines were seeded on coverslips and transiently transfected with cDNA encoding human TfnR. (B and C) Twenty-four hours after transfection, cells were serum-starved and allowed to uptake Alexa 488–labeled human Tfn as described in Materials and Methods. Cells were then fixed and incubated with anti-HA antibodies. Subsequently, samples were incubated with Alexa 594–labeled secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. Scale bars, 10 μm. RE, recycling endosomes.
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Figure 7: A patch of three amino acid residues in μ1B is necessary for AP-1B localization. (A) Schematic diagram of the C-terminus of μ1B and model of the C-terminus of μ1B threaded according to the atomic structure of μ2 using Swiss-Pdb Viewer (DeepView) software (www.expasy.org/spdbv; Guex and Peitsch, 1997). Residues highlighted in yellow are involved in cargo binding, and residues highlighted in green are residues important for membrane recruitment of AP-1B. Amino acid residues of the putative membrane-recruitment patch show regions of high similarity across species for μ1B and dissimilarity in other μ subunits. In this region, basic residues are shown in blue and acidic residues in red. Site-directed mutagenesis was used to replace residues in μ1B with corresponding residues from μ1A (underlined residues). (B) MDCK cells were seeded on collagen/fibronectin-coated coverslips and transiently transfected with cDNAs encoding μ1B-HA (top) or μ1Bloca-HA (bottom) and human TfnR. (C) LLC- PK1::μ1B-HA (top) and LLC-PK1::μ1Bloca-HA (bottom) cell lines were seeded on coverslips and transiently transfected with cDNA encoding human TfnR. (B and C) Twenty-four hours after transfection, cells were serum-starved and allowed to uptake Alexa 488–labeled human Tfn as described in Materials and Methods. Cells were then fixed and incubated with anti-HA antibodies. Subsequently, samples were incubated with Alexa 594–labeled secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. Scale bars, 10 μm. RE, recycling endosomes.

Mentions: To better understand the differences in membrane recruitment between AP-1A and AP-1B, we examined in more detail the C-terminus of μ1B. Based on crystal structures, the C-termini of adaptor μ chains contain two subdomains (Owen and Evans, 1998; Heldwein et al., 2004). The more N-terminally positioned subdomain A (amino acids 155-272 and 382-423 in μ1) is mainly involved in forming the binding pocket for the tyrosine-based sorting signals (Owen and Evans, 1998). The second, C-terminal subdomain B of μ2 is involved in binding to PI(4,5)P2. Subdomain B corresponds to amino acids 273-381 in μ1 (Collins et al., 2002; Figure 7A). In contrast, the N-terminus of the μ chain is needed for its incorporation into the AP complex (Collins et al., 2002; Heldwein et al., 2004).


Phosphatidylinositol 3,4,5-trisphosphate localization in recycling endosomes is necessary for AP-1B-dependent sorting in polarized epithelial cells.

Fields IC, King SM, Shteyn E, Kang RS, Fölsch H - Mol. Biol. Cell (2009)

A patch of three amino acid residues in μ1B is necessary for AP-1B localization. (A) Schematic diagram of the C-terminus of μ1B and model of the C-terminus of μ1B threaded according to the atomic structure of μ2 using Swiss-Pdb Viewer (DeepView) software (www.expasy.org/spdbv; Guex and Peitsch, 1997). Residues highlighted in yellow are involved in cargo binding, and residues highlighted in green are residues important for membrane recruitment of AP-1B. Amino acid residues of the putative membrane-recruitment patch show regions of high similarity across species for μ1B and dissimilarity in other μ subunits. In this region, basic residues are shown in blue and acidic residues in red. Site-directed mutagenesis was used to replace residues in μ1B with corresponding residues from μ1A (underlined residues). (B) MDCK cells were seeded on collagen/fibronectin-coated coverslips and transiently transfected with cDNAs encoding μ1B-HA (top) or μ1Bloca-HA (bottom) and human TfnR. (C) LLC- PK1::μ1B-HA (top) and LLC-PK1::μ1Bloca-HA (bottom) cell lines were seeded on coverslips and transiently transfected with cDNA encoding human TfnR. (B and C) Twenty-four hours after transfection, cells were serum-starved and allowed to uptake Alexa 488–labeled human Tfn as described in Materials and Methods. Cells were then fixed and incubated with anti-HA antibodies. Subsequently, samples were incubated with Alexa 594–labeled secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. Scale bars, 10 μm. RE, recycling endosomes.
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Figure 7: A patch of three amino acid residues in μ1B is necessary for AP-1B localization. (A) Schematic diagram of the C-terminus of μ1B and model of the C-terminus of μ1B threaded according to the atomic structure of μ2 using Swiss-Pdb Viewer (DeepView) software (www.expasy.org/spdbv; Guex and Peitsch, 1997). Residues highlighted in yellow are involved in cargo binding, and residues highlighted in green are residues important for membrane recruitment of AP-1B. Amino acid residues of the putative membrane-recruitment patch show regions of high similarity across species for μ1B and dissimilarity in other μ subunits. In this region, basic residues are shown in blue and acidic residues in red. Site-directed mutagenesis was used to replace residues in μ1B with corresponding residues from μ1A (underlined residues). (B) MDCK cells were seeded on collagen/fibronectin-coated coverslips and transiently transfected with cDNAs encoding μ1B-HA (top) or μ1Bloca-HA (bottom) and human TfnR. (C) LLC- PK1::μ1B-HA (top) and LLC-PK1::μ1Bloca-HA (bottom) cell lines were seeded on coverslips and transiently transfected with cDNA encoding human TfnR. (B and C) Twenty-four hours after transfection, cells were serum-starved and allowed to uptake Alexa 488–labeled human Tfn as described in Materials and Methods. Cells were then fixed and incubated with anti-HA antibodies. Subsequently, samples were incubated with Alexa 594–labeled secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. Scale bars, 10 μm. RE, recycling endosomes.
Mentions: To better understand the differences in membrane recruitment between AP-1A and AP-1B, we examined in more detail the C-terminus of μ1B. Based on crystal structures, the C-termini of adaptor μ chains contain two subdomains (Owen and Evans, 1998; Heldwein et al., 2004). The more N-terminally positioned subdomain A (amino acids 155-272 and 382-423 in μ1) is mainly involved in forming the binding pocket for the tyrosine-based sorting signals (Owen and Evans, 1998). The second, C-terminal subdomain B of μ2 is involved in binding to PI(4,5)P2. Subdomain B corresponds to amino acids 273-381 in μ1 (Collins et al., 2002; Figure 7A). In contrast, the N-terminus of the μ chain is needed for its incorporation into the AP complex (Collins et al., 2002; Heldwein et al., 2004).

Bottom Line: We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)].Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane.In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell-specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits micro1A or micro1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of micro1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

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