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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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PH-Akt-GFP colocalizes with TfnR. MDCK cells grown on coverslips were transiently transfected with cDNAs encoding RFP-2xFYVE (A), PTB-ARH-RFP (B), or PH-Akt-GFP (C). Six hours after transfection (A) or 24 h after transfection (B and C), cells were fixed and incubated with anti-TfnR antibodies (H68.4) followed by incubation with Alexa 488– or Alexa 594–conjugated secondary antibodies. Specimens were analyzed by confocal microscopy, and representative images are shown. Scale bars, 10 μm.
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Figure 1: PH-Akt-GFP colocalizes with TfnR. MDCK cells grown on coverslips were transiently transfected with cDNAs encoding RFP-2xFYVE (A), PTB-ARH-RFP (B), or PH-Akt-GFP (C). Six hours after transfection (A) or 24 h after transfection (B and C), cells were fixed and incubated with anti-TfnR antibodies (H68.4) followed by incubation with Alexa 488– or Alexa 594–conjugated secondary antibodies. Specimens were analyzed by confocal microscopy, and representative images are shown. Scale bars, 10 μm.

Mentions: To test for the distribution of PI(3)P in MDCK cells, we cotransfected coverslip grown cells with cDNA encoding a tandem FYVE domain chimera tagged with mRFP. Tandem FYVE domain constructs are used to increase the binding affinity of this domain to PI(3)P (Gillooly et al., 2000). Six hours after transfection, cells were stained for endogenous TfnR. As expected, we found the FYVE domain localizing to peripheral endosomal structures (Figure 1A). We confirmed by Tfn uptake for 7 min that these structures are early endosomes (not shown). Importantly, we did not observe colocalization between the FYVE domain and TfnR in perinuclear recycling endosomes (Figure 1A). Next we expressed mRFP-tagged PTB-ARH in MDCK cells grown on coverslips. We found PTB-ARH-RFP primarily localizing to the plasma membrane (Figure 1B). Finally, we transiently expressed the PH domain of Akt fused to GFP (PH-Akt-GFP) in coverslip-grown MDCK cells. The PH domain of Akt is known to bind relatively specifically to PI(3,4,5)P3 in vitro with a threefold less binding affinity to PI(3,4)P2 (James et al., 1996) and is commonly used to monitor PI(3,4,5)P3 localization in vivo (Di Paolo and De Camilli, 2006). We found PH-Akt-GFP localizing to the plasma membrane as well as to TfnR-positive recycling endosomes (Figure 1C).


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)

PH-Akt-GFP colocalizes with TfnR. MDCK cells grown on coverslips were transiently transfected with cDNAs encoding RFP-2xFYVE (A), PTB-ARH-RFP (B), or PH-Akt-GFP (C). Six hours after transfection (A) or 24 h after transfection (B and C), cells were fixed and incubated with anti-TfnR antibodies (H68.4) followed by incubation with Alexa 488– or Alexa 594–conjugated secondary antibodies. Specimens were analyzed by confocal microscopy, and representative images are shown. Scale bars, 10 μm.
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Related In: Results  -  Collection

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Figure 1: PH-Akt-GFP colocalizes with TfnR. MDCK cells grown on coverslips were transiently transfected with cDNAs encoding RFP-2xFYVE (A), PTB-ARH-RFP (B), or PH-Akt-GFP (C). Six hours after transfection (A) or 24 h after transfection (B and C), cells were fixed and incubated with anti-TfnR antibodies (H68.4) followed by incubation with Alexa 488– or Alexa 594–conjugated secondary antibodies. Specimens were analyzed by confocal microscopy, and representative images are shown. Scale bars, 10 μm.
Mentions: To test for the distribution of PI(3)P in MDCK cells, we cotransfected coverslip grown cells with cDNA encoding a tandem FYVE domain chimera tagged with mRFP. Tandem FYVE domain constructs are used to increase the binding affinity of this domain to PI(3)P (Gillooly et al., 2000). Six hours after transfection, cells were stained for endogenous TfnR. As expected, we found the FYVE domain localizing to peripheral endosomal structures (Figure 1A). We confirmed by Tfn uptake for 7 min that these structures are early endosomes (not shown). Importantly, we did not observe colocalization between the FYVE domain and TfnR in perinuclear recycling endosomes (Figure 1A). Next we expressed mRFP-tagged PTB-ARH in MDCK cells grown on coverslips. We found PTB-ARH-RFP primarily localizing to the plasma membrane (Figure 1B). Finally, we transiently expressed the PH domain of Akt fused to GFP (PH-Akt-GFP) in coverslip-grown MDCK cells. The PH domain of Akt is known to bind relatively specifically to PI(3,4,5)P3 in vitro with a threefold less binding affinity to PI(3,4)P2 (James et al., 1996) and is commonly used to monitor PI(3,4,5)P3 localization in vivo (Di Paolo and De Camilli, 2006). We found PH-Akt-GFP localizing to the plasma membrane as well as to TfnR-positive recycling endosomes (Figure 1C).

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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