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The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway.

Noakes CJ, Lee G, Lowe M - Mol. Biol. Cell (2011)

Bottom Line: The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1.IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN).The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.

ABSTRACT
Mutation of the inositol polyphosphate 5-phosphatase OCRL1 results in two disorders in humans, namely Lowe syndrome (characterized by ocular, nervous system, and renal defects) and type 2 Dent disease (in which only the renal symptoms are evident). The disease mechanisms of these syndromes are poorly understood. Here we identify two novel OCRL1-binding proteins, termed inositol polyphosphate phosphatase interacting protein of 27 kDa (IPIP27)A and B (also known as Ses1 and 2), that also bind the related 5-phosphatase Inpp5b. The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1. IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN). The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane. Our results identify IPIP27A and B as key players in endocytic trafficking and strongly suggest that defects in this process are responsible for the pathology of Lowe syndrome and Dent disease.

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Related in: MedlinePlus

Depletion of IPIP27A impairs transferrin recycling. (A) Control, IPIP27A and/or B RNAi-treated HeLa cells were allowed to internalize Alexa 488-conjugated transferrin for 30 min at 37ºC, washed, and incubated for an additional 0, 15, or 30 min at 37ºC prior to fixation and analysis by fluorescence microscopy. Scale bar = 10 μm. (B) Quantitation of cell-associated Alexa 488–conjugated transferrin at various times of incubation. Results are from three experiments with 20 cells counted per experiment, and are shown in arbitrary units (AU) as the mean + standard deviation. **p < 0.001, ***p < 0.0005.
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Figure 6: Depletion of IPIP27A impairs transferrin recycling. (A) Control, IPIP27A and/or B RNAi-treated HeLa cells were allowed to internalize Alexa 488-conjugated transferrin for 30 min at 37ºC, washed, and incubated for an additional 0, 15, or 30 min at 37ºC prior to fixation and analysis by fluorescence microscopy. Scale bar = 10 μm. (B) Quantitation of cell-associated Alexa 488–conjugated transferrin at various times of incubation. Results are from three experiments with 20 cells counted per experiment, and are shown in arbitrary units (AU) as the mean + standard deviation. **p < 0.001, ***p < 0.0005.

Mentions: The simplest explanation for the redistribution of TfR into clustered perinuclear or peripheral endosomes upon IPIP27A or B depletion is that the IPIPs are required for trafficking of the receptor out of these compartments. To test this hypothesis, we directly analyzed recycling of fluorescently labeled transferrin that had been internalized into IPIP-depleted cells. Depletion of IPIP27A or B alone or together had little effect on the amount of transferrin internalized into cells over a 30-min time period, indicating that the IPIPs are not essential for transferrin endocytosis (Figure 6A). The recycling of internalized transferrin was clearly impaired, however, upon depletion of either IPIP alone or when they were codepleted (Figure 6, A and B). Transferrin was retained in clustered perinuclear endosomes upon depletion of IPIP27A and in more peripheral endosomes when IPIP27B was depleted, consistent with the redistribution of TfR observed under these conditions (Figures 5C and 6A).


The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway.

Noakes CJ, Lee G, Lowe M - Mol. Biol. Cell (2011)

Depletion of IPIP27A impairs transferrin recycling. (A) Control, IPIP27A and/or B RNAi-treated HeLa cells were allowed to internalize Alexa 488-conjugated transferrin for 30 min at 37ºC, washed, and incubated for an additional 0, 15, or 30 min at 37ºC prior to fixation and analysis by fluorescence microscopy. Scale bar = 10 μm. (B) Quantitation of cell-associated Alexa 488–conjugated transferrin at various times of incubation. Results are from three experiments with 20 cells counted per experiment, and are shown in arbitrary units (AU) as the mean + standard deviation. **p < 0.001, ***p < 0.0005.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046058&req=5

Figure 6: Depletion of IPIP27A impairs transferrin recycling. (A) Control, IPIP27A and/or B RNAi-treated HeLa cells were allowed to internalize Alexa 488-conjugated transferrin for 30 min at 37ºC, washed, and incubated for an additional 0, 15, or 30 min at 37ºC prior to fixation and analysis by fluorescence microscopy. Scale bar = 10 μm. (B) Quantitation of cell-associated Alexa 488–conjugated transferrin at various times of incubation. Results are from three experiments with 20 cells counted per experiment, and are shown in arbitrary units (AU) as the mean + standard deviation. **p < 0.001, ***p < 0.0005.
Mentions: The simplest explanation for the redistribution of TfR into clustered perinuclear or peripheral endosomes upon IPIP27A or B depletion is that the IPIPs are required for trafficking of the receptor out of these compartments. To test this hypothesis, we directly analyzed recycling of fluorescently labeled transferrin that had been internalized into IPIP-depleted cells. Depletion of IPIP27A or B alone or together had little effect on the amount of transferrin internalized into cells over a 30-min time period, indicating that the IPIPs are not essential for transferrin endocytosis (Figure 6A). The recycling of internalized transferrin was clearly impaired, however, upon depletion of either IPIP alone or when they were codepleted (Figure 6, A and B). Transferrin was retained in clustered perinuclear endosomes upon depletion of IPIP27A and in more peripheral endosomes when IPIP27B was depleted, consistent with the redistribution of TfR observed under these conditions (Figures 5C and 6A).

Bottom Line: The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1.IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN).The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.

ABSTRACT
Mutation of the inositol polyphosphate 5-phosphatase OCRL1 results in two disorders in humans, namely Lowe syndrome (characterized by ocular, nervous system, and renal defects) and type 2 Dent disease (in which only the renal symptoms are evident). The disease mechanisms of these syndromes are poorly understood. Here we identify two novel OCRL1-binding proteins, termed inositol polyphosphate phosphatase interacting protein of 27 kDa (IPIP27)A and B (also known as Ses1 and 2), that also bind the related 5-phosphatase Inpp5b. The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1. IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN). The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane. Our results identify IPIP27A and B as key players in endocytic trafficking and strongly suggest that defects in this process are responsible for the pathology of Lowe syndrome and Dent disease.

Show MeSH
Related in: MedlinePlus