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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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Shiga toxin trafficking in IPIP27-depleted cells. (A) RNAi-treated HeLa cells were incubated on ice to allow surface binding of STxB-Cy3 (0 min) prior to warming to 37ºC for 20 or 45 min. Cells were labeled with antibodies to TfR or EEA1 (green) and Golgin-97 (blue) prior to analysis by immunofluorescence microscopy. Bar = 10 μm. (B) Quantitation of STxB-Cy3 transport to the TGN, expressed as the percentage of cells with endosomal STxB-Cy3 at t = 45 min. Results are from three experiments with 50 cells counted per experiment and are shown as the mean + standard deviation. *p < 0.01; **p < 0.001.
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Figure 8: Shiga toxin trafficking in IPIP27-depleted cells. (A) RNAi-treated HeLa cells were incubated on ice to allow surface binding of STxB-Cy3 (0 min) prior to warming to 37ºC for 20 or 45 min. Cells were labeled with antibodies to TfR or EEA1 (green) and Golgin-97 (blue) prior to analysis by immunofluorescence microscopy. Bar = 10 μm. (B) Quantitation of STxB-Cy3 transport to the TGN, expressed as the percentage of cells with endosomal STxB-Cy3 at t = 45 min. Results are from three experiments with 50 cells counted per experiment and are shown as the mean + standard deviation. *p < 0.01; **p < 0.001.

Mentions: Another way to measure endosome-to-TGN trafficking is to use the Shiga toxin B-subunit (STxB). STxB is internalized via non-clathrin-mediated endocytosis and delivered to early endosomes, from where it is transported to the TGN in a clathrin-dependent manner (Saint-Pol et al., 2004). We exploited this assay to determine whether the IPIPs are required for trafficking of other cargoes from endosomes to the TGN. In control cells, STxB was efficiently transported to the Golgi apparatus after a 45-min internalization (Figure 8, A and B). In contrast, in cells depleted of IPIP27A or B, a significant amount of STxB was retained within cytoplasmic puncta corresponding to endosomes at 45-min internalization (Figure 8, A and B). IPIP27 depletion therefore impairs endosome-to-TGN trafficking of STxB. Thus, IPIP27A and B are required for efficient endosome-to-TGN trafficking of both CIMPR and STxB.


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)

Shiga toxin trafficking in IPIP27-depleted cells. (A) RNAi-treated HeLa cells were incubated on ice to allow surface binding of STxB-Cy3 (0 min) prior to warming to 37ºC for 20 or 45 min. Cells were labeled with antibodies to TfR or EEA1 (green) and Golgin-97 (blue) prior to analysis by immunofluorescence microscopy. Bar = 10 μm. (B) Quantitation of STxB-Cy3 transport to the TGN, expressed as the percentage of cells with endosomal STxB-Cy3 at t = 45 min. Results are from three experiments with 50 cells counted per experiment and are shown as the mean + standard deviation. *p < 0.01; **p < 0.001.
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Related In: Results  -  Collection

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Figure 8: Shiga toxin trafficking in IPIP27-depleted cells. (A) RNAi-treated HeLa cells were incubated on ice to allow surface binding of STxB-Cy3 (0 min) prior to warming to 37ºC for 20 or 45 min. Cells were labeled with antibodies to TfR or EEA1 (green) and Golgin-97 (blue) prior to analysis by immunofluorescence microscopy. Bar = 10 μm. (B) Quantitation of STxB-Cy3 transport to the TGN, expressed as the percentage of cells with endosomal STxB-Cy3 at t = 45 min. Results are from three experiments with 50 cells counted per experiment and are shown as the mean + standard deviation. *p < 0.01; **p < 0.001.
Mentions: Another way to measure endosome-to-TGN trafficking is to use the Shiga toxin B-subunit (STxB). STxB is internalized via non-clathrin-mediated endocytosis and delivered to early endosomes, from where it is transported to the TGN in a clathrin-dependent manner (Saint-Pol et al., 2004). We exploited this assay to determine whether the IPIPs are required for trafficking of other cargoes from endosomes to the TGN. In control cells, STxB was efficiently transported to the Golgi apparatus after a 45-min internalization (Figure 8, A and B). In contrast, in cells depleted of IPIP27A or B, a significant amount of STxB was retained within cytoplasmic puncta corresponding to endosomes at 45-min internalization (Figure 8, A and B). IPIP27 depletion therefore impairs endosome-to-TGN trafficking of STxB. Thus, IPIP27A and B are required for efficient endosome-to-TGN trafficking of both CIMPR and STxB.

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