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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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Depletion of IPIP27A alters endosomal morphology. (A) IPIP27A and B were depleted separately or together from HeLa cells for 72 h, and depletion was monitored by Western blotting with the indicated antibodies. (B) Examples of early endosomal morphology in RNAi-treated cells as assessed by EEA1 staining and quantification of the phenotype. Results are from three experiments with 100 cells counted per experiment, and are shown as the mean + standard deviation. (C) Immunofluorescence microscopy of the RNAi-treated cells after labeling with antibodies to EEA1 and Golgin-97 or TfR. Scale bar = 10 μm.
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Figure 5: Depletion of IPIP27A alters endosomal morphology. (A) IPIP27A and B were depleted separately or together from HeLa cells for 72 h, and depletion was monitored by Western blotting with the indicated antibodies. (B) Examples of early endosomal morphology in RNAi-treated cells as assessed by EEA1 staining and quantification of the phenotype. Results are from three experiments with 100 cells counted per experiment, and are shown as the mean + standard deviation. (C) Immunofluorescence microscopy of the RNAi-treated cells after labeling with antibodies to EEA1 and Golgin-97 or TfR. Scale bar = 10 μm.

Mentions: To determine the functional role of IPIP27A and B, they were depleted separately or in combination using RNA interference. Depletion of both IPIPs was specific and efficient (Figure 5A). Importantly we obtained the same results using two independent oligos for each protein (details of small interfering RNA [siRNA] oligos are in Materials and Methods). Depletion of IPIP27A altered endosome morphology, resulting in enlarged EEA1-positive early endosomes that clustered in the perinuclear region (Figure 5, B and C). The Golgi also appeared more compact in the IPIP27A-depleted cells (Figure 5C). The clustered EEA1-positive endosomes contained TfR, which was redistributed to these structures (Figure 5C). Depletion of IPIP27B did not induce endosomal clustering, whereas the depletion of both IPIPs together resulted in a slightly weaker phenotype (Figure 5, A and B). Depletion of IPIP27B, however, did affect the steady-state distribution of TfR, which accumulated in endosomes located in the cell periphery (Figure 5C). In the double-depleted cells, TfR gave a phenotype resembling a combination of that observed with the single knock-downs (Figure 5C). These observations indicate a role for IPIP27A in endosomal organization and suggest that the IPIPs participate in TfR trafficking.


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 alters endosomal morphology. (A) IPIP27A and B were depleted separately or together from HeLa cells for 72 h, and depletion was monitored by Western blotting with the indicated antibodies. (B) Examples of early endosomal morphology in RNAi-treated cells as assessed by EEA1 staining and quantification of the phenotype. Results are from three experiments with 100 cells counted per experiment, and are shown as the mean + standard deviation. (C) Immunofluorescence microscopy of the RNAi-treated cells after labeling with antibodies to EEA1 and Golgin-97 or TfR. Scale bar = 10 μm.
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Related In: Results  -  Collection

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Figure 5: Depletion of IPIP27A alters endosomal morphology. (A) IPIP27A and B were depleted separately or together from HeLa cells for 72 h, and depletion was monitored by Western blotting with the indicated antibodies. (B) Examples of early endosomal morphology in RNAi-treated cells as assessed by EEA1 staining and quantification of the phenotype. Results are from three experiments with 100 cells counted per experiment, and are shown as the mean + standard deviation. (C) Immunofluorescence microscopy of the RNAi-treated cells after labeling with antibodies to EEA1 and Golgin-97 or TfR. Scale bar = 10 μm.
Mentions: To determine the functional role of IPIP27A and B, they were depleted separately or in combination using RNA interference. Depletion of both IPIPs was specific and efficient (Figure 5A). Importantly we obtained the same results using two independent oligos for each protein (details of small interfering RNA [siRNA] oligos are in Materials and Methods). Depletion of IPIP27A altered endosome morphology, resulting in enlarged EEA1-positive early endosomes that clustered in the perinuclear region (Figure 5, B and C). The Golgi also appeared more compact in the IPIP27A-depleted cells (Figure 5C). The clustered EEA1-positive endosomes contained TfR, which was redistributed to these structures (Figure 5C). Depletion of IPIP27B did not induce endosomal clustering, whereas the depletion of both IPIPs together resulted in a slightly weaker phenotype (Figure 5, A and B). Depletion of IPIP27B, however, did affect the steady-state distribution of TfR, which accumulated in endosomes located in the cell periphery (Figure 5C). In the double-depleted cells, TfR gave a phenotype resembling a combination of that observed with the single knock-downs (Figure 5C). These observations indicate a role for IPIP27A in endosomal organization and suggest that the IPIPs participate in TfR trafficking.

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