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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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IPIP27 depletion causes lysosomal hydrolase missorting. (A) Processing of newly synthesized cathepsin-D in RNAi-treated HeLa cells. Following pulse-labeling with [35S]methionine/cysteine and incubation at 37ºC for the indicated times, cathepsin-D was immunoprecipitated from the cells (intracellular) or medium (extracellular) and detected by autoradiography. (B) Quantitation of cathepsin-D processing and secretion. Results are from three experiments and expressed in arbitrary units (AU) as the mean + standard deviation. **p < 0.001. (C) Hexosaminidase activity in IPIP27 RNAi-treated HeLa cell extracts or the medium following incubation at 37ºC for 6 h was assayed as indicated in Materials and Methods. Results are expressed as the mean + standard error of the mean from four experiments. *p < 0.01; **p < 0.001; ***p < 0.0005. (D) Immunofluorescence microscopy of IPIP27-depleted HeLa cells labeled with antibodies to LAMP1. Bar = 10 μm. (E) Immunofluorescence microscopy of control or IPIP27-depleted cells that have internalized Alexa 488-conjugated EGF (green) for 4 h at 37ºC prior to fixation and labeling with an antibody to LAMP1. Bar = 10 μm.
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Figure 9: IPIP27 depletion causes lysosomal hydrolase missorting. (A) Processing of newly synthesized cathepsin-D in RNAi-treated HeLa cells. Following pulse-labeling with [35S]methionine/cysteine and incubation at 37ºC for the indicated times, cathepsin-D was immunoprecipitated from the cells (intracellular) or medium (extracellular) and detected by autoradiography. (B) Quantitation of cathepsin-D processing and secretion. Results are from three experiments and expressed in arbitrary units (AU) as the mean + standard deviation. **p < 0.001. (C) Hexosaminidase activity in IPIP27 RNAi-treated HeLa cell extracts or the medium following incubation at 37ºC for 6 h was assayed as indicated in Materials and Methods. Results are expressed as the mean + standard error of the mean from four experiments. *p < 0.01; **p < 0.001; ***p < 0.0005. (D) Immunofluorescence microscopy of IPIP27-depleted HeLa cells labeled with antibodies to LAMP1. Bar = 10 μm. (E) Immunofluorescence microscopy of control or IPIP27-depleted cells that have internalized Alexa 488-conjugated EGF (green) for 4 h at 37ºC prior to fixation and labeling with an antibody to LAMP1. Bar = 10 μm.

Mentions: Our results indicate that CIMPR trafficking from endosomes to the TGN is impaired by IPIP27 depletion. We should therefore expect reduced levels of CIMPR in the TGN, with consequent missorting of newly synthesized lysosomal hydrolases. To test this expectation, we monitored processing of the lysosomal hydrolase cathepsin-D, which is synthesized as a 53-kDa precursor that undergoes cleavage to 47 kDa intermediate and 31 kDa mature forms upon delivery to endosomes and then lysosomes, respectively (Davidson, 1995). Depletion of IPIP27A or B resulted in a dramatic reduction in the amount of cathepsin D processing, indicating reduced delivery to endosomes and lysosomes (Figure 9, A and B). There was also increased secretion of the cathepsin-D precursor, consistent with having reduced levels of CIMPR in the TGN. Interestingly, this was most apparent for IPIP27B depletion, suggesting a stronger defect in CIMPR recycling. We observed a similar result when analyzing the sorting of another lysosomal hydrolase hexosaminidase. Depletion of either IPIP caused increased secretion of hexosaminidase compared with controls, with a concomitant decrease in the amount of cell-associated hexosaminidase activity, and again the effect was more pronounced upon IPIP27B depletion (Figure 9C).


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)

IPIP27 depletion causes lysosomal hydrolase missorting. (A) Processing of newly synthesized cathepsin-D in RNAi-treated HeLa cells. Following pulse-labeling with [35S]methionine/cysteine and incubation at 37ºC for the indicated times, cathepsin-D was immunoprecipitated from the cells (intracellular) or medium (extracellular) and detected by autoradiography. (B) Quantitation of cathepsin-D processing and secretion. Results are from three experiments and expressed in arbitrary units (AU) as the mean + standard deviation. **p < 0.001. (C) Hexosaminidase activity in IPIP27 RNAi-treated HeLa cell extracts or the medium following incubation at 37ºC for 6 h was assayed as indicated in Materials and Methods. Results are expressed as the mean + standard error of the mean from four experiments. *p < 0.01; **p < 0.001; ***p < 0.0005. (D) Immunofluorescence microscopy of IPIP27-depleted HeLa cells labeled with antibodies to LAMP1. Bar = 10 μm. (E) Immunofluorescence microscopy of control or IPIP27-depleted cells that have internalized Alexa 488-conjugated EGF (green) for 4 h at 37ºC prior to fixation and labeling with an antibody to LAMP1. Bar = 10 μm.
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Figure 9: IPIP27 depletion causes lysosomal hydrolase missorting. (A) Processing of newly synthesized cathepsin-D in RNAi-treated HeLa cells. Following pulse-labeling with [35S]methionine/cysteine and incubation at 37ºC for the indicated times, cathepsin-D was immunoprecipitated from the cells (intracellular) or medium (extracellular) and detected by autoradiography. (B) Quantitation of cathepsin-D processing and secretion. Results are from three experiments and expressed in arbitrary units (AU) as the mean + standard deviation. **p < 0.001. (C) Hexosaminidase activity in IPIP27 RNAi-treated HeLa cell extracts or the medium following incubation at 37ºC for 6 h was assayed as indicated in Materials and Methods. Results are expressed as the mean + standard error of the mean from four experiments. *p < 0.01; **p < 0.001; ***p < 0.0005. (D) Immunofluorescence microscopy of IPIP27-depleted HeLa cells labeled with antibodies to LAMP1. Bar = 10 μm. (E) Immunofluorescence microscopy of control or IPIP27-depleted cells that have internalized Alexa 488-conjugated EGF (green) for 4 h at 37ºC prior to fixation and labeling with an antibody to LAMP1. Bar = 10 μm.
Mentions: Our results indicate that CIMPR trafficking from endosomes to the TGN is impaired by IPIP27 depletion. We should therefore expect reduced levels of CIMPR in the TGN, with consequent missorting of newly synthesized lysosomal hydrolases. To test this expectation, we monitored processing of the lysosomal hydrolase cathepsin-D, which is synthesized as a 53-kDa precursor that undergoes cleavage to 47 kDa intermediate and 31 kDa mature forms upon delivery to endosomes and then lysosomes, respectively (Davidson, 1995). Depletion of IPIP27A or B resulted in a dramatic reduction in the amount of cathepsin D processing, indicating reduced delivery to endosomes and lysosomes (Figure 9, A and B). There was also increased secretion of the cathepsin-D precursor, consistent with having reduced levels of CIMPR in the TGN. Interestingly, this was most apparent for IPIP27B depletion, suggesting a stronger defect in CIMPR recycling. We observed a similar result when analyzing the sorting of another lysosomal hydrolase hexosaminidase. Depletion of either IPIP caused increased secretion of hexosaminidase compared with controls, with a concomitant decrease in the amount of cell-associated hexosaminidase activity, and again the effect was more pronounced upon IPIP27B depletion (Figure 9C).

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