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Differential Clathrin Binding and Subcellular Localization of OCRL1 Splice Isoforms

2009; Elsevier BV; Volume: 284; Issue: 15 Linguagem: Inglês

10.1074/jbc.m807442200

1083-351X

Rawshan Choudhury, Christopher Noakes, Edward A. McKenzie, Corinne Kox, Martin Lowe,

Protein Kinase Regulation and GTPase Signaling

Mutation of the inositol polyphosphate 5-phosphatase OCRL1 causes the X-linked disorder oculocerebrorenal syndrome of Lowe, characterized by defects in the brain, kidneys, and eyes. OCRL1 exists as two splice isoforms that differ by a single exon encoding 8 amino acids. The longer protein, termed isoform a, is the only form in brain, whereas both isoforms are present in all other tissues. The significance of OCRL1 splicing is currently unclear. Given its proximity to a clathrin-binding site, we hypothesized that splicing may alter the clathrin binding properties of OCRL1. Here we show that this is indeed the case. OCRL1 isoform a binds clathrin with higher affinity than isoform b and is significantly more enriched in clathrin-coated trafficking intermediates. We also identify a second clathrin-binding site in OCRL1 that contributes to clathrin binding of both isoforms. Association of OCRL1 with clathrin-coated intermediates requires membrane association through interaction with Rab GTPases but not binding to the clathrin adaptor AP2. Expression of OCRL1 isoform a lacking the 5-phosphatase domain impairs transferrin endocytosis, whereas an equivalent version of isoform b does not. Our results suggest that OCRL1 exists as two functional pools, one participating in clathrin-mediated trafficking events such as endocytosis and another that is much less or not involved in this process. Mutation of the inositol polyphosphate 5-phosphatase OCRL1 causes the X-linked disorder oculocerebrorenal syndrome of Lowe, characterized by defects in the brain, kidneys, and eyes. OCRL1 exists as two splice isoforms that differ by a single exon encoding 8 amino acids. The longer protein, termed isoform a, is the only form in brain, whereas both isoforms are present in all other tissues. The significance of OCRL1 splicing is currently unclear. Given its proximity to a clathrin-binding site, we hypothesized that splicing may alter the clathrin binding properties of OCRL1. Here we show that this is indeed the case. OCRL1 isoform a binds clathrin with higher affinity than isoform b and is significantly more enriched in clathrin-coated trafficking intermediates. We also identify a second clathrin-binding site in OCRL1 that contributes to clathrin binding of both isoforms. Association of OCRL1 with clathrin-coated intermediates requires membrane association through interaction with Rab GTPases but not binding to the clathrin adaptor AP2. Expression of OCRL1 isoform a lacking the 5-phosphatase domain impairs transferrin endocytosis, whereas an equivalent version of isoform b does not. Our results suggest that OCRL1 exists as two functional pools, one participating in clathrin-mediated trafficking events such as endocytosis and another that is much less or not involved in this process. Oculocerebrorenal syndrome of Lowe is a rare X-linked disorder affecting primarily the brain, eyes, and kidneys (1Lowe C.U. Terrey M. Mac L.E. AMA Am. J. Dis. Child. 1952; 83: 164-184PubMed Google Scholar, 2Nussbaum R. Suchy S.F. Scriver C.R. Beauder A.L. Sly W.S. Valle D. Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill Inc., New York2001: 6257-6266Google Scholar). Lowe syndrome is caused by mutation of OCRL1, a ubiquitously expressed inositol polyphosphate 5-phosphatase that preferentially hydrolyzes phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (reviewed in Ref. 3Lowe M. Traffic. 2005; 6: 711-719Crossref PubMed Scopus (138) Google Scholar). OCRL1 is localized to the trans-Golgi network, early endosomes, plasma membrane ruffles, and clathrin-coated trafficking intermediates (4Olivos-Glander I.M. Janne P.A. Nussbaum R.L. Am. J. Hum. Genet. 1995; 57: 817-823PubMed Google Scholar, 5Faucherre A. Desbois P. Nagano F. Satre V. Lunardi J. Gacon G. Dorseuil O. Hum. Mol. Genet. 2005; 14: 1441-1448Crossref PubMed Scopus (51) Google Scholar, 6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar, 7Ungewickell A. Ward M.E. Ungewickell E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13501-13506Crossref PubMed Scopus (106) Google Scholar, 8Erdmann K.S. Mao Y. McCrea H.J. Zoncu R. Lee S. Paradise S. Modregger J. Biemesderfer D. Toomre D. De Camilli P. Dev. Cell. 2007; 13: 377-390Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). In addition to a central 5-phosphatase domain, OCRL1 also has C-terminal ASH and RhoGAP-like domains. The ASH domain binds members of the Rab family of small GTPases, which are required for the correct targeting of OCRL1 to the TGN 4The abbreviations used are: TGN, trans-Golgi network; GST, glutathione S-transferase; GFP, green fluorescent protein; NRK, normal rat kidney; MES, 4-morpholineethanesulfonic acid. and endosomes (9Hyvola N. Diao A. McKenzie E. Skippen A. Cockcroft S. Lowe M. EMBO J. 2006; 25: 3750-3761Crossref PubMed Scopus (124) Google Scholar). The RhoGAP-like domain appears to lack catalytic activity and rather serves to bind Rac and Cdc42, which may help anchor OCRL1 to the membrane (8Erdmann K.S. Mao Y. McCrea H.J. Zoncu R. Lee S. Paradise S. Modregger J. Biemesderfer D. Toomre D. De Camilli P. Dev. Cell. 2007; 13: 377-390Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 10Faucherre A. Desbois P. Satre V. Lunardi J. Dorseuil O. Gacon G. Hum. Mol. Genet. 2003; 12: 2449-2456Crossref PubMed Scopus (91) Google Scholar, 11Lichter-Konecki U. Farber L.W. Cronin J.S. Suchy S.F. Nussbaum R.L. Mol. Genet. Metab. 2006; 89: 121-128Crossref PubMed Scopus (36) Google Scholar). A recent study also suggested the RhoGAP-like domain may bind ARF1 and ARF6 (11Lichter-Konecki U. Farber L.W. Cronin J.S. Suchy S.F. Nussbaum R.L. Mol. Genet. Metab. 2006; 89: 121-128Crossref PubMed Scopus (36) Google Scholar). The C-terminal region of OCRL1 interacts with APPL1, another Rab5 effector that participates in signaling from endocytic membranes (8Erdmann K.S. Mao Y. McCrea H.J. Zoncu R. Lee S. Paradise S. Modregger J. Biemesderfer D. Toomre D. De Camilli P. Dev. Cell. 2007; 13: 377-390Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). In addition, OCRL1 binds directly to the terminal domain of clathrin heavy chain, which occurs via a type I clathrin box with the sequence LIDLE that is present on a loop protruding from the globular RhoGAP-like domain (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar, 7Ungewickell A. Ward M.E. Ungewickell E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13501-13506Crossref PubMed Scopus (106) Google Scholar, 8Erdmann K.S. Mao Y. McCrea H.J. Zoncu R. Lee S. Paradise S. Modregger J. Biemesderfer D. Toomre D. De Camilli P. Dev. Cell. 2007; 13: 377-390Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Binding studies have indicated that a second clathrin-binding site exists in OCRL1, consistent with its ability to polymerize clathrin in vitro, but it has yet to be identified (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar). OCRL1 can also bind via an N-terminal FEDNF motif to the appendage domain of α-adaptin, a subunit of the AP2 plasma membrane clathrin adaptor complex (7Ungewickell A. Ward M.E. Ungewickell E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13501-13506Crossref PubMed Scopus (106) Google Scholar). Together the specificity of OCRL1 5-phosphatase activity, its subcellular localization, and its known interaction partners suggest it could participate in a number of processes, including endocytosis, trafficking at the TGN/endosome interface, signaling from the plasma membrane, endosomes, and/or the TGN or regulation of actin dynamics at these locations (reviewed in Ref. 3Lowe M. Traffic. 2005; 6: 711-719Crossref PubMed Scopus (138) Google Scholar; see also Refs. 6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar, 8Erdmann K.S. Mao Y. McCrea H.J. Zoncu R. Lee S. Paradise S. Modregger J. Biemesderfer D. Toomre D. De Camilli P. Dev. Cell. 2007; 13: 377-390Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Suchy S.F. Nussbaum R.L. Am. J. Hum. Genet. 2002; 71: 1420-1427Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). It is also conceivable that OCRL1 could participate in other processes such as cell polarization or housekeeping through removal of ectopic phosphatidylinositol 4,5-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate from endomembranes. Although OCRL1 is ubiquitously expressed, the manifestations of Lowe syndrome are restricted to only a few tissues. This may be due to functional compensation by the related 5-phosphatase INPP5B, which shares the same domain organization and substrate specificity as OCRL1 (8Erdmann K.S. Mao Y. McCrea H.J. Zoncu R. Lee S. Paradise S. Modregger J. Biemesderfer D. Toomre D. De Camilli P. Dev. Cell. 2007; 13: 377-390Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 13Jefferson A.B. Majerus P.W. J. Biol. Chem. 1995; 270: 9370-9377Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 14Schmid A.C. Wise H.M. Mitchell C.A. Nussbaum R. Woscholski R. FEBS Lett. 2004; 576: 9-13Crossref PubMed Scopus (110) Google Scholar). INPP5B also has a similar cellular localization and interaction partner profile as OCRL1, with some differences, the most notable of which is its lack of clathrin binding and absence from clathrin-coated structures (8Erdmann K.S. Mao Y. McCrea H.J. Zoncu R. Lee S. Paradise S. Modregger J. Biemesderfer D. Toomre D. De Camilli P. Dev. Cell. 2007; 13: 377-390Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 15Shin H.W. Hayashi M. Christoforidis S. Lacas-Gervais S. Hoepfner S. Wenk M.R. Modregger J. Uttenweiler-Joseph S. Wilm M. Nystuen A. Frankel W.N. Solimena M. De Camilli P. Zerial M. J. Cell Biol. 2005; 170: 607-618Crossref PubMed Scopus (293) Google Scholar, 16Williams C. Choudhury R. McKenzie E. Lowe M. J. Cell Sci. 2007; 120: 3941-3951Crossref PubMed Scopus (43) Google Scholar). Knock-out studies in mice support the idea that OCRL1 and INPP5B can functionally compensate for loss of the other protein (17Janne P.A. Suchy S.F. Bernard D. MacDonald M. Crawley J. Grinberg A. Wynshaw-Boris A. Westphal H. Nussbaum R.L. J. Clin. Investig. 1998; 101: 2042-2053Crossref PubMed Scopus (137) Google Scholar). OCRL1 exists as two alternatively spliced forms, termed a and b (18Nussbaum R.L. Orrison B.M. Janne P.A. Charnas L. Chinault A.C. Hum. Genet. 1997; 99: 145-150Crossref PubMed Scopus (85) Google Scholar, 19Johnson J.M. Castle J. Garrett-Engele P. Kan Z. Loerch P.M. Armour C.D. Santos R. Schadt E.E. Stoughton R. Shoemaker D.D. Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1174) Google Scholar). Isoform a contains an additional exon lacking in isoform b that encodes 8 amino acids adjacent to the LIDLE clathrin box. Isoform a is present in all tissues, whereas isoform b is present in all tissues apart from the brain (19Johnson J.M. Castle J. Garrett-Engele P. Kan Z. Loerch P.M. Armour C.D. Santos R. Schadt E.E. Stoughton R. Shoemaker D.D. Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1174) Google Scholar). Isoform a is therefore the only form in brain, but in most other tissues it appears to be the minor form. The significance of OCRL1 splicing is currently unknown. Given the proximity of the alternatively spliced exon to the clathrin box, we hypothesized that it could influence binding of OCRL1 to clathrin. Here we show that this is indeed the case and that it is clathrin binding that determines the amount of OCRL1 that enters clathrin-coated transport intermediates. Isoform a binds clathrin with higher affinity than isoform b and is significantly more enriched in clathrin-coated intermediates. We also identify a second clathrin-binding site in the N terminus of OCRL1 that is important for clathrin binding of both OCRL1 isoforms. Expression of an OCRL1 isoform a construct lacking the 5-phosphatase domain delays transferrin endocytosis, whereas an equivalent version of isoform b does not, consistent with distinct functional roles for the two variants of OCRL1. This is likely of significance in the disease state because the brain, one of the major tissues affected in Lowe syndrome, only expresses isoform a. Materials and Antibodies-All reagents were from Sigma or Merck unless stated otherwise. Protease inhibitors (mixture set III) were from Calbiochem and used at 1:250. Rabbit KINS anti-OCRL1 and sheep anti-GST antibodies were described previously (9Hyvola N. Diao A. McKenzie E. Skippen A. Cockcroft S. Lowe M. EMBO J. 2006; 25: 3750-3761Crossref PubMed Scopus (124) Google Scholar). Mouse anti-clathrin heavy chain X22 for immunofluorescence studies was a kind gift from Prof. Liz Smythe (University of Sheffield, UK). Mouse antibody against clathrin heavy chain for Western blotting was purchased from BD Transduction Laboratories. Mouse anti-transferrin receptor antibody was from Zymed Laboratories Inc.. Sheep anti-golgin-84 and rabbit anti-GM130 antibodies have been described previously (20Diao A. Rahman D. Pappin D.J. Lucocq J. Lowe M. J. Cell Biol. 2003; 160: 201-212Crossref PubMed Scopus (193) Google Scholar). Mouse anti-CI-MPR was purchased from Affinity Bioreagents. Rabbit anti-CI-MPR was a kind gift from Prof. Paul Luzio (University of Cambridge, UK). Fluorophore-conjugated (Alexa 594 and Alexa 488) and horseradish peroxidase-conjugated secondary antibodies were purchased from Molecular Probes and Tago Immunologicals, respectively. Molecular Biology and Yeast Two-hybrid Experiments-All constructs were made using standard molecular biology techniques. Primer sequences are available on request. GFP- and His/protein S-tagged human OCRL1 isoform b (GenBank™ accession number NP_001578) constructs were described previously (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar, 9Hyvola N. Diao A. McKenzie E. Skippen A. Cockcroft S. Lowe M. EMBO J. 2006; 25: 3750-3761Crossref PubMed Scopus (124) Google Scholar). DNA encoding full-length isoform a (GenBank™ accession number NP_000267) was cloned from a human liver cDNA library and cloned into pEGFP-C1 (Clontech) for expression of GFP-tagged protein in mammalian cells, pGBKT7 (Stratagene) for yeast two-hybrid experiments, and pBAC2 (Novagen) for expression of His/protein S-tagged recombinant protein in insect cells. OCRL1 isoforms a and b were cloned into a modified version of pcDNA3.1 for N-terminal tagging with mCherry. The OCRL1 LIDIA and LIDLE clathrin box sequences were deleted or point mutated, and the FEDNF α-adaptin-binding site was mutated to AEANF using the QuikChange method (Stratagene). Constructs encoding GST-tagged clathrin terminal domain (residues 1-579), α-adaptin appendage domain, and Rab and Rac GTPases have been described previously (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar, 9Hyvola N. Diao A. McKenzie E. Skippen A. Cockcroft S. Lowe M. EMBO J. 2006; 25: 3750-3761Crossref PubMed Scopus (124) Google Scholar, 16Williams C. Choudhury R. McKenzie E. Lowe M. J. Cell Sci. 2007; 120: 3941-3951Crossref PubMed Scopus (43) Google Scholar). An oligonucleotide encoding residues 403-413 of human APPL1 was ligated into the BamHI and EcoRI sites of pGEX4T-2 for preparation of recombinant protein in Escherichia coli. All constructs were verified by DNA sequencing using the ABI Prism Big Dye Terminator Cycle Sequencing kit (Applied Biosystems). Plasmid for expression of mCherry-tagged wild-type Rab5 in mammalian cells was kindly provided by Prof. Philip Woodman (University of Manchester, UK). Yeast two-hybrid analysis was performed according to the Clontech manual as described previously (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar). The pGADT7-clathrin terminal domain was a kind gift from Prof. Harald Stenmark (Norwegian Radium Hospital, Oslo, Norway). The pGADT7-α-adaptin appendage domain has been described previously (16Williams C. Choudhury R. McKenzie E. Lowe M. J. Cell Sci. 2007; 120: 3941-3951Crossref PubMed Scopus (43) Google Scholar). Cell Culture, Transfection, and Transferrin Uptake-Adherent HeLa and NRK cells were grown at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. HeLa and NRK cells were transiently transfected with FuGENE 6 (Roche Diagnostics) and JetPEI (Polyplus), respectively, according to the manufacturer's instructions and incubated for 16-20 h before analysis. Transferrin uptake was performed by washing cells three times with warm uptake medium (L-15 containing 2 mg/ml bovine serum albumin) before adding warm uptake medium containing 5 μg/ml Texas Red- or Alexa 594-conjugated transferrin. Cells were incubated for 2, 5, 15, or 30 min at 37 °C and fixed directly into 3% paraformaldehyde at room temperature. Quantitation of uptake was performed using ImageJ by measuring the mean fluorescence intensity of each cell. Semi-quantitative analysis was also performed by comparing fluorescence intensity of transfected cells to neighboring nontransfected cells. Immunofluorescence Microscopy-Cells were fixed in 3% paraformaldehyde in phosphate-buffered saline, and immunofluorescence microscopy was performed as described previously (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar). Preparation of Cell Extracts and Pulldown Experiments-Recombinant GST-tagged proteins were expressed in E. coli and purified using standard techniques. Extracts were prepared from HeLa cells expressing GFP-tagged OCRL1 by washing the cells twice in ice-cold phosphate-buffered saline followed by extraction in HNMT (20 mm Hepes, pH 7.4, 0.1 m NaCl, 5 mm MgCl2, 0.25% Triton X-100) containing protease inhibitors (1 ml of HNMT/10-cm dish) for 15 min on ice. Extracts were clarified before use by spinning at 15,000 rpm in a microcentrifuge for 10 min. Binding was performed by incubating 250 μl of extract with 20-100 μg of GST-tagged bait protein bound to GSH-Sepharose for 5 h at 4 °C. Binding with purified proteins was performed by incubating 0.5 μg of His/S-tagged OCRL1 in 100 μl of HNMT containing 5 μg of bovine serum albumin with 2 μg of GST-tagged bait bound to 10 μl of GSH-Sepharose for 3 h at 4 °C. Binding with in vitro translated proteins was performed by incubating 5 μl of in vitro translated protein (made using the Promega coupled TnT kit according to the manufacturer's instructions) with 2 μg of GST-tagged bait bound to 10 μl of GSH-Sepharose in 100 μl of HNMT for 3 h at 4 °C. After washing three times with HNMT, proteins were eluted by boiling in SDS sample buffer and analyzed by Western blotting with appropriate antibodies or by autoradiography. Binding experiments were performed 2-4 times per experiment. Representative examples of each experiment are shown in the figures. Preparation of Clathrin-coated Vesicles-Clathrin-coated vesicles were isolated from HeLa cells expressing GFP-tagged OCRL1 according to Ref. 21Hirst J. Miller S.E. Taylor M.J. von Mollard G.F. Robinson M.S. Mol. Biol. Cell. 2004; 15: 5593-5602Crossref PubMed Scopus (79) Google Scholar. Cells from a confluent 10-cm dish were washed twice with ice-cold phosphate-buffered saline and scraped into 1 ml of buffer C (0.1 m MES, pH 6.5, 0.2 mm EGTA, 0.5 mm MgCl2) containing protease inhibitors before passing 22 times through a ball-bearing homogenizer (8.01-mm ball inside 8.02-mm barrel). The homogenate was centrifuged at 3,900 × g in a Beckman TLS55, and the low speed supernatant was incubated with 50 μg/ml RNase A for 30 min on ice. The membranes were pelleted by spinning at 50,000 rpm for 30 min in a Beckman TLA55 rotor and resuspended in 300 μl of buffer C by passing 10-15 times through a 25-gauge needle. The resuspended pellet (high speed pellet) was mixed with an equal volume of 12.5% Ficoll, 12.5% sucrose (in buffer C) and centrifuged for 25 min at 20,000 rpm in a TLA55 rotor. The supernatant was diluted with 4 volumes of buffer C, and the clathrin-coated vesicles were pelleted by spinning at 50,000 rpm for 30 min in a TLA55 rotor. The clathrin-coated vesicles were resuspended in a total volume of 30 μl of buffer C, snap-frozen in liquid nitrogen, and stored at -80 °C until use. Differential Clathrin Binding of OCRL1 Isoforms-OCRL1 isoform a contains eight amino acids adjacent to the clathrin box LIDLE that are missing in isoform b (Fig. 1A). We hypothesized that this could affect binding of OCRL1 to the terminal domain of clathrin heavy chain. This was tested by expressing GFP-tagged versions of the OCRL1 isoforms in HeLa cells and performing pulldown experiments. As shown in Fig. 1B, GFP-OCRL isoform a bound to clathrin more strongly than isoform b. In contrast, binding to other known OCRL1 binding partners, including the α-adaptin appendage domain, GTP-locked Rab5 and -6 and Rac1, and APPL1, was unaffected. To confirm the effect on clathrin binding was a property of OCRL1 itself and not because of additional interactions, binding was repeated using purified recombinant proteins. Recombinant OCRL1 isoform a bound clathrin significantly better than isoform b, whereas binding to α-adaptin was the same for each isoform (Fig. 1C). Differential Subcellular Localization of OCRL1 Isoforms-To determine whether the differential clathrin binding of OCRL1 isoforms a and b affects their localization in cells, both isoforms were expressed as GFP-tagged fusions in NRK cells, and their localization was analyzed by fluorescence microscopy. Both proteins predominantly localized to the perinuclear region (Fig. 2A). Double labeling confirmed co-localization with the Golgi apparatus in this region, as expected from previous studies (data not shown) (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar, 7Ungewickell A. Ward M.E. Ungewickell E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13501-13506Crossref PubMed Scopus (106) Google Scholar). In addition there was punctate staining dispersed throughout the cell, corresponding most likely to endocytic structures and clathrin-coated transport intermediates, as observed previously (see below) (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar, 7Ungewickell A. Ward M.E. Ungewickell E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13501-13506Crossref PubMed Scopus (106) Google Scholar, 8Erdmann K.S. Mao Y. McCrea H.J. Zoncu R. Lee S. Paradise S. Modregger J. Biemesderfer D. Toomre D. De Camilli P. Dev. Cell. 2007; 13: 377-390Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Strikingly, OCRL1 isoform a was significantly more abundant in the cytoplasmic puncta, and there was less diffuse cytosolic staining compared with isoform b (Fig. 2A). These differences were observed at all expression levels and also seen in HeLa cells (supplemental Fig. S1), indicating they are not a consequence of protein overexpression or dependent upon the cell type used. To further verify these observations, OCRL1 isoforms a and b fused to different fluorescent tags were co-expressed in the same cell. Again, OCRL1 isoform a was more obviously in puncta in the cytoplasm and less diffuse, irrespective of the tag used (Fig. 2B). To ascertain whether the OCRL1 isoform a-containing puncta correspond to trafficking intermediates, double labeling with various markers was performed. As expected, there was extensive overlap with clathrin, consistent with significant enrichment of OCRL1 isoform a in clathrin-coated transport intermediates (Fig. 3, top row; see also Fig. 7). We also observed partial overlap of GFP-OCRL1 isoform a with internalized transferrin and overexpressed Rab5, consistent with localization to endocytic structures (Fig. 3). Some of puncta contained CI-MPR, a cargo receptor found in clathrin-coated vesicles shuttling between the TGN and endosomes. Thus, OCRL1 isoform a is abundant in clathrin-coated transport intermediates within the endocytic pathway and those that traffic between the TGN and endosomes. Although previous studies have localized OCRL1 isoform b to similar structures (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar, 7Ungewickell A. Ward M.E. Ungewickell E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13501-13506Crossref PubMed Scopus (106) Google Scholar), its abundance there is markedly reduced compared with that of isoform a.FIGURE 7Association of OCRL1 isoforms with clathrin-coated vesicles. Clathrin-coated vesicles were partially purified from HeLa cells expressing the indicated GFP-OCRL1 constructs. A, equal amounts of total protein of homogenate (Hom), low speed supernatant (LSS), high speed supernatant (HSS), membrane fraction (HSP), and clathrin-coated vesicle (CCV) fractions isolated from cells expressing GFP-OCRL1 isoform a or b were analyzed by silver staining or Western blotting with antibodies to clathrin heavy chain (CHC), OCRL1 (to detect GFP-OCRL1), or Golgin-84. B and C, equal total protein amounts of fractions isolated from cells expressing the indicated clathrin box mutants were blotted for clathrin heavy chain and GFP-tagged OCRL1 as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of a Second Clathrin-binding Site in OCRL1-We previously found that deletion of the LIDLE clathrin box in OCRL1 isoform b did not abrogate clathrin binding indicating the presence of a second clathrin-binding site in the protein (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar). Yeast two-hybrid experiments suggested this may reside within the ASH domain, but this region lacks known clathrin-binding motifs (6Choudhury R. Diao A. Zhang F. Eisenberg E. Saint-Pol A. Williams C. Konstantakopoulos A. Lucocq J. Johannes L. Rabouille C. Greene L.E. Lowe M. Mol. Biol. Cell. 2005; 16: 3467-3479Crossref PubMed Scopus (155) Google Scholar). We therefore inspected the OCRL1 sequence further and identified an LIDIA sequence within the N-terminal region of mammalian OCRL1 (amino acids 73-77 in human OCRL1) that is similar to the type I clathrin box consensus (L(L/I)(D/E/N)(L/F)(D/E)). Interestingly, in zebrafish OCRL1 the corresponding sequence is LIDID, which matches the consensus perfectly. We therefore deleted this sequence in both human OCRL1 isoforms either alone or in conjunction with LIDLE and monitored binding to clathrin in pulldown assays. As shown in Fig. 4A, deletion of LIDLE had little effect on clathrin binding of OCRL1 isoform b, consistent with our previous findings. In contrast deletion of LIDIA resulted in a dramatic decrease in clathrin binding of isoform b. Deletion of both LIDLE and LIDIA gave a similar result. Deletion of LIDLE from OCRL1 isoform a reduced clathrin binding to a level comparable with that of isoform b, whereas deletion of LIDIA resulted in an even greater decrease in binding. Deletion of both sequences reduced binding to very low levels. None of the mutations affected binding to the α-adaptin appendage domain. Similarly, deletion of LIDLE from OCRL1 isoforms a and b had no effect on binding to APPL1 or GTP-locked Rab5, Rab6, or Rac1 (Fig. 4B). To further corroborate these results, binding was also analyzed in the yeast two-hybrid system. Binding to clathrin still occurred when either clathrin-binding site in OCRL1 isoforms a or b was deleted, whereas deletion of both sites completely abolished the interaction (Fig. 4C). Taken together these results indicate that LIDIA is a major clathrin-binding site in both OCRL1 isoforms, and that the difference in clathrin binding between isoforms a and b is because of the LIDLE sequence, which in isoform b contributes little to the interaction with clathrin. Given that LIDIA does not exactly match the consensus for a type I clathrin box, containing an alanine at position 5 instead of an acidic residue, we performed mutagenesis of this motif to determine