Regulated Membrane Recruitment of Dynamin-2 Mediated by Sorting Nexin 9

The endocytic proteins sorting nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase. The endocytic proteins sorting nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase. The dynamins are essential proteins in various vesicle-scission reactions in the cell. In addition, they are implicated in several other processes, such as in signaling and actin dynamics (for recent reviews see Refs. 1Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell. Biol. 2004; 5: 133-147Crossref PubMed Scopus (1099) Google Scholar and 2Schafer D.A. Traffic. 2004; 5: 463-469Crossref PubMed Scopus (127) Google Scholar). The best described process in which dynamin participates is the formation of clathrin-coated vesicles at the cell surface. Clathrin-mediated endocytosis is characterized by discrete molecular events occurring at the cytoplasmic side of the plasma membrane, leading to the sequestration of ligands from the cell surface (for reviews see Refs. 3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (421) Google Scholar, 4Kirchhausen T. Annu. Rev. Biochem. 2000; 69: 699-727Crossref PubMed Scopus (496) Google Scholar, 5Mousavi S.A. Malerod L. 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The sequential steps for the formation of a clathrin-coated vesicle involve the recruitment of a number of different proteins from the cytosol, including clathrin and adaptor protein 2 complex (AP-2), 1The abbreviations used are: AP-2, adaptor protein 2 complex; BAR, Bin/Amphiphysin/Rvs; DRB, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole; Dyn2, dynamin-2; FSBA, 5′-(4-fluorosulfonylbenzoyl)-adenosine; PC, phosphatidylcholine; PH, pleckstrin homology; PI, phosphatidylinositol; PRD, proline-rich domain; PX, Phox homology; SH3, Src homology 3; siRNA, small interfering RNA; SNX9, sorting nexin 9; GTPγS, guanosine 5′-3-O-(thio)triphosphate; AMP-PNP, adenosine 5′-(β,γ-imido)triphosphate; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; BSA, bovine serum albumin; PBS, phosphate-buffered saline. 1The abbreviations used are: AP-2, adaptor protein 2 complex; BAR, Bin/Amphiphysin/Rvs; DRB, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole; Dyn2, dynamin-2; FSBA, 5′-(4-fluorosulfonylbenzoyl)-adenosine; PC, phosphatidylcholine; PH, pleckstrin homology; PI, phosphatidylinositol; PRD, proline-rich domain; PX, Phox homology; SH3, Src homology 3; siRNA, small interfering RNA; SNX9, sorting nexin 9; GTPγS, guanosine 5′-3-O-(thio)triphosphate; AMP-PNP, adenosine 5′-(β,γ-imido)triphosphate; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; BSA, bovine serum albumin; PBS, phosphate-buffered saline. resulting in the invagination of the membrane and creation of a bud. Subsequently, in a dynamin-dependent process the neck of the bud is constricted, and the vesicle is released from the membrane. The events appear to be strictly coordinated and regulated to ensure directionality in the process.Dynamin is a large GTPase that in its GTP-bound state has the property of self-assembly, which in vitro results in the formation of rings and spirals (6Hinshaw J.E. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (654) Google Scholar, 7Zhang P. Hinshaw J.E. Nat. Cell Biol. 2001; 3: 922-926Crossref PubMed Scopus (207) Google Scholar). In the presence of membranes, purified dynamin forms coat-like structures able to spontaneously deform the membrane into tubules (8Sweitzer S.M. Hinshaw J.E. Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 9Takei K. Haucke V. Slepnev V. Farsad K. Salazar M. Chen H. De Camilli P. Cell. 1998; 94: 131-141Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). A pleckstrin homology (PH) domain is responsible for interactions with phosphoinositides in the lipid bilayer. At the carboxyl terminus a proline-rich domain (PRD) is located, which is the target for a number of Src homology 3 (SH3)-containing proteins proposed to aid in the function of dynamin (10Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (354) Google Scholar, 11Simpson F. Hussain N.K. Qualmann B. Kelly R.B. Kay B.K. McPherson P.S. Schmid S.L. Nat. Cell Biol. 1999; 1: 119-124Crossref PubMed Scopus (232) Google Scholar, 12Hill E. van Der K. Downes C.P. Smythe E. J. Cell Biol. 2001; 152: 309-323Crossref PubMed Scopus (108) Google Scholar). Despite its importance, surprisingly little is known about how dynamin is targeted to the site of action. It is believed that the lipid interaction of the PH domain is too weak to alone be responsible for the initial recruitment of dynamin from the cytosol to the plasma membrane (13Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (613) Google Scholar). Instead, this interaction may come into play after oligomerization of dynamin already at the membrane (14Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Truncation of the PRD in the neuronal isoform dynamin-1 (15Shpetner H.S. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1996; 271: 13-16Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) or in the ubiquitously expressed isoform dynamin-2 (Dyn2) (16Szaszak M. Gaborik Z. Turu G. McPherson P.S. Clark A.J. Catt K.J. Hunyady L. J. Biol. Chem. 2002; 277: 21650-21656Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) resulted in impaired endocytosis by mislocalization of the respective protein, and it is suggested that interactions with SH3-containing proteins, such as the amphiphysins in brain, may be important for correct targeting (17Wigge P. McMahon H.T. Trends Neurosci. 1998; 21: 339-344Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 18McPherson P.S. Cell. Signal. 1999; 11: 229-238Crossref PubMed Scopus (89) Google Scholar, 19Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (580) Google Scholar).Sorting nexin 9 (SNX9), a member of the diverse sorting nexin family of proteins (for a review see Ref. 20Worby C.A. Dixon J.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 919-931Crossref PubMed Scopus (331) Google Scholar), was previously suggested by us to be involved in the endocytic process as an accessory factor (21Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar, 22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). SNX9 has binding sites for both clathrin and AP-2 in a low complexity region and binds Dyn2 by an SH3 domain in the amino terminus. SNX9 has its own membrane binding activity, mediated by a carboxyl-terminal region containing a Phox homology (PX) domain and a Bin/Amphiphysin/Rvs (BAR) domain (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 23Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. Science. 2004; 303: 495-499Crossref PubMed Scopus (1334) Google Scholar, 24Habermann B. EMBO Rep. 2004; 5: 250-255Crossref PubMed Scopus (239) Google Scholar). Endogenous SNX9 was found to partially co-localize with AP-2 and Dyn2 at the plasma membrane, and overexpression in K562 and HeLa cells of truncated versions of SNX9 inhibited the uptake of transferrin (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar).In the cytosol of K562 cells, most of SNX9 was found to be present in a native complex together with Dyn2 and an unidentified protein of 41 kDa (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The present study was undertaken to explore the role of this complex and to investigate if the membrane recruitment of SNX9 and Dyn2 is coordinated. We show that SNX9 mediates the targeting of Dyn2 to the membrane, and we present evidence that the membrane binding activity of SNX9 is regulated by a reversible interaction with the glycolytic enzyme fructose-1,6-bisphosphate aldolase.EXPERIMENTAL PROCEDURESAntibodies—Anti-GST antibodies were generated by immunizing a rabbit with recombinant GST, and the serum was affinity-purified by using immobilized antigen. Goat anti-aldolase was from Chemicon International. Mouse monoclonal anti-α-tubulin was from Zymed Laboratories Inc. Secondary reagents used in immunofluorescence were Alexa Fluor 488-labeled anti-rabbit antibodies and Alexa Fluor 568-labeled anti-mouse antibodies (Molecular Probes). All other antibodies were as described (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Affinity-purified anti-SNX9 antibodies were used in all experiments.Cytosol and Proteins—Cytosol was prepared from K562 cells or from HeLa cells for the experiments shown in Figs. 3B and 4A, by centrifugation at 70,000 × g for 30 min after a rapid freeze/thaw cycle exactly as described (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). For recruitment experiments, the cytosol was desalted on Micro Bio-Spin P6 columns (Bio-Rad) equilibrated with KSHM buffer (100 mm potassium acetate, 85 mm sucrose, 20 mm HEPES-KOH, pH 7.4, and 1 mm magnesium acetate). A high molecular weight fraction was isolated from cytosol by velocity sedimentation (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), resulting in a 7-fold enrichment of SNX9. Immunoprecipitations were carried out as described (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The 41-kDa protein was identified by MALDI-TOF after trypsin cleavage of the Coomassie-stained band from SDS-PAGE (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Immunodepleted cytosols were obtained after incubation with immobilized antibodies, and after a brief centrifugation the supernatants were collected.Fig. 4Identification of aldolase as a component of the SNX9-Dyn2 complex and analysis of its binding specificity.A, cytosols (1 mg of protein) prepared from K562 cells and HeLa cells were immunoprecipitated with antibodies against SNX9 or with preimmune IgG. After SDS-PAGE, the presence of Dyn2, SNX9, and aldolase was detected by immunoblotting. B, depiction of recombinant constructs used in the present investigation. All constructs were expressed as fusion proteins with GST located amino terminally. At bottom is shown the amino acid sequence of the region in SNX9 designated LC4. Tryptophans found to be important for binding of aldolase are in boldface type. Acidic residues (see "Discussion") are underlined. C–E, K562 cytosol (20 mg/ml) (C) or purified aldolase (0.25 μm)(D and E) was incubated on ice with immobilized GST fusion proteins or with GST alone, and bound aldolase was analyzed by SDS-PAGE and immunoblotting. LC-S165/S169 is a mutant of LC in which tryptophan at position 165 and 169 were changed to serines. E, GST-SNX9 was incubated with aldolase in the presence of fructose 1,6-bisphosphate (FBP), glyceraldehyde 3-phosphate (G3P), fructose 6-phosphate (F6P), or without additions (–). All additives were at 100 μm.View Large Image Figure ViewerDownload (PPT)Recombinant full-length and parts of SNX9 expressed as GST fusion proteins were as described (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). cDNA encoding LC4PXBAR was amplified by PCR using appropriate primers and cDNA encoding full-length SNX9. The GST fusion protein was expressed in Escherichia coli after cloning into the pGEX-5X-1 vector (Amersham Biosciences) and purified as described previously (21Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar). Aldolase (rabbit muscle) was purchased from Sigma. Pull-down experiments with GST fusion proteins were performed as described previously (21Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar).Recruitment to Permeabilized Cells and Liposomes—K562 cells, opened by a freeze/thaw cycle (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), were pelleted by centrifugation at 850 × g for 5 min. The cell pellet was washed and incubated on ice for 1–2 h in 15 ml of KSHM buffer. The cells were finally pelleted and resuspended at 50 × 106 cells/ml in KSHM buffer containing 0.8% BSA. The cells were >99% permeabilized as judged by trypan blue staining.Recruitment reactions were performed in a total volume of 40 μl of cytosol (final concentration 5 mg/ml), and when used ATP (1 mm) and an ATP-generating mixture (8 mm creatine phosphate and 50 μg/ml creatine kinase), GTPγS (100 μm), sodium orthovanadate (1 mm), and other additions as indicated in the figure legends were adjusted to 30 μl with KSHM buffer and mixed on ice. When no cytosol was used, the reactions were supplemented with BSA to the same protein concentration. The nonhydrolyzable ATP analogue AMP-PNP was used at 1 mm. Cells (0.5 × 106) were added in 10 μl, and the reactions were started by transferring the tubes to a 37 °C water bath or kept on ice. After incubation for 20 min, the cells were pelleted by centrifugation at 10,000 × g for 5 min, rinsed with 200 μl of KSHM buffer without resuspension, and again centrifuged. The pellets were solubilized in 25 μl of 1% Nonidet P-40 in PBS, and after centrifugation the supernatants were prepared for SDS-PAGE.Liposomes were prepared by sonication as described (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Liposomes (50 nmol of lipid in 10 μl of KSHM buffer) were added to 30 μl of KSHM buffer containing either GST fusion protein in 0.5 mg/ml BSA or cytosol (5 mg/ml) with additions as indicated in the figure legends. After incubation at 37 °C or on ice for 20 min, the liposomes were pelleted by centrifugation at 20,000 × g for 5 min. Liposomes were rinsed with 200 μl of KSHM buffer, centrifuged, and finally prepared for SDS-PAGE.Phosphorylation with [γ-32P]ATP—SNX9 was enriched from cytosol by velocity sedimentation (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Reactions with liposomes were performed as above in the presence of 0.2 MBq of [γ-32P]ATP. After washings, liposomes were solubilized in 1% Nonidet P-40 in PBS containing 1 mm orthovanadate, and SNX9 was immunoprecipitated. Immunoprecipitates were washed using high stringency conditions with buffers containing SDS/Nonidet P-40 and high salt (25Carlsson S.R. Fukuda M. J. Biol. Chem. 1986; 261: 12779-12786Abstract Full Text PDF PubMed Google Scholar). For the experiment shown in Fig. 6B, the reaction was performed without liposomes, and the immunoprecipitates were washed in 0.5% Nonidet P-40 in 20 mm HEPES-KOH, pH 7.4.Fig. 6Phosphorylation of SNX9 correlates with the release of aldolase.A, high molecular weight fraction from cytosol enriched for SNX9 was incubated with phosphoinositide-containing liposomes (composition as in Fig. 2B) at 37 °C for 20 min in the presence of a small amount of cytosol (0.5 mg/ml), ATP, [γ-32P]ATP, GTPγS, and orthovanadate. After washings, liposomes were solubilized in detergent and subjected to immunoprecipitation. The material was separated by SDS-PAGE, and the dried gel was exposed to x-ray film. B, the SNX9-enriched cytosol fraction was immunoprecipitated with anti-SNX9 antibodies either directly (Before activation) or after incubation for 20 min at 37 °C in the presence of ATP, [γ-32P]ATP, GTPγS, and orthovanadate (After activation). Immunoprecipitates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride filter, which was first subjected to autoradiography and then analyzed for the presence of SNX9 and aldolase by immunoblotting. C, immobilized GST or GST-SNX9 was incubated for 20 min with cytosol (20 mg/ml) on ice or at 37 °C in the presence of ATP and an ATP-regenerating mixture, or with AMP-PNP as indicated. After washings, aldolase bound to the beads was analyzed by SDS-PAGE and immunoblotting. D, immobilized GST fusion proteins (500 ng), or GST alone, were incubated with cytosol (2 mg/ml), ATP, and [γ-32P]ATP for 20 min at 37 °C. After washings, the fusion proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride filter, and subjected to autoradiography followed by immunoblotting with anti-GST antibodies. The same exposures are shown for all immunoblot and autoradiography insets, respectively. E, cytosol (5 mg/ml) was incubated on ice together with kinase inhibitors staurosporine (20 μm), 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) (200 μm), herbimycin A (100 μm), genistein (400 μm), and 5′-(4-fluorosulfonylbenzoyl)-adenosine (FSBA)(1 mm), or without inhibitor (–) in the presence of GTPγS and orthovanadate. After 2 h, permeabilized K562 cells were added together with ATP and an ATP-regeneration mixture. The AMP-PNP sample was without ATP. The samples were incubated for 20 min at 37 °C, and bound SNX9 was analyzed by SDS-PAGE and immunoblotting.View Large Image Figure ViewerDownload (PPT)Phosphorylation of GST fusion proteins was performed after binding to glutathione-Sepharose (Amersham Biosciences), by incubation for 20 min at 37 °C in 20 μl of 20 mm HEPES-KOH, pH 7.4, containing 5 mm magnesium acetate, 40 μg of cytosol, 1 mm ATP, and 0.06 MBq of [γ-32P]ATP. Beads were washed in 0.1% Nonidet P-40 in PBS and prepared for SDS-PAGE.siRNA Knockdowns—HeLa cells were transfected twice with small interfering RNA (siRNA) and cultured essentially as described by Motley et al. (26Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (550) Google Scholar), except that each transfection was scaled down 4-fold. Six hours prior to analysis at day 3, cells were trypsinized and plated on 35-mm dishes or on coverslips for immunofluorescence studies. For the latter experiments, transfected cells were mixed with control cells to facilitate analysis and viewed using a Nikon Eclipse E800 microscope. To assay the efficiency of transfection, cells from 35-mm dishes were trypsinized, washed, and lysed in 1% Nonidet P-40 in PBS containing protease inhibitors and analyzed by immunoblotting. For analysis of membrane-bound and cytosolic pools of Dyn2, trypsinized cells from 50-mm dishes were washed, and cytosol and a 70,000 × g pellet (membranes) were prepared after a freeze/thaw-cycle as described (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and analyzed by immunoblotting.Two independent siRNAs were used to knock down the expression of SNX9. The SNX9a target was AAGAGAGUCAGCAUCAUGUCU and the SNX9b target was AACCUACUAACACUAAUCGAU. As a control we used a scrambled version of the SNX9a sequence (UAGGUACUCUGAGCAGAUC). The siRNAs were synthesized as option C siRNAs by Dharmacon, Inc., or by a similar protocol by Thermo Electron Corp.RESULTSWe first tested the membrane binding activity of cytosolic SNX9 and Dyn2 by incubations with permeabilized and washed K562 cells, followed by detection of bound SNX9 and Dyn2 by immunoblotting. The results in Fig. 1A show that SNX9 in cytosol required incubation at 37 °C in the presence of ATP for efficient binding, whereas incubation on ice resulted in much less cell-associated SNX9. In addition to ATP and heat, GTPγS and orthovanadate were required for full activity, and the omission of one these reagents gave intermediate binding. Under optimal conditions, essentially 100% of SNX9 was bound to the membranes. Dyn2 followed the binding pattern of SNX9 in terms of conditions, with the exception that Dyn2 had an absolute requirement for GTPγS. At maximum, ∼25% of Dyn2 bound to the membranes. This figure fits with the estimated proportion of cytosolic Dyn2 that is in complex with SNX9 (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar).Fig. 1Recruitment of SNX9 and Dyn2 to permeabilized cells. K562 cells were permeabilized by freezing/thawing, and the cytosol was washed out. Cells were incubated for 20 min at 37 °C, or on ice, under various conditions as indicated. After washings, the cells were analyzed for bound SNX9 and Dyn2 by SDS-PAGE and immunoblotting. A, requirements for binding of proteins from cytosol. Cytosol (5 mg/ml), or BSA as a control, was mixed with indicated reagents. The lane marked by * shows the direct analysis of 25% of cytosol added to the samples. B, requirements for binding of recombinant SNX9. Samples were incubated with purified GST-SNX9 (800 ng), with or without cells and indicated reagents, in the presence of BSA. C, immunodepleted cytosols (5 mg/ml), with or without added recombinant GST fusion proteins (800 ng) as indicated, were incubated at 37 °C together with ATP, GTPγS, orthovanadate, and permeabilized cells.View Large Image Figure ViewerDownload (PPT)Because SNX9 is present in cytosol as a complex, it was of interest to see how purified, recombinant SNX9 behaved when added to cell membranes. Fig. 1B shows that purified GST-SNX9 bound to cell membranes and that the binding did not require incubation at 37 °C or ATP or any of the other factors that was obligate for cytosolic SNX9. This finding indicates that the resting complex of SNX9 in the cytosol is inactive for membrane binding because of the lack of an exposed membrane-binding site. It also shows that the incubation with cytosol has no crucial influence on the properties of the membrane itself for binding of SNX9.To investigate if the membrane binding activities of SNX9 and Dyn2 are linked, cytosol was separately immunodepleted of SNX9 and Dyn2, or mock-depleted, and used in the recruitment assay. Although most of SNX9 is present in complex with Dyn2 in the cytosol, in the immunodepletion procedure substantial amounts of SNX9 are left behind in the cytosol after removal of Dyn2. This is due to the fact that the complex is labile and partly dissociates, especially when incubated with antibodies against Dyn2 (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). This allows for analysis of the recruitment dependence on the immunodepleted protein. As seen in Fig. 1C, Dyn2 depletion had no major effect on the binding of SNX9 to membranes, whereas SNX9 was necessary for the binding of Dyn2. Because we had found that purified SNX9 can bind Dyn2 in the cytosol (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), it was of interest to see if SNX9-depleted cytosol can be reconstituted with purified SNX9 for the binding of Dyn2 to membranes. Fig. 1C shows that full-length GST-SNX9, but not a variant that lacks the membrane-binding part (GST-SH3LC), can efficiently reconstitute the binding of Dyn2 to membranes. The combined results show that Dyn2 is recruited to the membrane through its interaction with SNX9 and that the membrane binding activity resides in the SNX9 molecule.If the binding of cytosolic SNX9 to membranes is mediated through a PX/BAR domain-lipid interaction, as found previously for purified SNX9 (22Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), the binding experiments should be possible to perform with pure lipids. Two different liposome preparations, one containing PC and PI (denoted PI) and one that in addition contained a mixture of phosphoinositides (denoted PIP), were incubated with cytosol on ice and/or at 37 °C together with ATP, GTPγS, and orthovanadate (Fig. 2A). Cytosolic SNX9 showed very little binding to liposomes when incubated on ice (Fig. 2A, lanes 1 and 2), in agreement with the results using cell membranes (see Fig. 1A). When incubated at 37 °C, extensive binding of SNX9 occurred (Fig. 2A, lanes 3 and 4), especially with liposomes containing phosphatidylinositols (lane 4). To see if this effect was because of modifications in the cytosol or on the membranes, the cytosol was first incubated at 37 °C and then mixed with liposomes and incubated on ice (Fig. 2A, lanes 5 and 6). The result shows that a major effect of the incubation at 37 °C is on the cytosol (Fig. 2A, compare lane 4 with lanes 2 and 6). This result shows again that the SNX9 complex in cytosol requires an activation step to expose the binding site for membranes (i.e. phosphoinositides). In addition to this effect, it appears that factors in the cytosol to some extent can modify the liposomes to become binders for activated SNX9 (Fig. 2A, compare lane 3 with lanes 1 and 5).Fig. 2Recruitment of cytosolic SNX9 and Dyn2 to liposomes.A, temperature and phosphoinositide requirements. Two different liposome preparations were incubated with ATP, GTPγS, and orthovanadate for 20 min at 37 °C, or on ice, together with normal cytosol or SNX9-depleted cytosol (5 mg/ml). One sample contained in addition 800 ng of GST-SNX9. Two samples (denoted by *) were incubated for 20 min at 37 °C before the addition of liposomes and were then incubated for another 20 min on ice. Bound proteins were analyzed by SDS-PAGE and immunoblotting. Liposomes denoted PI contained 50% PC and 50% PI, and liposomes denoted PIP contained 50% PC and 50% of an equal mixture of PI, PI(3)P, PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3. B, nucleotide and orthovanadate requirements. Cytosol (5 mg/ml) and indicated reagents were incubated with liposomes, or without liposomes, for 20 min at 37 °C or on ice. Bound proteins were analyzed by SDS-PAGE and immunoblotting. The lane marked by * shows the direct analysis of 25% of cytosol added to the samples. Liposomes contained 50% PC, 40% PI, and 10% of an equal mixture of the same phosphoinositides as in A.View Large Image Figure ViewerDownload (PPT)Cytosolic Dyn2 showed the same binding pattern to liposomes as SNX9 did (Fig. 2A), again arguing for the dependence of SNX9 for membrane binding of Dyn2. To test directly for this in the liposome system, we incubated phosphoinositide-containing liposomes with SNX9-depleted cytosol, with or without the addition of purified GST-SNX9 (Fig. 2A, lanes 7 and 8). The results clearly show that Dyn2 binds to liposomes only if SNX9 is present, establishing a role for SNX9 as a molecule that links Dyn2 to phosphoinositide membranes. The nucleotide and orthovanadate requirements for the activation step of the SNX9-Dyn2 complex in cytosol were confirmed in the liposome assay (Fig. 2B). Furthermore, replacing ATP with AMP-PNP in the mixture showed that both SNX9 and Dyn2 required ATP hydrolysis for efficient binding to the liposomes.The importance of SNX9 for the localization of Dyn2 in living cells was tested by RNA interference experiments in HeLa cells. Transfection with the small inhibitory duplex RNA (SNX9a and SNX9b) against two different regions in SNX9 mRNA l