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Interaction of the Mammalian Endosomal Sorting Complex Required for Transport (ESCRT) III Protein hSnf7-1 with Itself, Membranes, and the AAA+ ATPase SKD1

2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês

10.1074/jbc.m413968200

1083-351X

Yuan Lin, Lisa A. Kimpler, Teresa V. Naismith, Joshua M. Lauer, Phyllis I. Hanson,

Autophagy in Disease and Therapy

SKD1/VPS4B is an AAA+ (ATPase associated with a variety of cellular activities) protein involved in multivesicular body (MVB) biogenesis. In this study, we show that the impairment in MVB biogenesis caused by the ATP hydrolysis-deficient mutant SKD1(E235Q) is accompanied by assembly of a large detergent-insoluble protein complex that includes normally soluble endogenous components of mammalian endosomal sorting complex required for transport (ESCRT) I and ESCRT-III complexes. Membrane-bound ESCRT-III complex has been proposed to be the substrate that recruits SKD1 to nascent MVBs. To explore this relationship, we studied interactions among the human ESCRT-III components hSnf7-1 and hVps24, membranes, and SKD1. We found that a significant portion of overexpressed hSnf7-1 associated with membranes where it formed a large protein complex that recruited SKD1 and perturbed normal MVB biogenesis. Overexpressed hVps24 also associated with membranes and perturbed endosome structure but only when fused to green fluorescent protein. Domain analysis revealed that the basic N-terminal half of hSnf7-1 localized to membranes and formed detergent-resistant polymers, some of which looked like filopodia extending into the lumen of swollen endosomes or out from the plasma membrane. The C-terminal acidic half of hSnf7-1 did not associate with membranes and was required for interaction of hSnf7-1 with SKD1. Together with earlier studies, our work suggests that a variety of ESCRT-III-containing polymers can assemble on membranes and recruit SKD1 during formation of the MVB. SKD1/VPS4B is an AAA+ (ATPase associated with a variety of cellular activities) protein involved in multivesicular body (MVB) biogenesis. In this study, we show that the impairment in MVB biogenesis caused by the ATP hydrolysis-deficient mutant SKD1(E235Q) is accompanied by assembly of a large detergent-insoluble protein complex that includes normally soluble endogenous components of mammalian endosomal sorting complex required for transport (ESCRT) I and ESCRT-III complexes. Membrane-bound ESCRT-III complex has been proposed to be the substrate that recruits SKD1 to nascent MVBs. To explore this relationship, we studied interactions among the human ESCRT-III components hSnf7-1 and hVps24, membranes, and SKD1. We found that a significant portion of overexpressed hSnf7-1 associated with membranes where it formed a large protein complex that recruited SKD1 and perturbed normal MVB biogenesis. Overexpressed hVps24 also associated with membranes and perturbed endosome structure but only when fused to green fluorescent protein. Domain analysis revealed that the basic N-terminal half of hSnf7-1 localized to membranes and formed detergent-resistant polymers, some of which looked like filopodia extending into the lumen of swollen endosomes or out from the plasma membrane. The C-terminal acidic half of hSnf7-1 did not associate with membranes and was required for interaction of hSnf7-1 with SKD1. Together with earlier studies, our work suggests that a variety of ESCRT-III-containing polymers can assemble on membranes and recruit SKD1 during formation of the MVB. Cells deliver endocytosed soluble and membrane-bound cargo to late endosomes en route to lysosomes. Newly synthesized lysosomal proteins also travel through the late endosome to reach the lysosome (1.Luzio J.P. Poupon V. Lindsay M.R. Mullock B.M. Piper R.C. Pryor P.R. Mol. Membr. Biol. 2003; 20: 141-154Crossref PubMed Scopus (126) Google Scholar). A critical feature of late endosomal function is the ability to invaginate membrane and selected proteins into the interior or lumen, giving late endosomes their alternate name of multivesicular body (MVB) 1The abbreviations used are: MVB, multivesicular body; ESCRT, endosomal sorting complex required for transport; AAA+, ATPase associated with a variety of cellular activities; DTT, dithiothreitol; GFP, green fluorescent protein; EGFP, enhanced GFP; SKD1, suppressor of K+ transport growth defect 1; Ni2+-NTA, nickel-nitrilotriacetic acid; GST, glutathione S-transferase; HEK, human embryonic kidney; LAMP-2, lysosome-associated membrane protein-2; EEA1, early endosome antigen 1; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; CHMP, charged multivesicular body protein; FL, full length. (2.Murk J.L. Humbel B.M. Ziese U. Griffith J.M. Posthuma G. Slot J.W. Koster A.J. Verkleij A.J. Geuze H.J. Kleijmeer M.J. Proc. Natl. Acad. Sci. U. S. 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There are at least 18 class E proteins known in yeast (4.Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1028) Google Scholar, 9.Shiflett S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10.Yeo S.C. Xu L. Ren J. Boulton V.J. Wagle M.D. Liu C. Ren G. Wong P. Zahn R. Sasajala P. Yang H. Piper R.C. Munn A.L. J. Cell Sci. 2003; 116: 3957-3970Crossref PubMed Scopus (82) Google Scholar). Interestingly all are soluble and cycle on and off the endosomal membrane. Studies of homologues of class E proteins in mammalian cells indicate that the function of these proteins is conserved from yeast to mammals (for reviews, see Refs. 11.Raiborg C. Rusten T.E. Stenmark H. Curr. Opin. Cell Biol. 2003; 15: 446-455Crossref PubMed Scopus (417) Google Scholar and 12.Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Elegant studies carried out over the last few years have established that class E proteins assemble into functional complexes. These include the S. cerevisiae endosomal sorting complex required for transport (ESCRT) complexes I, II, and III (13.Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar, 14.Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 15.Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar) as well as a Vps27-Hse1 complex (16.Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (285) Google Scholar). Current models suggest that these complexes act sequentially to select and move cargo into the lumen of the MVB, although the actual reactions that promote vesicle budding and release into the endosomal lumen have not been defined. The proposed MVB pathway begins when the Vps27-Hse1 complex binds phosphatidylinositol 3-phosphate and ubiquitin-conjugated proteins on the endosomal membrane (using FYVE and ubiquitin-interacting motif domains). Vps27 then recruits ESCRT-I (Vps23, Vps28, and Vps37) via interactions between it and Vps23 (17.Katzmann D.J. Stefan C.J. Babst M. Emr S.D. J. Cell Biol. 2003; 162: 413-423Crossref PubMed Scopus (369) Google Scholar). This complex is next thought to engage the ESCRT-II complex (Vps22, Vps25, and Vps36) (14.Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 18.Hierro A. Sun J. Rusnak A.S. Kim J. Prag G. Emr S.D. Hurley J.H. 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Cell. 2004; 15: 4337-4346Crossref PubMed Scopus (129) Google Scholar). One class E protein, Vps4, is an AAA+ (ATPase associated with a variety of cellular activities) ATPase (23.Babst M. Sato T.K. Banta L.M. Emr S.D. EMBO J. 1997; 16: 1820-1831Crossref PubMed Scopus (350) Google Scholar, 24.Vale R.D. J. Cell Biol. 2000; 150: F13-F19Crossref PubMed Google Scholar). It is not a part of the ESCRT machinery, but interfering with its activity causes ESCRT components to accumulate in a large detergent-resistant complex on the class E compartment membrane (14.Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 15.Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar, 25.Babst M. Wendland B. Estepa E.J. Emr S.D. 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Mammalian homologues of Vps4, VPS4A and SKD1 (suppressor of K+ transport growth defect 1, also known as VPS4B), appear to play a comparable role to their yeast counterpart in regulating MVB biogenesis. Expressing ATP hydrolysis-deficient mutants of either isoform in cultured cells causes the mammalian equivalent of the class E phenotype: vacuolation of endosomes and impairment of late endosomal trafficking (28.Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell. 2000; 11: 747-763Crossref PubMed Scopus (176) Google Scholar, 29.Bishop N. Woodman P. Mol. Biol. Cell. 2000; 11: 227-239Crossref PubMed Scopus (214) Google Scholar, 30.Fujita H. Yamanaka M. Imamura K. Tanaka Y. Nara A. Yoshimori T. Yokota S. Himeno M. J. Cell Sci. 2003; 116: 401-414Crossref PubMed Scopus (110) Google Scholar, 31.Sachse M. Strous G.J. Klumperman J. J. Cell Sci. 2004; 117: 1699-1708Crossref PubMed Scopus (57) Google Scholar). We will use VPS4 to refer generally to mammalian isoforms and VPS4A or SKD1 to refer to a specific one. Interestingly expressing ATP hydrolysis-deficient VPS4 also blocks budding of enveloped viruses such as human immunodeficiency virus, type 1, suggesting that the machinery for generating vesicles inside the MVB is generally involved in budding and fission of vesicles leaving the cytosol (32.Garrus J.E. von Schwedler U.K. Pornillos O.W. Morham S.G. Zavitz K.H. Wang H.E. Wettstein D.A. Stray K.M. Cote M. Rich R.L. Myszka D.G. Sundquist W.I. Cell. 2001; 107: 55-65Abstract Full Text Full Text PDF PubMed Scopus (1180) Google Scholar, 33.von Schwedler U.K. Stuchell M. Muller B. Ward D.M. Chung H.Y. Morita E. Wang H.E. Davis T. He G.P. Cimbora D.M. Scott A. Krausslich H.G. Kaplan J. Morham S.G. Sundquist W.I. Cell. 2003; 114: 701-713Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). How VPS4 and other class E proteins cooperate with each other and cargo proteins to promote this budding and fission, however, is far from clear. Based on what is known about other AAA+ ATPases (34.Ogura T. Wilkinson A.J. Genes Cells. 2001; 6: 575-597Crossref PubMed Scopus (849) Google Scholar), it has been proposed that VPS4 disassembles a stable protein complex of membrane-bound ESCRT proteins. While all class E proteins are candidate substrates, it seems unlikely that VPS4 acts directly on so many structurally diverse proteins. Instead VPS4 may modify one or a few components that hold the ESCRT machinery together. Several observations suggest that components of the ESCRT-III complex are the most likely substrates for VPS4. In yeast, inactive Vps4 fails to bind membranes in cells that lack subunits of the ESCRT-III complex (including Vps2 and Vps24) (15.Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. 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To further define the nature of interactions between mammalian ESCRT-III proteins and VPS4 and to more generally gain insight into the role of these proteins in formation of the MVB, we characterized human homologues of two ESCRT-III components, hSnf7-1 and hVps24, and their interactions with the VPS4 isoform SKD1. We found that many of the properties attributed to a heteropolymeric ESCRT-III complex in yeast (membrane binding, polymer assembly, and interaction with VPS4) could be recapitulated in mammalian cells by simple overexpression of hSnf7-1. Domain analysis allowed us to assign membrane binding and polymerization to the N-terminal basic half of the protein and interaction with SKD1 to the acidic C-terminal half. We propose that the properties and interactions of hSnf7-1 shed light on the likely organization of ESCRT-III polymers in the MVB pathway. Plasmids and Mutagenesis—To express SKD1 in Escherichia coli, mouse SKD1 cDNA (a kind gift from Dr. Carol Vandenberg (University of California)) was inserted into pHO4d (41.Hanson P.I. Roth R. Morisaki H. Jahn R. Heuser J.E. Cell. 1997; 90: 523-535Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar) between NcoI and EcoRI sites with amino acids PNSG between the C terminus of SKD1 and the His6-Myc tag. The E235Q mutation was generated by QuikChange™ site-directed mutagenesis (Stratagene). To express SKD1 wild type or E235Q in mammalian cells, the corresponding cDNA was inserted into pEGFP-N1 (Clontech) between XhoI and BamHI sites. The resulting proteins have amino acids ADPPVAT between SKD1 and EGFP. To create tetracycline-regulated constructs, the entire SKD1-green fluorescent protein (GFP) fusion was amplified and inserted into pcDNA4/TO (Invitrogen) using BamHI and XhoI sites. To clone human homologues of Snf7 and Vps24, we amplified proteins identified in a data base search from human melanoma cDNA (a gift from Dr. Helen Piwnica-Worms, Washington University). hVps24 was amplified using primers complementary to neuroendocrine differentiation factor (GenBank™ accession number AF219226), while human Snf7 was amplified with primers complementary to HSPC134 (GenBank™ accession number AF161483). Our Snf7 clone differed by three amino acids from HSPC134 and is identical to the more recently described hSnf7-1 (GenBank™ accession number AAQ91193). DNA fragments were inserted into pGEX4T-1 (Amersham Biosciences, GST tag) and pET28a (Novagen (Madison, WI), His6 and T7 tags) using BamHI and XhoI sites for expression in E. coli. For expression in mammalian cells, hSnf7-1 and hVps24 were inserted into three vectors. Untagged constructs were generated by cloning into pcDNA3.1(+) (Invitrogen) between BamHI and XhoI sites. Myc-tagged constructs were generated by cloning into pcDNA3.1/Myc-His(–)A (Invitrogen) between XbaI and HindIII sites with a linker of KLGP between hSnf7-1/hVps24 and the Myc epitope. GFP-tagged constructs were created by cloning into pEGFP-N1 (Clontech) between NheI and HindIII sites with a linker of KLRILQSTVPRARDPPVAT between hSnf7-1 or hVps24 and EGFP. To generate hSnf7-1 fragments, DNA corresponding to amino acids 1–222 (hSnf7 full length (FL)), 1–116 (hSnf7-N), or 117–222 (hSnf7-C) was amplified and inserted into pGEX4T-1 and pET28a between BamHI and XhoI sites for expression in E. coli and into FLAG-pcDNA3.1(+) (a gift from Dr. Kenneth Johnson, Washington University) between BamHI and XhoI sites for expression in mammalian cells. The cloning resulted in a linker of GS between the N-terminal FLAG epitope and the hSnf7-1 protein. Sequences of all constructs were confirmed by sequencing using ABI big dye reagents at the Nucleic Acid Chemistry Laboratory (Washington University). Protein Expression and Purification—SKD1 was expressed in BL21(DE3) E. coli grown at 30 °C. Cells were harvested 4 h after induction with isopropyl β-d-thiogalactopyranoside, lysed by sonication in 30 mm HEPES, pH 7.4, 400 mm KCl, 1 mm dithiothreitol (DTT), 3 mm MgCl2, 2 mm ATP, and 5% glycerol and centrifuged at 20,000 × g. Supernatant was incubated with Ni2+-NTA-agarose resin (Qiagen), and bound SKD1 was eluted in buffer containing 160 mm imidazole. It was further purified over a Superdex 200 HR 10/30 gel filtration column (Amersham Biosciences) in 50 mm HEPES, pH 7.4, 150 mm KCl, 1 mm DTT, 2 mm MgCl2, 5% glycerol, and 0.5 mm ATP. His6-hSnf7-1 (full length, N, and C) was expressed in BL21-CodonPlus(DE3)-RIL (Stratagene, San Diego, CA) E. coli grown at room temperature. Cells were harvested 2 h after induction with isopropyl β-d-thiogalactopyranoside and lysed by sonication in 20 mm Tris, pH 7.4, 200 mm NaCl, and 1 mm DTT. His6-hSnf7-1 N and C fragments were purified using Ni2+-NTA resin as above. C fragment was further purified over a Mono Q HR 5/5 column (Amersham Biosciences). To purify full-length His6-hSnf7-1, the pellet obtained after centrifuging lysed bacteria was resuspended and sonicated in buffer containing 1.5% sarkosyl. Homogenate was then treated with 3.5% Triton X-100 and recentrifuged, and His6-hSnf7-1 was purified using Ni2+-NTA resin as above. GST-hSnf7-1 (full-length and C) and GST-hVps24 were purified using glutathione-Sepharose (Amersham Biosciences) according to the manufacturer's recommendations. Peak protein fractions were snap frozen in liquid nitrogen. Protein concentration was determined by Bradford assay using bovine serum albumin as a standard (Bio-Rad). RNA Blot Hybridization—DNA encoding hVps24 or hSnf7-1 was excised from pGEX4T-1 constructs with BamHI and XhoI for use as a hybridization probe. DNA fragments were labeled by random priming (RediPrime™ II kit (Amersham Biosciences)) according to the manufacturer's recommendations. Human multiple tissue Northern blots (Clontech) were hybridized with these probes in ExpressHyb solution at 68 °C for 1 h according to the manufacturer's recommendations and visualized by autoradiography. Cell Culture and Transfection—T-REx™ HEK293 cells (Invitrogen) expressing tetracycline repressor were grown in Dulbecco's modified Eagle's medium supplemented with 10% tetracycline-free fetal bovine serum (Hyclone), 2 mml-glutamine, and 5 μg/ml blasticidin. COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mml-glutamine. All cells were grown in a 5% CO2 incubator at 37 °C. Cells were transfected using Lipofectamine (Invitrogen) according to the manufacturer's recommendations. Transiently transfected COS-7 cells were analyzed 18–24 h following transfection. Tetracycline-inducible SKD1 stable cell lines were generated essentially as described previously (42.Dalal S. Rosser M.F. Cyr D.M. Hanson P.I. Mol. Biol. Cell. 2004; 15: 637-648Crossref PubMed Scopus (151) Google Scholar). Cell lines were maintained in T-REx HEK293 medium containing 125 μg/ml Zeocin. To induce protein expression, tetracycline (0.5 μg/ml) was added for the indicated length of time. If not otherwise specified, standard conditions for inducing SKD1 expression were 4–5 h of tetracycline. Antibodies—Anti-SKD1, anti-hSnf7-1, and anti-hVps24 antibodies were raised in rabbits (RW2, R7119, and R7122 (Bethyl Laboratory Inc.)) using recombinant SKD1-His6, GST-hSnf7-1, and His6-hVps24 purified from E. coli as antigens. All were affinity-purified using antigen immobilized on nitrocellulose strips. Mouse anti-CD63 and anti-LAMP-2 were from Developmental Studies Hybridoma Bank (University of Iowa); rabbit and mouse (M2) anti-FLAG were from Sigma; mouse anti-ubiquitin FK2 specific for ubiquitin-conjugated proteins was from Affiniti Research Products (Exeter, UK); mouse anti-EEA1 was from BD Transduction Laboratories; rabbit anti-giantin was from Covance (Berkeley, CA); mouse anti-β-COP was fromSigma; mouse anti-Myc was generated using the 9E10 hybridoma cell line (43.Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2221) Google Scholar); rabbit anti-Myc was from Cell Signaling Technology (Beverly, MA); rabbit anti-GFP (B5) was as described previously (42.Dalal S. Rosser M.F. Cyr D.M. Hanson P.I. Mol. Biol. Cell. 2004; 15: 637-648Crossref PubMed Scopus (151) Google Scholar); mouse anti-α-SNAP (Cl77.2) was from Synaptic Systems (Göttingen, Germany); mouse anti-Tsg101 (4A-10) was from GeneTex (San Antonio, TX); mouse T7 tag antibody was from Novagen; and Alexa Fluor 488 and Alexa Fluor 568 goat anti-mouse or goat anti-rabbit IgG were from Molecular Probes (Eugene, OR). Immunostaining and Microscopy—For imaging, cells were plated onto plain (COS-7 cells) or collagen-coated (HEK293 T-REx cells) glass coverslips. Cells were fixed in phosphate-buffered saline (140 mm NaCl, 15 mm phosphate, pH 7.4) containing 4% paraformaldehyde. GFP fusion proteins were imaged directly. For antibody staining, fixed cells were permeabilized in phosphate-buffered saline containing 0.2% Triton X-100, blocked with 5% goat serum, and incubated with primary and secondary antibodies. Cells were examined using a Zeiss Axioplan2 microscope coupled to a Radiance plus confocal laser system (Bio-Rad) with 488 and 543 nm laser lines. Images were merged and assembled using MetaMorph® Imaging System 6 (Universal Imaging Corp.) and Adobe Photoshop 7 (Adobe Systems, San Jose, CA). Cell Fractionation—For the fractionation shown in Fig. 1C, SKD1(E235Q)-GFP stable cells induced for 4 h were lysed with a ball-bearing homogenizer in 20 mm HEPES, pH 7.5, 1 mm EDTA, 1 mm EGTA, 5 mm ATP, 0.25 m sucrose, 1 mm phenylmethylsulfonyl fluoride, and Complete™ protease inhibitor (Roche Applied Science). Lysates were cleared of nuclei by centrifugation at 500 × g for 3 min, layered on top of a continuous sucrose gradient (0.25–1.75 m in the same buffer), and centrifuged at 100,000 × g for 18 h in a Beckman SW41 rotor. Fractions were collected, separated by SDS-PAGE, and analyzed by Western blotting with the indicated antibodies. For Fig. 2, SKD1(E235Q)-GFP stable cells induced for 4 h were lysed with a ball-bearing homogenizer in 20 mm HEPES, pH 7.5, 150 mm NaCl, 2 mm MgCl2, 5 mm ATP, and 1 mm DTT. Cell lysates were precleared (1000 × g for 5 min) to remove nuclei and unlysed cells and then centrifuged at 100,000 × g for 30 min to collect membranes. To test solubilization conditions, P100 membranes were resuspended in homogenization buffer containing the indicated reagents for 30 min on ice or at 30 °C and then repelleted. For Fig. 4A, cell lysates were precleared as above and centrifuged at 20,000 × g for 10 min to yield supernatant and pellet.Fig. 4Endogenous hSnf7-1, hVps24, and Tsg101 associate with SKD1(E235Q)-GFP.A, postnuclear supernatants from uninduced or induced SKD1(E235Q) HEK293 cells were centrifuged at 20,000 × g for 15 min. The distribution of the proteins indicated was analyzed by Western blotting. B, magnetic bead immunoisolation of SKD1(E235Q)-GFP and associated proteins from sonicated Triton X-100-treated SKD1(E235Q)-GFP HEK293 cell lysates. Proteins bound to the indicated beads (CNT is control rabbit antibody; GFP is rabbit anti-GFP) were separated by SDS-PAGE, and proteins of interest were detected with specific antibodies. S, supernatant; P, pellet; Ab, antibody; sup., supernatant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For Figs. 5C and 7C, transfected COS-7 cells were lysed with a ball-bearing homogenizer in 20 mm HEPES, pH 7.5, 1 mm EDTA, 1 mm EGTA, 0.25 m sucrose, 1 mm phenylmethylsulfonyl fluoride, and Complete protease inhibitor. Lysates were cleared of nuclei (500 × g, 3 min), layered on top of a continuous sucrose gradient (0.25–1.75 m), and processed as above. For Fig. 5D, lysates were centrifuged at 20,000 × g, and resulting pellets were resuspended in buffer containing 1% Triton X-100 for 30 min prior to a second centrifugation at 20,000 × g.Fig. 7The N-terminal half of hSnf7-1 associates with membranes and binds phosphoinositides.A, stick diagram of hSnf7-1 showing its bipartite charge distribution and predicted coiled-coil motifs (black boxes, predicted by COILS (61.Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3522) Google Scholar)). aa, amino acid. B, cellular distribution of hSnf7-1 N and C fragments. COS-7 cells shown were transfected with FLAG-tagged hSnf7-N or hSnf7-C and immunostained with FLAG antibody. The scale bar represents 10 μm. C, distribution of hSnf7-N in homogenates of transfected COS-7 cells separated on a linear 0.25–1.75 m sucrose gradient. D, purified hSnf7-1-FL, -N, and -C proteins used for PIP blot overlay assays separated by SDS-PAGE and visualized by staining with Coomassie Blue. E, PIP Strips™ showing phosphoinositide binding by purified hSnf7-1-FL, -N, and -C. Bound hSnf7-1 was detected by Western blotting. Lipids are spotted as follows: 1, lysophosphatidic acid; 2, lysophosphocholine; 3, phosphatidylinositol; 4, phosphatidylinositol 3-phosphate; 5, phosphatidylinositol 4-phosphate; 6, phosphatidylinositol 5-phosphate; 7, phosphatidylethanolamine; 8, phosphatidylcholine; 9, sphingosine-1-phosphate; 10, phosphatidylinositol 3,4-bisphosphate; 11, phosphatidylinositol 3,5-bisphosphate; 12, phosphatidylinositol 4,5-bisphosphate; 13, phosphatidylinositol 3,4,5-