Structure-function analysis of stomatin Rainer Prohaska Max F. Perutz Laboratories (MFPL) Medical University of Vienna Vienna, Austria Overhydrated Hereditary Stomatocytosis http://emedicine.medscape.com/article/955921-media Missing band 7 membrane protein in OHSt SDS-PAGE NEPHGE vs. SDS-PAGE 7.2b stomatin Lande et al. (1982) J.Clin.Invest. 70, 1273 Stewart et al. (1992) Blood 79, 1593 Schematic structure of stomatin Palmitoyl- Palmitoyl- Acetyl- 287 N C P CRAC PHB/SPFH-domain Coiled-coil ORA I Intramembrane Oligomerisation PHB: Prohibitin homology domain and Lipid Raft- SPFH: Stomatin, Prohibitin, Flotillin, HflK/C Association motif cDNA sequence: Hiebl-Dirschmied et al. (1991) BBA 1090, 123 Stewart et al. (1992) Blood 79, 1593 Phosphorylation: Salzer et al. (1993) BBA 1151,149 Palmitoylation: Snyers et al. (1999) FEBS Lett. 449, 101 Schematic model of stomatin structure out in CC Intact human RBCs +/- proteinase K Stomatin and immunoblot flotillins are monotopic membrane proteins Ulrich Salzer Stomatin and flotillins are oligomeric lipid raft (DRM) proteins DRMs Sedimentation analysis band 3 band 4.1 marker band 4.2 immunoblot SDS-PAGE Silver staining Salzer & Prohaska (2001) Blood 97,1141 Stomatin • 31 kDa RBC integral membrane protein (band 7.2b) with a monotopic, oligomeric structure; associated with lipid rafts. • Missing in OHSt RBCs ( “K+/Na+ leak”). Regulator of an ion channel? RhAG mutation Bruce et al. (2009) Blood 113, 1350. mRNA is present in OHSt erythroid precursors. Protein is made but degraded upon enucleation Fricke et al. (2005) Br.J.Haematol. 131, 265. • Widely distributed in mammalian tissues. • Subcellular localisation: plasma membrane (apical), late endosomal compartment (MVBs, lysosomes), lipid droplets, phagosomes, neutrophil granules, platelet α-granules. • K.o. mouse does not show stomatocytosis, nor another major phenotype Zhu et al. (1999) Blood 93, 2404. • Stomatin orthologues are present in all kingdoms of life. The stomatin family • Human stomatins • Stomatin (STOM, EPB72) ►ubiquitous • SLP1 (STOML1, hUNC24) ►brain, heart LTP-D • SLP2 (STOML2) ►ubiquitous • SLP3 (STOML3, SRO) ►sensory neurons • Podocin (NPHS2) ►kidney podocytes • C. elegans stomatins • sto-1, sto-2, sto-3, sto-4, sto-5, sto-6, stl-1 • mec-2, unc-1, unc-24 ►neurons Stomatin localisation to PM, recycling endosomes, and lysosomes A431 carcinoma Stomatin Rab11 cells Stomatin Nieman-Pick C1 Stefanie Rungaldier Stomatin and SLP-1 co-localise to the late endosomal compartment Stomatin is localised to the plasma membrane (arrows) and late endosomes of HeLa cells, whereas SLP-1 is exclusively localised to LEs Mario Mairhofer Localisation of Stable transfectants in A431 carcinoma cells Mutation CLSM localization localization stomatin mutants Stom (1-288) Wt PM, LE normal Stom (22-288) ∆N PM, LE normal (GFP-tagged) Stom (1-263) ∆C PM, LE normal Stom (C30S) IM PM, LE normal Stom (P47S) IM Cytoplasmic, ER aberrant Stom (I57A) CRAC PM, LE normal out Stom (Y60A) CRAC Cytoplasmic aberrant Stom (R62A) CRAC PM, LE normal Stom (C87S) PHB Cytoplasmic aberrant Stom (D89A) PHB Cytoplasmic aberrant Stom (F91A) PHB PM, LE normal CC in Stom (R97A) PHB Cytoplasmic aberrant Stom (K198A) PHB LE, cytoplasmic aberrant Stom (P200A) CC PM, LE, cytoplasmic aberrant Stom (∆204-241) ∆CC PM, LE normal Stom (P245A) CC PM, LE, cytoplasmic aberrant Stom (F269A) ORA PM, LE normal Stom (P270A) ORA LE, cytoplasmic aberrant Stefanie Rungaldier Stomatin in exovesicles Ca2+-induced vesiculation of red cells actin (TRITC-phalloidin) RBCs treated with Ca2+ and ionophore stomatin A23187 (mcAb GARP-50) Ulrich Salzer Segregation of stomatin and flotillins upon (micro-)vesiculation Protein Ratio MV/Gh 4.0 Gh: Ghosts MV: Microvesicles 2.0 0.2 0.4 0.5 0.4 Ulrich Salzer Model of lipid and protein uptake into the malarial vacuole Murphy et al. (2007) Blood 110, 2132 Structural considerations The crystal structure of the P. horikoshii stomatin core domain PHB/SPFH domain CC domain Anti-parallel coiled-coil interaction Yokoyama et al. (2008) J.Mol.Biol. 376, 868 Oligomerisation? Hypothetical model of stomatin multimers + … N-terminal IM domain … C-terminal ORA/PDZ domain … PHB domains triangular platform … kinked α-helices Lipid raft association? Model of stomatin-raft aggregation Small rafts Aggregated raft Scaffolding platform? Stomatins interact with transporters and channels • MEC-2 / ENaC (Epithelial sodium channels) • Stomatin / ASICs (Acid sensing ion channels) • Podocin / TRPC6 (Transient receptor potential channel 6) • UNC-1 / UNC-9 (Innexin gap junction) • Stomatin / Pannexin-1 (gap junction) • Stomatin / GLUT1 (Glucose and DHA transporter) Integral scaffolding proteins? Hypothetical model of transporter- or channel-stomatin-complexes ENaC ASIC SLP1 GLUT1 cholesterol Hypothetical model of lateral GLUT1-stomatin interaction Stomatin GLUT1 trimer Taken from: Mueckler & Makepeace (2009) Taken from: Yokoyama et al. (2008) Biochem. 48, 5934 J.Mol.Biol. 376, 868 Crosslinking of RBC membrane proteins Isolation of EGS-cross-linked stomatin-complexes Protein positions (without cross -linking) spectrin ankyrin band 3 4.1 4.2 GPA dimer Anti-stomatin- flotillins Sepharose column actin GAPDH 10% SDS-PAGE GPA monomer stomatin Silver staining F 1 2 3 4 5 M 1 2 3 4 5 M F Elution fractions Elution fractions Rungaldier et al. (2013) BBA-Mem. 1828, 956 Semiquantitative MS/MS analysis of EGS-cross-linked stomatin complexes 140,000.00 AvTIC units 120,000.00 (Average Total Ion Current) EGS 100,000.00 80,000.00 Stomatin Band 3 60,000.00 GLUT1 40,000.00 20,000.00 0.00 >500 kDa 300 kDa 130 kDa MS/MS identification of EGS-cross-linked stomatin-associated proteins 140,000.00 400,000.00 AvTIC EGS units 350,000.00 120,000.00 300,000.00 100,000.00 >500 kDa 250,000.00 500 kDa 80,000.00 >500 kDa 200,000.00 300 kDa 60,000.00 130 kDa 150,000.00 40,000.00 100,000.00 20,000.00 50,000.00 0.00 0.00 Isolation of DSS-cross-linked stomatin-complexes EGS DSS Anti-stomatin- Sepharose column 10% SDS-PAGE Silver staining Elution fractions Elution fractions Rungaldier et al. (2013) BBA-Mem. 1828, 956 MS/MS analysis of DSS-cross-linked stomatin complexes 180,000.00 180,000.00 AvTIC units 160,000.00 160,000.00 140,000.00 140,000.00 120,000.00 120,000.00 100,000.00 100,000.00 Stomatin >500 kDa Band 3 80,000.00 80,000.00 500 kDa GLUT1 300 kDA 60,000.00 60,000.00 40,000.00 40,000.00 20,000.00 20,000.00 0.00 0.00 >500 kDa 500 kDa 300 kDA SLC4A1 SLC2A1 SLC40A1 SLC14A1 DRMs from cross-linked membranes DRMs non-DRMs oligomers EGS dimer WB: stomatin oligomers DSS dimer Gradient fractions Sucrose density gradient ultracentrifugation Isolation of DSS-cross-linked stomatin-complexes from DRMs 500 kDa 250 kDa 300 kDa Immunoaffinity 130 kDa purification by 10% SDS-PAGE anti-stomatin- Silver staining Sepharose 100 kDa 70 kDa 55 kDa Elution fractions MS/MS-analysis of DSS-cross-linked stomatin-complexes from DRMs Band 3, GLUT1, Aquaporin-1, Ferroportin-1, Lutheran-BG-GP/laminin receptor, Aldolase A, Glyceraldehyde-3-phosphate dehydrogenase 250,000.00 250,000.00 AvTIC AvTIC units units 200,000.00 200,000.00 150,000.00 150,000.00 Stomatin 500 kDa Band 3 GLUT1 300 kDa 100,000.00 AQP1 100,000.00 50,000.00 50,000.00 0.00 0.00 500 kDa 300 kDa MS/MS analysis of high-molecular stomatin-complexes from DRMs 90,000.00 AvTIC units 80,000.00 DSS 70,000.00 60,000.00 50,000.00 Stomatin Band 3 40,000.00 GLUT1 30,000.00 20,000.00 10,000.00 0.00 800 kDa 600 kDa 500 kDa 400 kDa 270 kDa 230 kDa 200 kDa Rungaldier et al. (2013) BBA-Mem. 1828, 956 Hypothetical model of the RBC membrane Rungaldier et al. (2013) BBA-Mem. 1828, 956 MFPL Inst. Biophysics, Univ. Linz Ellen Umlauf Gerhard Schütz Luc Snyers Peter Hinterdorfer Mario Mairhofer Ulrich Salzer CNRS, Univ. Montpellier Stefanie Rungaldier Naomi Taylor Edina Csaszar Multiprotein complexes in the red cell membrane Salomao et al. (2008) Proc.Natl.Acad.Sci.USA 105, 8026 Isolation and analysis of cross-linked stomatin-complexes • Cross-linking of RBC membranes (EGS or DSS) • Quenching and solubilisation with TX-100 or SDS/TX-100 • ► +/- Isolation of DRMs • Solution onto anti-stomatin affinity column • Extensive washing, elution with glycine/HCl • SDS-PAGE / MS-compatible silver staining • Excision of bands, trypsinisation, LC-MS/MS High-molecular stomatin-complexes from DRMs 4–12% gradient SDS-PAGE Silver staining Elution fractions Erythrocyte DRMs Low density high density Immuno- blot Gradient fractions Stomatin Only 10% of fr. 6-9 GLUT1 were loaded for GLUT1 Density gradient Flotillin 2 ultracentrifugation Glycophorin A Band 3 Rungaldier et al. (2013) BBA-Mem. 1828, 956 FRAP analysis of stomatin mobility Wt Stom oligomeric DRM-protein Stom(1-262) monomeric non-DRM protein Umlauf et al. (2006) J.Biol.Chem. 281, 23349 Stomatin domain dimer Brand et al. (2012) EMBO J. 31, 3635 The SPFH/PHB-superfamily Stomatin, Prohibitin, Flotillin, HflK/C Reggie Tavernarakis et al. (1999) TIBS 24, 425 .