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 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

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 (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 • (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

(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