Binding of [³H]photocholesterol to wildtype and mutant stomatin.

<p>COS-7 cells were transiently transfected with WT or mutant stomatin constructs. Subsequently they were incubated with a photoactivatable, radioactive cholesterol derivative ([³H]photocholesterol) and irradiated with UV light to crosslink [³H]photocholesterol to respective binding proteins. The cells were solubilized and stomatin was immunoprecipitated by monoclonal anti-stomatin antibody GARP-50. (<b>A</b>) SDS-PAGE and autoradiography revealed cholesterol-binding to WT and mutant stomatin. (<b>B</b>) The expression level of the constructs was determined by immunoblotting with monoclonal anti-stomatin antibody GARP-50.</p

Binding of [³H]photocholesterol to wildtype and mutant stomatin.

<p>COS-7 cells were transiently transfected with WT or mutant stomatin constructs. Subsequently they were incubated with a photoactivatable, radioactive cholesterol derivative ([³H]photocholesterol) and irradiated with UV light to crosslink [³H]photocholesterol to respective binding proteins. The cells were solubilized and stomatin was immunoprecipitated by monoclonal anti-stomatin antibody GARP-50. (<b>A</b>) SDS-PAGE and autoradiography revealed cholesterol-binding to WT and mutant stomatin. (<b>B</b>) The expression level of the constructs was determined by immunoblotting with monoclonal anti-stomatin antibody GARP-50.</p

Densitometric analysis of [³H]photocholesterol-binding to wildtype and mutant stomatin.

<p>Densitometric analysis of [³H]photocholesterol-binding to wildtype and mutant stomatin.</p

Structure-function analysis of human stomatin: A mutation study

<div><p>Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic “hairpin” structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton.</p></div

Comparison of stomatin structural changes and functional consequences referring to wildtype.

<p>Comparison of stomatin structural changes and functional consequences referring to wildtype.</p

FRAP-analysis of GFP-tagged wildtype and mutant stomatin.

<p>A431 cells stably expressing GFP-tagged WT or mutant stomatin at the plasma membrane were analyzed by FRAP measurements. N ≥ 20. The data for mobile fractions and recovery halftimes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178646#pone.0178646.t004" target="_blank">Table 4</a>.</p

Molecular size distribution of GFP-tagged wildtype and mutant stomatin.

<p>Molecular size distribution of GFP-tagged wildtype and mutant stomatin.</p

Lateral mobility of plasma membrane-bound GFP-tagged wildtype and mutant stomatin.

<p>Effects of cholesterol and cytochalasin D.</p

Schematic structure of GFP-tagged wildtype and mutant stomatin.

<p>(<b>A</b>) Schematic model of wildtype (WT) stomatin, composed of the N-terminal region (N-ter), <u>i</u>ntra<u>m</u>embrane domain (IM), <u>c</u>holesterol <u>r</u>ecognition/interaction <u>a</u>mino acid <u>c</u>onsensus (CRAC)-like motif (CL), prohibitin homology domain (PHB), also known as <u>s</u>tomatin, <u>p</u>rohibitin, <u>f</u>lotillin, <u>H</u>flK/C (SPFH) domain, coiled-coil domain (CC), <u>o</u>ligomerization and lipid <u>r</u>aft-<u>a</u>ssociation domain (ORA), and C-terminal domain (C-ter). Palmitate residues bound to Cys-30 and Cys-87 are symbolized by zigzag lines. Stomatin mutants are shown that are deleted at the N-terminus (ΔN), C-terminus (ΔC), and coiled-coil domain (ΔCC), respectively. The positions of exchanged amino acid residues in point mutants are marked. Exchange of Cys-30 or Cys-87 for Ser abolished palmitate bonding. (<b>B</b>) Hypothetical model of a monomeric wildtype stomatin in association with a biological membrane. Sidedness is marked by “in” (cytoplasmic) and “out” (extracellular or luminal). The color code denotes the domains as illustrated in (<b>A</b>). The green ball at the N-terminal region symbolizes the phosphorylation site at Ser-10; the “P” at the kink within the hydrophobic IM domain marks residue Pro-47, which is responsible for the monotopic membrane protein structure. The model is roughly drawn according to known and estimated sizes; the N-terminal region is α-helical (E. Umlauf, unpublished results), the PHB/SPFH core domain is 5 nm in length and 2 nm in height, while the coiled-coil domain is 6 nm long [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178646#pone.0178646.ref047" target="_blank">47</a>]. CARC denotes a reversed CRAC motif; there are three CARC motifs, two overlapping with the CRAC-like (CL) and one overlapping with the ORA motif. Schematic models of the most remarkable mutants are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178646#pone.0178646.s004" target="_blank">S4 Fig</a>.</p

Ability of GFP-tagged stomatin mutants to target the plasma membrane, form oligomers, and/or associate with DRMs.

<p>Ability of GFP-tagged stomatin mutants to target the plasma membrane, form oligomers, and/or associate with DRMs.</p