Fig 1.
Schematic structure of GFP-tagged wildtype and mutant stomatin.
(A) Schematic model of wildtype (WT) stomatin, composed of the N-terminal region (N-ter), intramembrane domain (IM), cholesterol recognition/interaction amino acid consensus (CRAC)-like motif (CL), prohibitin homology domain (PHB), also known as stomatin, prohibitin, flotillin, HflK/C (SPFH) domain, coiled-coil domain (CC), oligomerization and lipid raft-association 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) 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 (A). 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 The model is roughly drawn according to known and estimated sizes; the N-terminal region is α-helical (E. Umlauf, unpublished results), the PHB/SPFH core domain is 5 nm in length and 2 nm in height, while the coiled-coil domain is 6 nm long [47]. 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 S4 Fig.
Fig 2.
Binding of [3H]photocholesterol to wildtype and mutant stomatin.
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. (A) SDS-PAGE and autoradiography revealed cholesterol-binding to WT and mutant stomatin. (B) The expression level of the constructs was determined by immunoblotting with monoclonal anti-stomatin antibody GARP-50.
Table 1.
Densitometric analysis of [3H]photocholesterol-binding to wildtype and mutant stomatin.
Fig 3.
Oligomerization and DRM-association of GFP-tagged wildtype and mutant stomatin.
A431 cells stably expressing GFP-tagged WT or mutant stomatin were solubilized and subjected to linear density gradient centrifugation to estimate molecular size (left panel) or step density gradient centrifugation to determine DRM-association (right panel). Gradient fractions were analyzed by SDS-PAGE and proteins were identified by immunoblotting with anti-GFP. The linear 15–50% sucrose gradient was verified by refractometry and calibrated by marker proteins. SDS-PAGE was performed by running molecular weight markers in parallel. GFP-tagged stomatin constructs showed values of about 70 kDa except for the Pro47Ser mutant, which was estimated at 80 kDa, as predicted due to glycosylation [40].
Table 2.
Molecular size distribution of GFP-tagged wildtype and mutant stomatin.
Table 3.
Distribution of GFP-tagged wildtype and mutant stomatin between DRMs and non-DRMs.
Fig 4.
FRAP-analysis of GFP-tagged wildtype and mutant stomatin.
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 Table 4.
Table 4.
Lateral mobility of plasma membrane-bound GFP-tagged wildtype and mutant stomatin.
Correlation of mobile fractions and recovery halftimes with the ability to oligomerize and/or associate with DRMs.
Fig 5.
Lateral mobility of GFP-tagged wildtype and mutant stomatin.
Effects of cholesterol and cytochalasin D. A431 cells stably expressing GFP-tagged WT or mutant stomatin at the plasma membrane were analyzed by FRAP measurements. The cells were either depleted of or loaded with cholesterol, treated with cytochalasin D (cytoD), or treated with a combination of both. N ≥ 20. The data for mobile fractions and recovery halftimes are given in Table 5.
Table 5.
Lateral mobility of plasma membrane-bound GFP-tagged wildtype and mutant stomatin.
Effects of cholesterol and cytochalasin D.
Table 6.
Ability of GFP-tagged stomatin mutants to target the plasma membrane, form oligomers, and/or associate with DRMs.
Table 7.
Comparison of stomatin structural changes and functional consequences referring to wildtype.