Structure-function analysis of human stomatin: A mutation study.

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.

Acanthocytosis and the c.680 A>G Mutation in the PANK2 Gene: A Study Enrolling a Cohort of PKAN Patients from the Dominican Republic.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of Neurodegeneration with Brain Iron Accumulation (NBIA) associated with mutations in the pantothenate kinase 2 gene (PANK2). Pantothenate kinases catalyze the rate-limiting step of coenzyme A synthesis and Pank2 is the only pantothenate kinase isoform in humans that is localized to mitochondria. Acanthocytosis, the occurrence of spiculated erythrocytes, is observed in about 10% of the PKAN patients. Therefore PKAN is also classified together with other rare neurodegenerative diseases like Chorea Acanthocytosis (ChAc) and McLeod syndrome (MLS) into the Neuroacanthocytosis (NA) syndromes. It has not been investigated yet whether acanthocytosis in PKAN is associated with a specific subset of Pank2 mutations. In this study, we analyzed acanthocytosis of a cohort of 25 PKAN patients from the Dominican Republic that are homozygous for the c.680 A>G mutation in the PANK2 gene as compared to control donors that are heterozygous or wild-type with respect to this mutation. 3D modeling of this mutation indicated that the replacement of a tyrosine by a cysteine at position 227 in Pank2 disrupts a polar interaction within the A domain of the enzyme. Mean acanthocyte count was elevated in the cohort of patients, however, acanthocytosis varied among the patients with nearly half of them showing high (>20%) or elevated acanthocytosis and the rest showing mild (6-10%) or no (<6%) acanthocytosis. Heterozygous control donors revealed a tendency to mild acanthocytosis. Based on the insight that Pank2 is a normal constituent of red blood cells and de novo biosynthesis of coenzyme A is likely to take place in the erythrocyte cytosol we propose a hypothetical model that accounts for the variability in the occurrence of acanthocytic cells in PKAN.

Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains.

The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins.

Brain, blood, and iron: perspectives on the roles of erythrocytes and iron in neurodegeneration.

The terms "neuroacanthocytosis" (NA) and "neurodegeneration with brain iron accumulation" (NBIA) both refer to groups of genetically heterogeneous disorders, classified together due to similarities of their phenotypic or pathological findings. Even collectively, the disorders that comprise these sets are exceedingly rare and challenging to study. The NBIA disorders are defined by their appearance on brain magnetic resonance imaging, with iron deposition in the basal ganglia. Clinical features vary, but most include a movement disorder. New causative genes are being rapidly identified; however, the mechanisms by which mutations cause iron accumulation and neurodegeneration are not well understood. NA syndromes are also characterized by a progressive movement disorder, accompanied by cognitive and psychiatric features, resulting from mutations in a number of genes whose roles are also basically unknown. An overlapping feature of the two groups, NBIA and NA, is the occurrence of acanthocytes, spiky red cells with a poorly-understood membrane dysfunction. In this review we summarise recent developments in this field, specifically insights into cellular mechanisms and from animal models. Cell membrane research may shed light upon the significance of the erythrocyte abnormality, and upon possible connections between the two sets of disorders. Shared pathophysiologic mechanisms may lead to progress in the understanding of other types of neurodegeneration.

Myristoylation of human LanC-like protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin.

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-beta-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.

Characterization of the stomatin domain involved in homo-oligomerization and lipid raft association.

The cytoplasmically oriented monotopic integral membrane protein stomatin forms high-order oligomers and associates with lipid rafts. To characterize the domains that are involved in oligomerization and detergent-resistant membrane (DRM) association, we expressed truncation and point mutants of stomatin and analyzed their size and buoyancy by ultracentrifugation methods. A small C-terminal region of stomatin that is largely hydrophobic, Ser-Thr-Ile-Val-Phe-Pro-Leu-Pro-Ile (residues 264-272), proved to be crucial for oligomerization, whereas the N-terminal domain (residues 1-20) and the last 12 C-terminal amino acids (residues 276-287) were not essential. The introduction of alanine substitutions in the region 264-272 resulted in the appearance of monomers. Remarkably, only three of these residues, Ile-Val-Phe (residues 266-268), were found to be indispensable for the DRM association. Interestingly, the exchange of Pro-269 and to some extent the residues 270-272, which are essential for oligomerization, did not affect the DRM association of stomatin. This suggests that the formation of oligomers is not necessary for the association of stomatin with DRMs. Internal deletions near the membrane anchoring domain resulted in the formation of intermediate size oligomers suggesting a conformational interdependence of large parts of the C-terminal region. Fluorescence recovery after photobleaching analysis of the tagged, monomeric, non-DRM mutant ST-(1-262)-green fluorescent protein and wild type stomatin StomGFP showed a significantly higher lateral mobility of the truncation mutant in the plasma membrane suggesting a membrane interaction of the respective C-terminal region also in vivo.

Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection.

Infection of human erythrocytes by the apicomplexan malaria parasite Plasmodium falciparum results in endovacuolar uptake of 4 host proteins that reside in erythrocyte detergent-resistant membranes (DRMs). Whether this vacuolar transport reflects selective uptake of host DRM proteins remains unknown. A further complication is that DRMs of vastly different protein and cholesterol contents have been isolated from erythrocytes. Here we show that isolated DRMs containing the highest cholesterol-to-protein ratio have low protein mass. Liquid chromatography, mass spectrometry, and antibody-based studies reveal that the major DRM proteins are band 3, flotillin-1 and -2, peroxiredoxin-2, and stomatin. Band 3 and stomatin, which reflect the bulk mass of erythrocyte DRM proteins, and all tested non-DRM proteins are excluded from the vacuolar parasite. In contrast, flotillin-1 and -2 and 8 minor DRM proteins are recruited to the vacuole. These data suggest that DRM association is necessary but not sufficient for vacuolar recruitment and there is active, vacuolar uptake of a subset of host DRM proteins. Finally, the 10 internalized DRM proteins show varied lipid and peptidic anchors indicating that, contrary to the prevailing model of apicomplexan vacuole formation, DRM association, rather than lipid anchors, provides the preferred criteria for protein recruitment to the malarial vacuole.

Flotillin-1/reggie-2 traffics to surface raft domains via a novel golgi-independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation.

Flotillins are lipid raft-associated proteins, which have been implicated in neuronal regeneration and insulin signaling. We now show that newly synthesized flotillin-1 reaches the plasma membrane via a Sar1-independent and brefeldin A-resistant targeting pathway. Consistent with post-translational membrane association of flotillin, protease sensitivity experiments suggest that flotillin-1 is not a transmembrane protein but is associated with the cytoplasmic face of the plasma membrane. The N terminus of flotillin contains a prohibitin-like domain (PHB), which shows homology to a number of proteins associated with raft domains including stomatin, podocin, and prohibitin. We show that the PHB domain of flotillin can efficiently target a heterologous protein, green fluorescent protein, to the plasma membrane. Another PHB-containing protein, stomatin, traffics to the plasma membrane via the conventional secretory pathway. Plasma membrane association of both full-length flotillin and the green fluorescent protein-tagged PHB domain of flotillin is dependent on palmitoylation and requires a conserved cysteine residue, Cys-34, in the PHB domain. The results identify a novel targeting mechanism for plasma membrane association of flotillin-1 involving a Golgi-independent trafficking pathway, the PHB domain, and palmitoylation.