Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid.

Cells regulate the biophysical properties of their membranes by coordinated synthesis of different classes of lipids. Here, we identified a highly dynamic feedback mechanism by which the budding yeast Saccharomyces cerevisiae can regulate phospholipid biosynthesis. Phosphatidic acid on the endoplasmic reticulum directly bound to the soluble transcriptional repressor Opi1p to maintain it as inactive outside the nucleus. After the addition of the lipid precursor inositol, this phosphatidic acid was rapidly consumed, releasing Opi1p from the endoplasmic reticulum and allowing its nuclear translocation and repression of target genes. Thus, phosphatidic acid appears to be both an essential ubiquitous metabolic intermediate and a signaling lipid.

Molecular characterization of peripherin-2 and rom-1 mutants responsible for digenic retinitis pigmentosa.

Peripherin-2 and Rom-1 are homologous tetraspanning membrane proteins that assemble into noncovalent tetramers and higher order disulfide-linked oligomers implicated in photoreceptor disc morphogenesis. Individuals who coinherit a L185P peripherin-2 mutation and a null or G113E rom-1 mutation are afflicted with retinitis pigmentosa, whereas individuals who inherit only one defective gene are normal. We examined the expression, subunit assembly, and disulfide-mediated oligomerization of L185P and L185A peripherin-2 and L188P Rom-1 by velocity sedimentation, co-immunoprecipitation, and cross-linking. These mutants formed noncovalent dimers under disulfide-reducing conditions but failed to assemble into core tetramers. Under nonreducing conditions, L185P dimers formed disulfide-linked tetramers but not higher order oligomers. L185P coassembled with wild-type peripherin-2 and Rom-1 to form tetramers and higher order disulfide-linked oligomers characteristic of the wild-type proteins. The G113E Rom-1 mutant expressed 20-fold lower than wild-type Rom-1, indicating that it behaves mechanistically as a null allele. We conclude that Leu(185) of peripherin-2 (Leu(188) of Rom-1) is critical for tetramer but not dimer formation and that the core tetramer has 2-fold symmetry. Peripherin-2-containing tetramers are required for higher order disulfide-linked oligomer formation. The level of these oligomers is critical for stable photoreceptor disc formation and the digenic retinitis pigmentosa disease phenotype.

Disulfide-mediated oligomerization of Peripherin/Rds and Rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration.

Peripherin/Rds is a tetraspanning membrane protein that has been implicated in photoreceptor outer segment morphogenesis and inherited retinal degenerative diseases. Together with the structurally related protein, Rom-1, it forms a complex along the rims of rod and cone disc membranes. We have compared the oligomeric structure of these proteins from nonreduced and dithiothreitol reduced membranes by velocity sedimentation, SDS-gel electrophoresis, immunoaffinity chromatography, and chemical cross-linking. Under reducing conditions peripherin/Rds and Rom-1 existed as homomeric and heteromeric core complexes devoid of intermolecular disulfide bonds. Under nonreducing conditions core complexes associated through intermolecular disulfide bonds to form oligomers. One intermediate-size oligomer contained monomers and disulfide-linked dimers of peripherin/Rds and Rom-1, while larger oligomers consisted only of disulfide-linked peripherin/Rds dimers when analyzed on nonreducing SDS gels. Consistent with this result, disc membranes contained twice as much peripherin/Rds as Rom-1. Peripherin/Rds individually expressed in COS-1 cells also formed disulfide-linked oligomers bridged through Cys-150 residues, whereas Rom-1 showed little tendency to form oligomers. These results indicate that peripherin/Rds and Rom-1 associate noncovalently to form multisubunit core complexes. Peripherin/Rds containing core complexes interact through specific intermolecular disulfide bonds to form oligomers which may play a crucial role in photoreceptor disc morphogenesis and retinal degenerative diseases.

Cysteine residues of photoreceptor peripherin/rds: role in subunit assembly and autosomal dominant retinitis pigmentosa.

Peripherin/rds is a tetraspanning membrane glycoprotein that is essential for the morphogenesis and stabilization of outer segments of vertebrate rod and cone photoreceptor cells. Mutations in the gene for peripherin/rds are responsible for retinal degeneration in the rds mouse and a variety of progressive human retinal degenerative diseases including autosomal dominant retinitis pigmentosa and macular dystrophy. Peripherin/rds associates with rom-1, a homologous subunit, to form a heterotetrameric complex. This study examines the importance of cysteine residues for the structure of peripherin/rds and its assembly with rom-1. Each of the 13 cysteine residues in bovine peripherin/rds was individually replaced with a serine residue by site-directed mutagenesis, and the resulting mutants were expressed individually or together with rom-1 in COS-1 cells. SDS-polyacrylamide gel electrophoresis, immunoprecipitation, and velocity sedimentation were carried out to evaluate the ability of these mutants to form disulfide-linked homodimers, associate with rom-1, and assemble into tetramers characteristic of wild-type peripherin/rds. Substitution of each of the six nonconserved cysteines had no apparent effect on dimer formation, folding, or subunit assembly. In contrast, replacement of any of the seven conserved cysteine residues predicted to lie within a 150 amino acid intradiscal loop significantly altered these properties. Six of these mutants, including a C214S mutant linked to autosomal dominant retinitis pigmentosa, were unable to fold normally, interact with rom-1, or self-assemble into tetramers but instead formed a mixture of large aggregates and a smaller component, most likely a dimer. The C150S mutant, on the other hand, was incapable of forming intermolecular disulfide bonds but did associate with rom-1 into a heterotetramer. These results suggest that (1) the conserved C150 residue is required for intermolecular disulfide bonding but not subunit assembly; (2) the six other conserved cysteine residues are crucial for proper folding and subunit assembly, possibly through formation of intramolecular disulfide bonds; and (3) the misfolding and defective subunit assembly of the C214S mutant is responsible for a form of monogenic autosomal dominant retinitis pigmentosa.