Mutations in elongation of very long-chain fatty acid-4 (ELOVL4) are associated with autosomal dominant Stargardt-like macular degeneration (STGD3), with a five base-pair (5 bp) deletion mutation resulting in the loss of 51 carboxy-terminal amino acids and truncation of the protein. In addition to the retina, Elovl4 is expressed in a limited number of mammalian tissues, including skin, with unknown function(s). We generated a knock-in mouse model with the 5-bp deletion in the Elovl4 gene. As anticipated, mice carrying this mutation in the heterozygous state (Elovl4(+/del)) exhibit progressive photoreceptor degeneration. Unexpectedly, homozygous mice (Elovl4(del/del)) display scaly, wrinkled skin, have severely compromised epidermal permeability barrier function, and die within a few hours after birth. Histopathological evaluation of the Elovl4(del/del) pups revealed no apparent abnormality(ies) in vital internal organs. However, skin histology showed an abnormally-compacted outer epidermis [stratum corneum (SC)], while electron microscopy revealed deficient epidermal lamellar body contents, and lack of normal SC lamellar membranes that are essential for permeability barrier function. Lipid analyses of epidermis from Elovl4(del/del) mice revealed a global decrease in very long-chain fatty acids (VLFAs) (i.e., carbon chain > or =C28) in both the ceramide/glucosylceramide and the free fatty-acid fractions. Strikingly, Elovl4(del/del) skin was devoid of the epidermal-unique omega-O-acylceramides, that are key hydrophobic components of the extracellular lamellar membranes in mammalian SC. These findings demonstrate that ELOVL4 is required for generating VLFA critical for epidermal barrier function, and that the lack of epidermal omega-O-acylceramides is incompatible with survival in a desiccating environment.

The SEC14 gene encodes an essential phosphatidylinositol (PtdIns) transfer protein required for formation of Golgi-derived secretory vesicles in yeast. Suppressor mutations that rescue temperature-sensitive sec14 mutants provide an approach for determining the role of Sec14p in secretion. One suppressor, sac1-22, causes accumulation of PtdIns(4)P. SAC1 encodes a phosphatase that can hydrolyze PtdIns(4)P and certain other phosphoinositides. These findings suggest that PtdIns(4)P is limiting in sec14 cells and that elevation of PtdIns(4)P production can suppress the secretory defect. Correspondingly, we found that PtdIns(4)P levels were decreased significantly in sec14-3 mutants shifted to 37°C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34°C) when carrying a plasmid overexpressing PIK1, encoding one of two essential PtdIns 4-kinases. This effect is specific because overexpression of the other PtdIns 4-kinase gene (STT4) or a PtdIns 3-kinase gene (VPS34) did not rescue sec14-3 cells. To further address Pik1p function in secretion, two different pik1 ts mutants were examined. Upon shift to restrictive temperature (37°C), the PtdIns(4)P levels dropped by about 60% in both pik1 ts strains within 1 h. During the same period, cells displayed a reduction (40 -50%) in release of a secreted enzyme (invertase). However, similar treatment did not effect maturation of a vacuolar enzyme (carboxypeptidase Y). These findings indicate that, first, PtdIns(4)P limitation is a major contributing factor to the secretory defect in sec14 cells; second, Sec14p function is coupled to the action of Pik1p, and; third, PtdIns(4)P has an important role in the Golgi-toplasma membrane stage of secretion.In eukaryotic cells, secreted proteins are synthesized on ribosomes targeted to the endoplasmic reticulum (ER), 1 translocated into the ER lumen, and transported through the secretory pathway (1). From the ER, secretory proteins are transported to the Golgi apparatus, through the subcompartments of the Golgi, and then to the cell surface or to certain intracellular organelles, all via small membrane-bound vesicles (transport vesicles) (2-4). Cargo proteins are packaged into transport vesicles that bud from one compartment and fuse with another. Mechanisms of vesicle budding and fusion are conserved from yeast to mammalian cells (3,5). Because of its tractability for genetic analysis, bakers' yeast (Saccharomyces cerevisiae) has proven to be a useful organism to identify gene products required for various events in secretion. Genetic screens, first applied by Schekman and co-workers (6), resulted in the isolation of temperature-sensitive sec mutants that displayed defects in different stages of secretion at the nonpermissive temperature. Characterization of the corresponding normal (SEC) genes has pinpointed many proteins necessary for secretory processes; and, a large number of gene products are now known to function at various steps in the secretory pathway (reviewed in Ref. 7). The SEC14 gene encodes a phosph...

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] is a key signaling molecule in animal cells. It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP 3 ), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively. In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P 2 binds to proteins that modify cytoskeletal architecture and phospholipid constituents. Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P 2 synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol. These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides. We also show data demonstrating that whole-plant IP 3 levels increase significantly within 1 min of stress initiation, and that IP 3 levels continue to increase for more than 30 min during stress application. Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments. Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P 2 is hydrolyzed to IP 3 . The accumulation of IP 3 occurs during a time frame similar to that observed for stress-induced calcium mobilization. These data also suggest that the majority of the PtdIns(4,5)P 2 synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP 3 signaling pathway.Phosphoinositides are a class of membrane phospholipids that serve numerous roles in eukaryotic cellular processes. The family of phosphoinositides includes phosphatidylinositol monophosphate species phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 4-phosphate [PtdIns(4)P], phosphatidylinositol bisphosphate species phosphatidylinositol 3,4-bisphosphate, phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P 2 ], and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], and the phosphatidylinositol trisphosphate species phosphatidylinositol 3,4,5-trisphosphate. PtdIns(3)P and PtdIns(4)P regulate vesicle-mediated protein transport to the vacuole/lysosome and protein secretion,

Objective Neurodegeneration with brain iron accumulation (NBIA) represents a distinctive phenotype of neurodegenerative disease for which several causative genes have been identified. The spectrum of neurologic disease associated with mutations in NBIA genes is broad, with phenotypes that range from infantile neurodegeneration and death in childhood to adult-onset parkinsonism-dystonia. Here we report the discovery of a novel gene that leads to a distinct form of NBIA. Methods Using autozygosity mapping and candidate gene sequencing, we identified mutations in the fatty acid hydroxylase gene FA2H, newly implicating abnormalities of ceramide metabolism in the pathogenesis of NBIA. Results Neuroimaging demonstrated T2 hypointensity in the globus pallidus, confluent T2 white matter hyperintensities, and profound pontocerebellar atrophy in affected members of two families. Phenotypically, affected family members exhibited spastic quadriparesis, ataxia, and dystonia with onset in childhood and episodic neurological decline. Analogous to what has been reported previously for PLA2G6, the phenotypic spectrum of FA2H mutations is diverse based on our findings and those of prior investigators, because FA2H mutations have been identified in both a form of hereditary spastic paraplegia (SPG35) and a progressive familial leukodystrophy. Interpretation These findings link white matter degeneration and NBIA for the first time and implicate new signaling pathways in the genesis of NBIA.

Phosphatidylinositol 4-kinase, Pik1, is essential for viability. GFP-Pik1 localized to cytoplasmic puncta and the nucleus. The puncta colocalized with Sec7-DsRed, a marker of trans-Golgi cisternae. Kap95 (importin-β) was necessary for nuclear entry, but not Kap60 (importin-α), and exportin Msn5 was required for nuclear exit. Frq1 (frequenin orthologue) also is essential for viability and binds near the NH2 terminus of Pik1. Frq1-GFP localized to Golgi puncta, and Pik1 lacking its Frq1-binding site (or Pik1 overexpressed in frq1Δ cells) did not decorate the Golgi, but nuclear localization was unperturbed. Pik1(Δ10-192), which lacks its nuclear export sequence, displayed prominent nuclear accumulation and did not rescue inviability of pik1Δ cells. A Pik1-CCAAX chimera was excluded from the nucleus and also did not rescue inviability of pik1Δ cells. However, coexpression of Pik1(Δ10-192) and Pik1-CCAAX in pik1Δ cells restored viability. Catalytically inactive derivatives of these compartment-restricted Pik1 constructs indicated that PtdIns4P must be generated both in the nucleus and at the Golgi for normal cell function.