Sterols and sphingolipids are limited to eukaryotic cells, and their interaction has been proposed to favor formation of lipid microdomains. Although there is abundant biophysical evidence demonstrating their interaction in simple systems, convincing evidence is lacking to show that they function together in cells. Using lipid analysis by mass spectrometry and a genetic approach on mutants in sterol metabolism, we show that cells adjust their membrane composition in response to mutant sterol structures preferentially by changing their sphingolipid composition. Systematic combination of mutations in sterol biosynthesis with mutants in sphingolipid hydroxylation and head group turnover give a large number of synthetic and suppression phenotypes. Our unbiased approach provides compelling evidence that sterols and sphingolipids function together in cells. We were not able to correlate any cellular phenotype we measured with plasma membrane fluidity as measured using fluorescence anisotropy. This questions whether the increase in liquid order phases that can be induced by sterol-sphingolipid interactions plays an important role in cells. Our data revealing that cells have a mechanism to sense the quality of their membrane sterol composition has led us to suggest that proteins might recognize sterol-sphingolipid complexes and to hypothesize the coevolution of sterols and sphingolipids.

Two functions of the p24 complex are described: one connects GPI-anchored proteins to COPII proteins at ER exit sites to facilitate their incorporation into ER-derived vesicles, and the other serves in quality control of GPI-anchored proteins to retrieve unremodeled GPI-anchored proteins from the Golgi back to the ER.

Glycosylphosphatidylinositol (GPI), covalently attached to many eukaryotic proteins, not only acts as a membrane anchor but is also thought to be a sorting signal for GPI-anchored proteins that are associated with sphingolipid and sterol-enriched domains. GPI anchors contain a core structure conserved among all species. The core structure is synthesized in two topologically distinct stages on the leaflets of the endoplasmic reticulum (ER). Early GPI intermediates are assembled on the cytoplasmic side of the ER and then are flipped into the ER lumen where a complete GPI precursor is synthesized and transferred to protein. The flipping process is predicted to be mediated by a protein referred as flippase; however, its existence has not been proven. Here we show that yeast Arv1p is an important protein required for the delivery of an early GPI intermediate, GlcN-acylPI, to the first mannosyltransferase of GPI synthesis in the ER lumen. We also provide evidence that ARV1 deletion and mutations in other proteins involved in GPI anchor synthesis affect inositol phosphorylceramide synthesis as well as the intracellular distribution and amounts of sterols, suggesting a role of GPI anchor synthesis in lipid flow from the ER.

Sphingolipids are a class of membrane lipids conserved from yeast to mammals which determine whether a cell dies or survives. Perturbations in sphingolipid metabolism cause apoptotic cell death. Recent studies indicate that reduced sphingolipid levels trigger the cell death, but little is known about the mechanisms. In the budding yeast Saccharomyces cerevisiae, we show that reduction in complex sphingolipid levels causes loss of viability, most likely due to the induction of mitochondria-dependent apoptotic cell death pathway, accompanied by changes in mitochondrial and endoplasmic reticulum morphology and endoplasmic reticulum stress. Elevated cytosolic free calcium is required for the loss of viability. These results indicate that complex sphingolipids are essential for maintaining endoplasmic reticulum homeostasis and suggest that perturbation in complex sphingolipid levels activates an endoplasmic reticulum stress-mediated and calcium-dependent pathway to propagate apoptotic signals to the mitochondria.

Sphingolipids are required for many cellular functions including response to heat shock. We analyzed the yeast lcb1-100 mutant, which is conditionally impaired in the first step of sphingolipid biosynthesis and shows a strong decrease in heat shock protein synthesis and viability. Transcription and nuclear export of heat shock protein mRNAs is not affected. However, lcb1-100 cells exhibited a strong decrease in protein synthesis caused by a defect in translation initiation under heat stress conditions. The essential lipid is sphingoid base, not ceramide or sphingoid base phosphates. Deletion of the eIF4E-binding protein Eap1p in lcb-100 cells restored translation of heat shock proteins and increased viability. The translation defect during heat stress in lcb1-100 was due at least partially to a reduced function of the sphingoid base-activated PKH1/2 protein kinases. In addition, depletion of the translation initiation factor eIF4G was observed in lcb1-100 cells and ubiquitin overexpression allowed partial recovery of translation after heat stress. Taken together, we have shown a requirement for sphingoid bases during the recovery from heat shock and suggest that this reflects a direct lipid-dependent signal to the cap-dependent translation initiation apparatus. INTRODUCTIONEukaryotic cells have developed several mechanisms to respond to rapid increases in temperature. On heat stress, cells reduce the rate of synthesis of proteins that were expressed before the heat shock and change their transcription profile dramatically to produce mainly heat shock responsive proteins (Gasch et al., 2000;Murray et al., 2004). In the budding yeast Saccharomyces cerevisiae changes in transcription upon heat stress are fairly well understood, involving control by two transcription factors, the heat shock factor Hsf1p and Msn2p/4p. Hsf1p binds to heat shock elements (HSEs) found in the promoter region of many heat shock protein genes (Wu, 1995). Genes that do not contain HSEs, but whose transcription is induced by heat and other stress signals, including osmotic shock, DNA damage, and oxidative stress, contain stress response elements (STREs) in their promoters. On these stresses, Msn2/4p shuttles from the cytosol to the nucleus and activates transcription through binding the STREs (Schmitt and McEntee, 1996;Gorner et al., 1998). After transcription, the corresponding mRNAs are exported from the nucleus and are translated (Stutz and Rosbash, 1998). Proteins encoded by heat stress responsive genes are responsible for the synthesis of the thermoprotectant trehalose (Singer and Lindquist, 1998), for the folding of proteins and for the degradation of unfolded and aggregated proteins (Imai et al., 2003;Riezman, 2004).In addition to the induction of heat shock proteins, yeast cells induce the de novo synthesis of free sphingoid bases, followed by ceramides and sphingolipids (Jenkins, 2003). The first steps in the biosynthesis of sphingolipids in animal cells and in yeast are similar, but differ in production of complex sphingolipids. ...

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is crucial for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that in yeast coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control a later stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.

Compensatory growth of organs after loss of their mass and/or function is controlled by hepatocyte growth factor (HGF), but the underlying regulatory mechanisms remain elusive. Here, we show that CUB domain-containing protein 1 (CDCP1) promotes HGF-induced compensatory renal growth. Using canine kidney cells as a model of renal tubules, we found that HGF-induced temporal up-regulation of Src activity and its scaffold protein, CDCP1, and that the ablation of CDCP1 robustly abrogated HGF-induced phenotypic changes, such as morphological changes and cell growth/proliferation. Mechanistic analyses revealed that up-regulated CDCP1 recruits Src into lipid rafts to activate STAT3 associated with the HGF receptor Met, and activated STAT3 induces the expression of matrix metalloproteinases and mitogenic factors. After unilateral nephrectomy in mice, the Met-STAT3 signaling is transiently up-regulated in the renal tubules of the remaining kidney, whereas CDCP1 ablation attenuates regenerative signaling and significantly suppresses compensatory growth. These findings demonstrate that CDCP1 plays a crucial role in controlling compensatory renal growth by focally and temporally integrating Src and Met signaling.