Nte1p-mediated deacylation of phosphatidylcholine functionally interacts with Sec14p

Deciphering the function of the essential yeast Sec14p protein has revealed a regulatory interface between cargo secretion from Golgi and lipid homeostasis. Abrogation of the CDP-choline (CDP-Cho) pathway for phosphatidylcholine (PC) synthesis allows for life in the absence of the otherwise essential Sec14p. Nte1p, the product of open reading frame YML059c, is an integral membrane phospholipase against CDP-Cho-derived PC producing intracellular glycerophosphocholine (GPCho) and free fatty acids. We monitored Nte1p activity through in vivo PC turnover measurements and observed that intracellular GPCho accumulation is decreased in a sec14(ts) strain shifted to 37 degrees C in 10 mm choline (Cho)-containing medium compared with a Sec14p-proficient strain. Overexpression of two Sec14p homologs Sfh2p and Sfh4p in sec14(ts) cells restored secretion and growth at the restrictive temperature but did not restore GPCho accumulation. Instead, newly synthesized PC was degraded by phospholipase D (Spo14p). Similar analysis performed in a sec14Delta background confirmed these observations. These results imply that the ability of Sfh2p and Sfh4p to restore secretion and growth is not through a shared function with Sec14p in the regulation of PC turnover via Nte1p. Furthermore, our analyses revealed a profound alteration of PC metabolism triggered by the absence of Sec14p: Nte1p unresponsiveness, Spo14p activation, and deregulation of Pct1p. Sfh2p- and Sfh4p-overexpressing cells coped with the absence of Sec14p by controlling the rate of phosphocholine formation, limiting the amount of Cho available for this reaction, and actively excreting Cho from the cell. Increased Sfh4p also significantly reduced the uptake of exogenous Cho. Beyond the new PC metabolic control features we ascribe to Sfh2p and Sfh4p we also describe a second role for Sec14p in mediating PC homeostasis. Sec14p acts as a positive regulator of Nte1p-mediated PC deacylation with the functional consequence of increased Nte1p activity increasing the permissive temperature for the growth of sec14(ts) cells.     

Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in mammalian cell membranes and is essential for cell viability. The levels of this lipid must be tightly controlled to maintain homeostasis. Therefore, changes in the rate of PtdCho synthesis are generally balanced by changes in PtdCho catabolism and vice versa. It is commonly accepted that the rate of PtdCho synthesis is regulated by CTP:phosphocholine cytidylyltransferase (CT). However, it is not certain if PtdCho mass is regulated by specific catabolic enzyme(s). Our goal is to determine if PtdCho homeostasis is regulated by a phospholipase A₂ (PLA₂). To this end, we have prepared Chinese hamster ovary (CHO) cell lines that overexpress CT. CT activity is 7–10-fold higher in the transfected cells than in parental CHO cells. This increase in CT activity is associated with increases in both PtdCho synthesis and PtdCho catabolism. Glycerophosphocholine is the PtdCho catabolite that accumulates in the transfected cells, which suggests that PtdCho turnover is mediated by a phospholipase A₂ (PLA₂). Indeed, higher levels of calcium-independent PLA₂ activity are measured in the cytosols of the CHO cells that overexpress CT, compared to parental CHO cells. The elevated calcium-independent PLA₂ activity is associated with increases in the expression of the 80-kDa calcium-independent PLA₂ (iPLA₂). Together, these data suggest that the 80-kDa iPLA₂ may be modulated in response to changes in PtdCho levels and therefore is involved in the regulation of PtdCho homeostasis in CHO cells.     

Evidence for an intrinsic toxicity of phosphatidylcholine to Sec14p-dependent protein transport from the yeast Golgi complex

Yeast phosphatidylinositol-transfer protein (Sec14p) is essential for Golgi secretory function and cell viability. This requirement of Sec14p is relieved by genetic inactivation of the cytidine diphosphate-choline pathway for phosphatidycholine (PtdCho) biosynthesis. Standard phenotypic analyses indicate that inactivation of the phosphatidylethanolamine (PtdEtn) pathway for PtdCho biosynthesis, however, does not rescue the growth and secretory defects associated with Sec14p deficiency. We now report inhibition of choline uptake from the media reveals an efficient "bypass Sec14p" phenotype associated with PtdEtn-methylation pathway defects. We further show that the bypass Sec14p phenotype associated with PtdEtn-methylation pathway defects resembles other bypass Sec14p mutations in its dependence on phospholipase D activity. Finally, we find that increased dosage of enzymes that catalyze phospholipase D-independent turnover of PtdCho, via mechanisms that do not result in a direct production of phosphatidic acid or diacylglycerol, effect a partial rescue of sec14-1(ts)-associated growth defects. Taken together, these data support the idea that PtdCho is intrinsically toxic to yeast Golgi secretory function.     

Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.     

Regulation of phosphatidylcholine biosynthesis by mitogenic growth factors

Phosphatidylcholine (PC) biosynthesis in cultured 3T3 fibroblasts was increased in varying degrees by these mitogenic growth factors: fetal bovine serum, insulin, 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor, vasopressin, fibroblast growth factor and insulin-like growth factors I and II. PC synthesis was increased 2-4-fold by 10% serum, up to 4-fold by growth factors alone, and up to 8-fold by combinations of two or more growth factors. Single growth factors had no effect on the incorporation of [3H]choline into the acid-soluble precursors of PC, while serum or combinations of two or more mitogens could increase the incorporation of [3H]choline into acid-soluble material by up to 2-fold. Serum was shown to increase choline phosphorylation, choline kinase activity and the size of the phosphocholine pool. These data were utilized to calculate the radioactive specific activity of phosphocholine. Serum did not increase phosphocholine specific activity above control values; thus the increased incorporation of labelled choline into PC after serum stimulation resulted from increased PC synthesis and not from a simple change in specific activity of precursor phosphocholine.     

Sec14 related proteins in yeast

Lipid transport between membranes of eukaryotic organisms represents an essential aspect of organelle biogenesis. This transport must be strictly selective and directional to assure specific lipid composition of individual membranes. Despite the intensive research effort in the last few years, our understanding of how lipids are sorted and moved within cells is still rather limited. Evidence indicates that at least some of the mechanisms generating and maintaining non-random distribution of lipids in cells are linked to the action of phosphatidylinositol transfer proteins (PITPs). The major PITP in yeast Saccharomyces cerevisiae, Sec14p, is essential in promoting Golgi secretory function by modulating of its membrane lipid composition. This review focuses on a group of five yeast proteins that share significant sequence homology with Sec14p. Based on this sequence identity, they were termed Sfh (Sec fourteen homologue) proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism, phosphoinositide mediated signaling and membrane trafficking.     

Regulation of lymphoid homeostasis by interleukin-15

Interleukin (IL)-15 is a member of the common gamma chain family of cytokines, and is closely related to IL-2. While these two cytokines share several important biological functions in vitro, recent mouse models have demonstrated unique roles for these two cytokines in supporting lymphoid homeostasis in vivo. IL-15 has been shown to regulate the homeostasis of both innate and adaptive immune cells, and this review will discuss several ways in which this pleiotropic cytokine may support lymphoid homeostasis.     

Regulation of neuronal bioenergy homeostasis by glutamate

Bioenergy homeostasis is crucial in maintaining normal cell function and survival and it is thus important to understand cellular mechanisms underlying its regulation. Neurons use a large amount of ATP to maintain membrane potential and synaptic communication, making the brain the most energy consuming organ in the body. Glutamate mediates a large majority of synaptic transmission which is responsible for the expression of neural plasticity and higher brain functions. Most of the energy cost is attributable to the glutamatergic system; under pathological conditions such as stroke and brain ischemia, neural energy depletion is accompanied by a massive release of glutamate. However, the specific cellular processes implicated in glutamate-dependent bioenergy dynamics are not well understood. We find that glutamate induces a rapid and dramatic reduction of ATP levels in neurons, through reduced ATP genesis and elevated consumption. ATP reduction depends on NMDA receptor activity, but is not a result of neuronal firing, gap junction-mediated leaking or intracellular signaling. Similar changes in ATP levels are also induced by synaptic glutamate accumulation following suppression of glutamate transporter activity. Furthermore, the glutamate-induced ATP down-regulation is blocked by the sodium pump inhibitor ouabain, suggesting the sodium pump as the primary energy consumer during glutamate stimulation. These data suggest the important role of glutamate in the control of cellular ATP homeostasis.     

Sec61β facilitates the maintenance of endoplasmic reticulum homeostasis by associating microtubules

Sec61β, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61β causes lethality, but its physiological role is unclear. Here, we show that Sec61β interacts directly with microtubules. Overexpression of Sec61β containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61β is critical for microtubule association. Depletion of Sec61β induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61β. Loss of Sec61β causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61β may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.     

Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle

Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.     

Sec14 family of lipid transfer proteins in yeasts

The hydrophobicity of lipids prevents their free movement across the cytoplasm. To achieve highly heterogeneous and precisely regulated lipid distribution in different cellular membranes, lipids are transported by lipid transfer proteins (LTPs) in addition to their transport by vesicles. Sec14 family is one of the most extensively studied groups of LTPs. Here we provide an overview of Sec14 family of LTPs in the most studied yeast Saccharomyces cerevisiae as well as in other selected non-Saccharomyces yeasts—Schizosaccharomyces pombe, Kluyveromyces lactis, Candida albicans, Candida glabrata, Cryptococcus neoformans, and Yarrowia lipolytica. Discussed are specificities of Sec14-domain LTPs in various yeasts, their mode of action, subcellular localization, and physiological function. In addition, quite few Sec14 family LTPs are target of antifungal drugs, serve as modifiers of drug resistance or influence virulence of pathologic yeasts. Thus, they represent an important object of study from the perspective of human health.     

Regulation of immune cell homeostasis by type I interferons

Although initially identified and best characterized for their role in innate antiviral defence, type I interferons (IFN-I) are also known to have an important impact on the adaptive immune response. In part, this is linked to another long-recognised property of IFN-I, namely their ability to modify cellular proliferation and survival. Here, we review the influence of IFN-I on immune cell homeostasis, focusing on their effects on T cells and antigen-presenting cells.