The leucine zipper domain of the transcriptional repressor Opi1 underlies a signal transduction mechanism regulating lipid synthesis.

In Saccharomyces cerevisiae, the transcriptional repressor Opi1 regulates the expression of genes involved in phospholipid synthesis responding to the abundance of the phospholipid precursor phosphatidic acid at the endoplasmic reticulum. We report here the identification of the conserved leucine zipper (LZ) domain of Opi1 as a hot spot for gain of function mutations and the characterization of the strongest variant identified, Opi1N150D. LZ modeling posits asparagine 150 embedded on the hydrophobic surface of the zipper and specifying dynamic parallel homodimerization by allowing electrostatic bonding across the hydrophobic dimerization interface. Opi1 variants carrying any of the other three ionic residues at amino acid 150 were also repressing. Genetic analyses showed that Opi1N150D variant is dominant, and its phenotype is attenuated when loss of function mutations identified in the other two conserved domains are present in cis. We build on the notion that membrane binding facilitates LZ dimerization to antagonize an intramolecular interaction of the zipper necessary for repression. Dissecting Opi1 protein in three polypeptides containing each conserved region, we performed in vitro analyses to explore interdomain interactions. An Opi11-190 probe interacted with Opi1291-404, the C terminus that bears the activator interacting domain (AID). LZ or AID loss of function mutations attenuated the interaction of the probes but was unaffected by the N150D mutation. We propose a model for Opi1 signal transduction whereby synergy between membrane-binding events and LZ dimerization antagonizes intramolecular LZ-AID interaction and transcriptional repression.

Bi-allelic variants in CHKA cause a neurodevelopmental disorder with epilepsy and microcephaly.

The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.

SLC25 Family Member Genetic Interactions Identify a Role for HEM25 in Yeast Electron Transport Chain Stability.

The SLC25 family member SLC25A38 (Hem25 in yeast) was recently identified as a mitochondrial glycine transporter that provides substrate to initiate heme/hemoglobin synthesis. Mutations in the human SLC25A38 gene cause congenital sideroblastic anemia. The full extent to which SLC25 family members coregulate heme synthesis with other mitochondrial functions is not clear. In this study, we surveyed 29 nonessential SLC25 family members in Saccharomyces cerevisiae for their ability to support growth in the presence and absence of HEM25 Six SLC25 family members were identified that were required for growth or for heme synthesis in cells lacking Hem25 function. Importantly, we determined that loss of function of the SLC25 family member Flx1, which imports FAD into mitochondria, together with loss of function of Hem25, resulted in inability to grow on media that required yeast cells to supply energy using mitochondrial respiration. We report that specific components of complexes of the electron transport chain are decreased in the absence of Flx1 and Hem25 function. In addition, we show that mitochondria from flx1Δ hem25Δ cells contain uncharacterized Cox2-containing high molecular weight aggregates. The functions of Flx1 and Hem25 provide a facile explanation for the decrease in heme level, and in specific electron transport chain complex components.

Lipid synthesis and membrane contact sites: a crossroads for cellular physiology.

Membrane contact sites (MCSs) are regions of close apposition between different organelles that contribute to the functional integration of compartmentalized cellular processes. In recent years, we have gained insight into the molecular architecture of several contact sites, as well as into the regulatory mechanisms that underlie their roles in cell physiology. We provide an overview of two selected topics where lipid metabolism intersects with MCSs and organelle dynamics. First, the role of phosphatidic acid phosphatase, Pah1, the yeast homolog of metazoan lipin, toward the synthesis of triacylglycerol is outlined in connection with the seipin complex, Fld1/Ldb16, and lipid droplet formation. Second, we recapitulate the different contact sites connecting mitochondria and the endomembrane system and emphasize their contribution to phospholipid synthesis and their coordinated regulation. A comprehensive view is emerging where the multiplicity of contact sites connecting different cellular compartments together with lipid transfer proteins functioning at more than one MCS allow for functional redundancy and cross-regulation.

Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia.

Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.

Choline transport activity regulates phosphatidylcholine synthesis through choline transporter Hnm1 stability.

Choline is a precursor for the synthesis of phosphatidylcholine through the CDP-choline pathway. Saccharomyces cerevisiae expresses a single high affinity choline transporter at the plasma membrane, encoded by the HNM1 gene. We show that exposing cells to increasing levels of choline results in two different regulatory mechanisms impacting Hnm1 activity. Initial exposure to choline results in a rapid decrease in Hnm1-mediated transport at the level of transporter activity, whereas chronic exposure results in Hnm1 degradation through an endocytic mechanism that depends on the ubiquitin ligase Rsp5 and the casein kinase 1 redundant pair Yck1/Yck2. We present details of how the choline transporter is a major regulator of phosphatidylcholine synthesis.

NTE1-encoded phosphatidylcholine phospholipase b regulates transcription of phospholipid biosynthetic genes.

The Saccharomyces cerevisiae NTE1 gene encodes an evolutionarily conserved phospholipase B localized to the endoplasmic reticulum (ER) that degrades phosphatidylcholine (PC) generating glycerophosphocholine and free fatty acids. We show here that the activity of NTE1-encoded phospholipase B (Nte1p) prevents the attenuation of transcription of genes encoding enzymes involved in phospholipid synthesis in response to increased rates of PC synthesis by affecting the nuclear localization of the transcriptional repressor Opi1p. Nte1p activity becomes necessary for cells growing in inositol-free media under conditions of high rates of PC synthesis elicited by the presence of choline at 37 degrees C. The specific choline transporter encoded by the HNM1 gene is necessary for the burst of PC synthesis observed at 37 degrees C as follows: (i) Nte1p is dispensable in an hnm1Delta strain under these conditions, and (ii) there is a 3-fold increase in the rate of choline transport via the Hnm1p choline transporter upon a shift to 37 degrees C. Overexpression of NTE1 alleviated the inositol auxotrophy of a plethora of mutants, including scs2Delta, scs3Delta, ire1Delta, and hac1Delta among others. Overexpression of NTE1 sustained phospholipid synthesis gene transcription under conditions that normally repress transcription. This effect was also observed in a strain defective in the activation of free fatty acids for phosphatidic acid synthesis. No changes in the levels of phosphatidic acid were detected under conditions of altered expression of NTE1. Consistent with a synthetic impairment between challenged ER function and inositol deprivation, increased expression of NTE1 improved the growth of cells exposed to tunicamycin in the absence of inositol. We describe a new role for Nte1p toward membrane homeostasis regulating phospholipid synthesis gene transcription. We propose that Nte1p activity, by controlling PC abundance at the ER, affects lateral membrane packing and that this parameter, in turn, impacts the repressing transcriptional activity of Opi1p, the main regulator of phospholipid synthesis gene transcription.

The Kap60-Kap95 karyopherin complex directly regulates phosphatidylcholine synthesis.

Phosphatidylcholine is the major phospholipid in eukaryotic cells. There are two main pathways for the synthesis of phosphatidylcholine: the CDP-choline pathway present in all eukaryotes and the phosphatidylethanolamine methylation pathway present in mammalian hepatocytes and some single celled eukaryotes, including the yeast Saccharomyces cerevisiae. In S. cerevisiae, the rate-determining step in the synthesis of phosphatidylcholine via the CDP-choline pathway is catalyzed by Pct1. Pct1 converts phosphocholine and CTP to CDP-choline and pyrophosphate. In this study, we determined that Pct1 is in the nucleoplasm and at endoplasmic reticulum and nuclear membranes. Pct1 directly interacts with the alpha-importin Kap60 via a bipartite basic region in Pct1, and this region of Pct1 was required for its entry into the nucleus. Pct1 also interacted with the beta-importin Kap95 in cell extracts, implying a model whereby Pct1 interacts with Kap60 and Kap95 with this tripartite complex transiting the nuclear pore. Exclusion of Pct1 from the nucleus by elimination of its nuclear localization signal or by decreasing Kap60 function did not affect the level of phosphatidylcholine synthesis. Diminution of Kap95 function resulted in almost complete ablation of phosphatidylcholine synthesis under conditions where Pct1 was extranuclear. The beta-importin Kap95 is a direct regulator membrane synthesis.

Phosphatidylcholine synthesis and its catabolism by yeast neuropathy target esterase 1.

Phosphatidylcholine (PtdCho) is the major phospholipid component of eukaryotic membranes and deciphering the molecular mechanisms regulating PtdCho homeostasis is necessary to fully understand many pathophysiological situations where PtdCho metabolism is altered. This concept is illustrated in this review by summarizing recent evidence on Nte1p, a yeast endoplasmic reticulum resident phospholipase B that deacylates PtdCho producing intracellular glycerophosphocholine. The mammalian and Drosophila homologues, neuropathy target esterase and swiss cheese, respectively, have been implicated in normal brain development with increased intracytoplasmic vesicularization and multilayered membrane stacks as cytological signatures of their absence. Consistent with a role in lipid and membrane homeostasis, Nte1p-mediated PtdCho deacylation is strongly affected by Sec14p, a component of the yeast secretory machinery characterized by its ability to interface between lipid metabolism and vesicular trafficking. The preference of Nte1p toward PtdCho produced through the CDP-choline pathway and the downstream production of choline by the Gde1p glycerophosphodiesterase for resynthesis of PtdCho by the CDP-choline pathway are also highlighted.

Identification of novel phospholipid binding proteins in Saccharomyces cerevisiae.

A proteomics approach was used to search for novel phospholipid binding proteins in Saccharomyces cerevisiae. Phospholipids were immobilized on a solid support and the lipids were probed with soluble yeast protein extracts. From this, the phosphatidic acid binding proteins were eluted and identified by mass spectrometry. Thirteen proteins were identified and 11 of these were previously unknown lipid binding proteins. The protein-lipid interactions identified would not have been predicted using bioinformatics approaches as none possessed a known lipid binding motif. A subset of the identified proteins was purified to homogeneity and determined to directly bind phospholipids immobilized on a solid support or organized into liposomes. This simple approach could be systematically applied to perform an exhaustive screen for soluble lipid binding proteins in S. cerevisiae or other organisms.

Glycerophosphocholine catabolism as a new route for choline formation for phosphatidylcholine synthesis by the Kennedy pathway.

In eukaryotes, neuropathy target esterase (Nte1p in yeast) deacylates phosphatidylcholine derived exclusively from the CDP-choline pathway to produce glycerophosphocholine (GroPCho) and release two fatty acids. The metabolic fate of GroPCho in eukaryotic cells is currently not known. Saccharomyces cerevisiae contains two open reading frames predicted to contain glycerophosphodiester phosphodiesterase domains, YPL110c and YPL206c. Pulse-chase experiments were conducted to monitor GroPCho metabolic fate under conditions known to alter CDP-choline pathway flux and consequently produce different rates of formation of GroPCho. From this analysis, it was revealed that GroPCho was metabolized to choline, with this choline serving as substrate for renewed synthesis of phosphatidylcholine. YPL110c played the major role in this metabolic pathway. To extend and confirm the metabolic studies, the ability of the ypl110cDelta and ypl206cDelta strains to utilize exogenous GroPCho or glycerophosphoinositol as the sole source of phosphate was analyzed. Consistent with our metabolic profiling, the ypl206cDelta strain grew on both substrates with a similar rate to wild type, whereas the ypl110cDelta strain grew very poorly on GroPCho and with moderately reduced growth on glycerophosphoinositol.