PtdSer Decarboxylase Research Articles

Phosphatidylserine synthase in the endoplasmic reticulum of Toxoplasma is essential for its lytic cycle in human cells

Glycerophospholipids have emerged as a significant contributor to the intracellular growth of pathogenic protist Toxoplasma gondii. Phosphatidylserine (PtdSer) is one such lipid, attributed to the locomotion and motility-dependent invasion and egress events in its acutely infectious tachyzoite stage. However, the de novo synthesis of PtdSer and the importance of the pathway in tachyzoites remain poorly understood. We show that a base-exchange-type PtdSer synthase (PSS) located in the parasite’s endoplasmic reticulum produces PtdSer, which is rapidly converted to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase (PSD) activity. The PSS-PSD pathway enables the synthesis of several lipid species, including PtdSer (16:0/18:1) and PtdEtn (18:2/20:4, 18:1/18:2 and 18:2/22:5). The PSS-depleted strain exhibited a lower abundance of the major ester-linked PtdEtn species and concurrent accrual of host-derived ether-PtdEtn species. Most phosphatidylthreonine (PtdThr) species—an exclusive natural analog of PtdSer, also made in the endoplasmic reticulum—were repressed. PtdSer species, however, remained largely unaltered, likely due to the serine-exchange reaction of PtdThr synthase in favor of PtdSer upon PSS depletion. Not least, the loss of PSS abrogated the lytic cycle of tachyzoites, impairing the cell division, motility, and egress. In a nutshell, our data demonstrate a critical role of PSS in the biogenesis of PtdSer and PtdEtn species and its physiologically essential repurposing for the asexual reproduction of a clinically relevant intracellular pathogen.

Aminoglycerophospholipid flipping and P4-ATPases in Toxoplasma gondii

Lipid flipping in the membrane bilayers is a widespread eukaryotic phenomenon that is catalyzed by assorted P4-ATPases. Its occurrence, mechanism, and importance in apicomplexan parasites have remained elusive, however. Here we show that Toxoplasma gondii, an obligate intracellular parasite with high clinical relevance, can salvage phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) but not phosphatidylcholine (PtdCho) probes from its milieu. Consistently, the drug analogs of PtdCho are broadly ineffective in the parasite culture. NBD-PtdSer imported to the parasite interior is decarboxylated to NBD-PtdEtn, while the latter is not methylated to yield PtdCho, which confirms the expression of PtdSer decarboxylase but a lack of PtdEtn methyltransferase activity and suggests a role of exogenous lipids in membrane biogenesis of T. gondii. Flow cytometric quantitation of NBD-probes endorsed the selectivity of phospholipid transport and revealed a dependence of the process on energy and protein. Accordingly, our further work identified five P4-ATPases (TgP4-ATPase1-5), all of which harbor the signature residues and motifs required for phospholipid flipping. Of the four proteins expressed during the lytic cycle, TgP4-ATPase1 is present in the apical plasmalemma; TgP4-ATPase3 resides in the Golgi network along with its noncatalytic partner Ligand Effector Module 3 (TgLem3), whereas TgP4-ATPase2 and TgP4-ATPase5 localize in the plasmalemma as well as endo/cytomembranes. Last but not least, auxin-induced degradation of TgP4-ATPase1-3 impaired the parasite growth in human host cells, disclosing their crucial roles during acute infection. In conclusion, we show selective translocation of PtdEtn and PtdSer at the parasite surface and provide the underlying mechanistic and physiological insights in a model eukaryotic pathogen.

Phosphatidylserine decarboxylase is critical for the maintenance of skeletal muscle mitochondrial integrity and muscle mass

ObjectivePhosphatidylethanolamine (PtdEtn) is a major phospholipid in mammals. It is synthesized via two pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum and the phosphatidylserine (PtdSer) decarboxylase (PSD) pathway in the mitochondria. While the CDP-ethanolamine pathway is considered the major route for PtdEtn synthesis in most mammalian tissues, little is known about the importance of the PSD pathway in vivo, especially in tissues enriched with mitochondria such as skeletal muscle. Therefore, we aimed to examine the role of the mitochondrial PSD pathway in regulating PtdEtn homeostasis in skeletal muscle in vivo. MethodsTo determine the functional significance of this pathway in skeletal muscle in vivo, an adeno-associated viral vector approach was employed to knockdown PSD expression in skeletal muscle of adult mice. Muscle lipid and metabolite profiling was performed using mass spectrometry. ResultsPSD knockdown disrupted muscle phospholipid homeostasis leading to an ∼25% reduction in PtdEtn and an ∼45% increase in PtdSer content. This was accompanied by the development of a severe myopathy, evident by a 40% loss in muscle mass as well as extensive myofiber damage as shown by increased DNA synthesis and central nucleation. In addition, PSD knockdown caused marked accumulation of abnormally appearing mitochondria that exhibited severely disrupted inner membrane integrity and reduced OXPHOS protein content. ConclusionsThe PSD pathway has a significant role in maintaining phospholipid homeostasis in adult skeletal muscle. Moreover, PSD is essential for maintenance of mitochondrial integrity and skeletal muscle mass.

An Assembly of Proteins and Lipid Domains Regulates Transport of Phosphatidylserine to Phosphatidylserine Decarboxylase 2 in Saccharomyces cerevisiae

Saccharomyces cerevisiae uses multiple biosynthetic pathways for the synthesis of phosphatidylethanolamine. One route involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), the transport of this lipid to endosomes, and decarboxylation by PtdSer decarboxylase 2 (Psd2p) to produce phosphatidylethanolamine. Several proteins and protein motifs are known to be required for PtdSer transport to occur, namely the Sec14p homolog PstB2p/Pdr17p; a PtdIns 4-kinase, Stt4p; and a C2 domain of Psd2p. The focus of this work is on defining the protein-protein and protein-lipid interactions of these components. PstB2p interacts with a protein encoded by the uncharacterized gene YPL272C, which we name Pbi1p (PstB2p-interacting 1). PstB2p, Psd2, and Pbi1p were shown to be lipid-binding proteins specific for phosphatidic acid. Pbi1p also interacts with the ER-localized Scs2p, a binding determinant for several peripheral ER proteins. A complex between Psd2p and PstB2p was also detected, and this interaction was facilitated by a cryptic C2 domain at the extreme N terminus of Psd2p (C2-1) as well the previously characterized C2 domain of Psd2p (C2-2). The predicted N-terminal helical region of PstB2p was necessary and sufficient for promoting the interaction with both Psd2p and Pbi1p. Taken together, these results support a model for PtdSer transport involving the docking of a PtdSer donor membrane with an acceptor via specific protein-protein and protein-lipid interactions. Specifically, our model predicts that this process involves an acceptor membrane complex containing the C2 domains of Psd2p, PstB2p, and Pbi1p that ligate to Scs2p and phosphatidic acid present in the donor membrane, forming a zone of apposition that facilitates PtdSer transfer.

The Obligate Intracellular Parasite Toxoplasma gondii Secretes a Soluble Phosphatidylserine Decarboxylase

Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (PtdSer) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent K(m) of 67 μM. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for ∼20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted ∼20% of its total PSD activity at 37 °C, and the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism.

An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship

Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best-known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca(2+) signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.

Elimination of the CDP-ethanolamine Pathway Disrupts Hepatic Lipid Homeostasis

Phosphoethanolamine cytidylyltransferase (ECT) catalyzes the rate-controlling step in a major pathway for the synthesis of phosphatidylethanolamine (PtdEtn). Hepatocyte-specific deletion of the ECT gene in mice resulted in normal appearing animals without overt signs of liver injury or inflammation. The molecular species of PtdEtn in the ECT-deficient livers were significantly altered compared with controls and matched the composition of the phosphatidylserine (PtdSer) pool, illustrating the complete reliance on the PtdSer decarboxylase pathway for PtdEtn synthesis. PtdSer structure was controlled by the substrate specificity of PtdSer synthase that selectively converted phosphatidylcholine molecular species containing stearate paired with a polyunsaturated fatty acid to PtdSer. There was no evidence for fatty acid remodeling of PtdEtn. The elimination of diacylglycerol utilization by the CDP-ethanolamine pathway led to a 10-fold increase in triacylglycerols in the ECT-deficient hepatocytes that became engorged with lipid droplets. Triacylglycerol accumulation was associated with a significant elevation in the expression of the transcription factors and target genes that drive de novo lipogenesis. The absence of the ECT pathway for diacylglycerol utilization at the endoplasmic reticulum triggers increased fatty acid synthesis to support the formation of triacylglycerols leading to liver steatosis.

Uptake and Utilization of Lyso-phosphatidylethanolamine by Saccharomyces cerevisiae

Phosphatidylethanolamine (PtdEtn) is synthesized by multiple pathways located in different subcellular compartments in yeast. Strains defective in the synthesis of PtdEtn via phosphatidylserine (PtdSer) synthase/decarboxylase are auxotrophic for ethanolamine, which must be transported into the cell and converted to phospholipid by the cytidinediphosphate-ethanolamine-dependent Kennedy pathway. We now demonstrate that yeast strains with psd1Delta psd2Delta mutations, devoid of PtdSer decarboxylases, import and acylate exogenous 1-acyl-2-hydroxyl-sn-glycero-3-phosphoethanolamine (lyso-PtdEtn). Lyso-PtdEtn supports growth and replaces the mitochondrial pool of PtdEtn much more efficiently than and independently of PtdEtn derived from the Kennedy pathway. Deletion of both the PtdSer decarboxylase and Kennedy pathways yields a strain that is a stringent lyso-PtdEtn auxotroph. Evidence for the specific uptake of lyso-PtdEtn by yeast comes from analysis of strains harboring deletions of the aminophospholipid translocating P-type ATPases (APLTs). Elimination of the APLTs, Dnf1p and Dnf2p, or their noncatalytic beta-subunit, Lem3p, blocked the import of radiolabeled lyso-PtdEtn and resulted in growth inhibition of lyso-PtdEtn auxotrophs. In cell extracts, lyso-PtdEtn is rapidly converted to PtdEtn by an acyl-CoA-dependent acyltransferase. These results now provide 1) an assay for APLT function based on an auxotrophic phenotype, 2) direct demonstration of APLT action on a physiologically relevant substrate, and 3) a genetic screen aimed at finding additional components that mediate the internalization, trafficking, and acylation of exogenous lyso-phospholipids.

Protein and lipid motifs regulate phosphatidylserine traffic in yeast

Phosphatidylserine (PtdSer) is synthesized in the endoplasmic reticulum and its subdomains associated with the mitochondria [MAM (mitochondria-associated membrane)] and subsequently transported to the loci of the PtdSer decarboxylases, Pds1p (phosphatidylserine decarboxylase 1 encoded by the PSD1 gene that complements psd1 mutations) in the mitochondria, and Psd2p (PtdSer decarboxylase 2 encoded by the PSD2 gene that complements psd2 mutations) in the Golgi. Decarboxylation of PtdSer to PtdEtn (phosphatidylethanolamine) can be used as a biochemical indicator of transport to these organelles, which is regulated by specific lipid and protein motifs. PtdSer transport to mitochondria is controlled by ubiquitination via the action of the ubiquitin ligase subunit Met30p (a ubiquitin ligase subunit encoded by the MET30 gene that complements the met30 mutation affecting methionine biosynthesis). Mutant strains with lesions in the MET30 gene are defective in PtdSer transport and show altered ubiquitination of specific target proteins, such as the transcription factor Met4p (a transcription factor encoded by the MET4 gene that complements the met4 mutation affecting methionine biosynthesis). Mutations to MET30 cause defects in both the MAM as a donor of PtdSer, and the mitochondria as an acceptor of PtdSer in the transport reaction. PtdSer transport to the locus of Psd2p is controlled by specific protein and lipid motifs. The C2 (Ca2+ and phospholipid-binding sequence) domain of Psd2p, and the lipid-binding protein PstB2p (PtdSer transport B pathway protein encoded by the PSTB2 gene that complements the pstB2 mutation affecting PtdSer transport), must be present on acceptor membranes for PtdSer transport to occur. In addition, the action of the PtdIns 4-kinase, Stt4p (PtdIns 4-kinase encoded by the STT4 gene that complements the stt4 mutation causing staurosporine and temperature-sensitive growth) is also required for PtdSer transport to the locus of Psd2p. Reconstitution of PtdSer transport to Psd2p using liposomes demonstrates that PtdSer-rich domains present in vesicles are preferred substrates for transport. In addition, the incorporation of phosphatidic acid into donor membranes enhances the rate of PtdSer transport. Collectively, these data support a model for PtdSer transport in which specific proteins and lipids are required on donor and acceptor membranes.

Protein and lipid motifs regulate phosphatidylserine traffic in yeast

Phosphatidylserine (PtdSer) is synthesized in the endoplasmic reticulum and its subdomains associated with the mitochondria [MAM (mitochondria-associated membrane)] and subsequently transported to the loci of the PtdSer decarboxylases, Pds1p (phosphatidylserine decarboxylase 1 encoded by the PSD1 gene that complements psd1 mutations) in the mitochondria, and Psd2p (PtdSer decarboxylase 2 encoded by the PSD2 gene that complements psd2 mutations) in the Golgi. Decarboxylation of PtdSer to PtdEtn (phosphatidylethanolamine) can be used as a biochemical indicator of transport to these organelles, which is regulated by specific lipid and protein motifs. PtdSer transport to mitochondria is controlled by ubiquitination via the action of the ubiquitin ligase subunit Met30p (a ubiquitin ligase subunit encoded by the MET30 gene that complements the met30 mutation affecting methionine biosynthesis). Mutant strains with lesions in the MET30 gene are defective in PtdSer transport and show altered ubiquitination of specific target proteins, such as the transcription factor Met4p (a transcription factor encoded by the MET4 gene that complements the met4 mutation affecting methionine biosynthesis). Mutations to MET30 cause defects in both the MAM as a donor of PtdSer, and the mitochondria as an acceptor of PtdSer in the transport reaction. PtdSer transport to the locus of Psd2p is controlled by specific protein and lipid motifs. The C2 (Ca2+ and phospholipid-binding sequence) domain of Psd2p, and the lipid-binding protein PstB2p (PtdSer transport B pathway protein encoded by the PSTB2 gene that complements the pstB2 mutation affecting PtdSer transport), must be present on acceptor membranes for PtdSer transport to occur. In addition, the action of the PtdIns 4-kinase, Stt4p (PtdIns 4-kinase encoded by the STT4 gene that complements the stt4 mutation causing staurosporine and temperature-sensitive growth) is also required for PtdSer transport to the locus of Psd2p. Reconstitution of PtdSer transport to Psd2p using liposomes demonstrates that PtdSer-rich domains present in vesicles are preferred substrates for transport. In addition, the incorporation of phosphatidic acid into donor membranes enhances the rate of PtdSer transport. Collectively, these data support a model for PtdSer transport in which specific proteins and lipids are required on donor and acceptor membranes.

Compartmentalization of Lipid Biosynthesis in Mycobacteria

The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.

Genetic analysis of intracellular aminoglycerophospholipid traffic.

Inter- and intramembrane phospholipid transport processes are central features of membrane biogenesis and homeostasis. Relatively recent successes in the molecular genetic analysis of aminoglycerophospholipid transport processes in both yeast and mammalian cells are now providing important new information defining specific protein and lipid components that participate in these reactions. Studies focused on phosphatidylserine (PtdSer) transport to the mitochondria reveal that the process is regulated by ubiquitination. In addition, a specific mutation disrupts PtdSer transport between mitochondrial membranes. Analysis of PtdSer transport from the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 demonstrates the requirement for a phosphatidylinositol-4-kinase, a phosphatidylinositol-binding protein, and the C2 domain of the decarboxylase. Examination of NBD-phosphatidylcholine transport demonstrates the involvement of the prevacuolar compartment and a requirement for multiple genes involved in regulating vacuolar protein sorting for transport of the lipid to the vacuole. In intramembrane transport, multiple genes are now identified including those encoding multidrug resistant protein family members, DNF family members, ATP binding cassette transporters, and pleiotropic drug resistance family members. The scramblase family constitutes a collection of putative transmembrane transporters that function in an ATP-independent manner. The genetic analysis of lipid traffic is uncovering new molecules involved in all aspects of the regulation and execution of the transport steps and also providing essential tools to critically test the involvement of numerous candidate molecules.

Reconstitution of Phosphatidylserine Transport from Chemically Defined Donor Membranes to Phosphatidylserine Decarboxylase 2 Implicates Specific Lipid Domains in the Process

Phosphatidylserine (PtdSer) is transported from its site of synthesis in the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 (Psd2p) in the Golgi/vacuole and decarboxylated to form phosphatidylethanolamine. Recent biochemical and genetic evidence has implicated the C2 domain of Psd2p and a membrane-bound form of the phosphatidylinositol binding/transfer protein, PstB2p, as essential for this transport process. We devised a reconstituted system in which chemically defined donor membranes function to transfer PtdSer to the biological acceptor membranes containing Psd2p. The transfer of PtdSer is poor when the donor membranes have a high degree of curvature but markedly enhanced when the membranes are relatively planar (> or =400-nm diameter). PtdSer transfer is also dependent upon both the bulk and the surface concentrations of the lipid, with pure PtdSer vesicles acting as the most efficient donors at all concentrations. The lipid transfer from donor membranes containing either 100% PtdSer or 50% PtdSer at a fixed concentration (e.g. 250 microM PtdSer) differs by a factor of 20. Surface dilution of PtdSer by choline, ethanolamine, glycerol, and inositol phospholipids markedly inhibits PtdSer transfer, whereas phosphatidic acid (PtdOH) stimulates the transfer. Most importantly, the transfer of PtdSer from liposomes to Psd2p fails to occur in acceptor membranes from strains lacking PstB2p or the C2 domain of Psd2p. These data support a model for PtdSer transport from planar domains highly enriched in PtdSer or in PtdSer plus PtdOH.

Evidence from engineering that decarboxylation of free serine is the major source of ethanolamine moieties in plants.

Plants form ethanolamine (Etn) moieties by decarboxylating serine or phosphatidylserine (PtdSer), and use them to make phosphatidylethanolamine, phosphatidylcholine, choline, and glycine betaine. Serine decarboxylation is mediated by a serine decarboxylase (SDC) that is unique to plants and has a characteristic N-terminal extension. This extension was shown to have little influence on function of the enzyme in vitro. To explore the importance of SDC and its extension in vivo, native or truncated versions of the Arabidopsis enzyme were expressed in tobacco. Transgene expression increased SDC activity by up to 10-fold and free Etn level up to 6-fold, but did not change levels of serine, choline, phosphocholine, or phosphatidyl bases. The truncated enzyme gave significantly higher Etn levels. These results show that SDC activity exerts substantial control over flux to Etn, and suggest that the enzyme's N-terminus may have a regulatory role. In complementary studies with Arabidopsis, we showed that a mutant with 9-fold elevated mitochondrial PtdSer decarboxylase activity had normal pools of serine, Etn, and Etn metabolites. Taken together, these data indicate that serine decarboxylation is the main source of Etn moieties in plants. The ability to enhance serine --> Etn flux should advance engineering of choline and glycine betaine accumulation.

Phosphatidylserine Transport to the Mitochondria Is Regulated by Ubiquitination

Mitochondrial membrane biogenesis requires the interorganelle transport of phospholipids. Phosphatidylserine (PtdSer) synthesized in the endoplasmic reticulum and related membranes (mitochondria-associated membrane (MAM)) is transported to the mitochondria by unknown gene products and decarboxylated to form phosphatidylethanolamine at the inner membrane by PtdSer decarboxylase 1 (Psd1p). We have designed a screen for strains defective in PtdSer transport (pstA mutants) between the endoplasmic reticulum and Psd1p that relies on isolating ethanolamine auxotrophs in suitable (psd2Delta) genetic backgrounds. Following chemical mutagenesis, we isolated an ethanolamine auxotroph that we designate pstA1-1. Using in vivo and in vitro phospholipid synthesis/transport measurements, we demonstrate that the pstA1-1 mutant is defective in PtdSer transport between the MAM and mitochondria. The gene that complements the growth defect and PtdSer transport defect of the pstA1-1 mutant is MET30, which encodes a substrate recognition subunit of the SCF (suppressor of kinetochore protein 1, cullin, F-box) ubiquitin ligase complex. Reconstitution of different permutations of MAM and mitochondria from wild type and pstA1-1 strains demonstrates that the MET30 gene product affects both organelles. These data provide compelling evidence that interorganelle PtdSer traffic is regulated by ubiquitination.

Enhancement of Transport-dependent Decarboxylation of Phosphatidylserine by S100B Protein in Permeabilized Chinese Hamster Ovary Cells

Phosphatidylethanolamine synthesis through the phosphatidylserine (PtdSer) decarboxylation pathway requires PtdSer transport from the endoplasmic reticulum or mitochondrial-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PtdSer decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence was identical to part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His(6)-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PtdSer decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PtdSer decarboxylase, the recombinant S100B protein did not stimulate PtdSer decarboxylase activity and exhibited no PtdSer decarboxylase activity. These results implicated the S100B protein in the transport of PtdSer to the mitochondrial inner membrane.

Characterization of Phosphatidylserine Transport to the Locus of Phosphatidylserine Decarboxylase 2 in Permeabilized Yeast

In yeast, nascent phosphatidylserine (PtdSer) can be transported to the mitochondria and Golgi/vacuole for decarboxylation to synthesize phosphatidylethanolamine (PtdEtn). In strains with a psd1Delta allele for the mitochondrial PtdSer decarboxylase, the conversion of nascent PtdSer to PtdEtn can serve as an indicator of lipid transport to the locus of PtdSer decarboxylase 2 (Psd2p) in the Golgi/vacuole. We have followed the metabolism of [(3)H]serine into PtdSer and PtdEtn to study lipid transport in permeabilized psd1Delta yeast. The permeabilized cells synthesize (3)H-PtdSer and, after a 20-min lag, decarboxylate it to form [(3)H]PtdEtn. Formation of [(3)H]PtdEtn is linear between 20 and 100 min of incubation and does not require ongoing PtdSer synthesis. PtdSer transport can be resolved into a two-component system using washed, permeabilized psd1Delta cells as donors and membranes isolated by ultracentrifugation as acceptors. With this system, the transport-dependent decarboxylation of nascent PtdSer is dependent upon the concentration of acceptor membranes, requires Mn(2+) but not nucleotides, and is inhibited by EDTA. High speed membranes isolated from a previously identified PtdSer transport mutant, pstB2, contain normal Psd2p activity but fail to reconstitute PtdSer transport and decarboxylation. Reconstitution with permutations of wild type and pstB2Delta donors and acceptors identifies the site of the mutant defect as the acceptor side of the transport reaction.

A New Gene Involved in the Transport-dependent Metabolism of Phosphatidylserine, PSTB2/PDR17, Shares Sequence Similarity with the Gene Encoding the Phosphatidylinositol/Phosphatidylcholine Transfer Protein,SEC14

A new yeast strain, designated pstB2, that is defective in the conversion of nascent phosphatidylserine (PtdSer) to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase 2, has been isolated. The pstB2 strain requires ethanolamine for growth. Incubation of cells with [(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrates a 50% increase in the labeling of PtdSer and a 72% decrease in PtdEtn formation in the mutant relative to the parental strain. The PSTB2 gene was isolated by complementation, and it restores ethanolamine prototrophy and corrects the defective lipid metabolism of the pstB2 strain. The PSTB2 gene is allelic to the pleiotropic drug resistance gene, PDR17, and is homologous to SEC14, which encodes a phosphatidylinositol/phosphatidylcholine transfer protein. The protein, PstB2p, displays phosphatidylinositol but not PtdSer transfer activity, and its overexpression causes suppression of sec14 mutants. However, overexpression of the SEC14 gene fails to suppress the conditional lethality of pstB2 strains. The transport-dependent metabolism of PtdSer to PtdEtn occurs in permeabilized wild type yeast but is dramatically reduced in permeabilized pstB2 strains. Fractionation of permeabilized cells demonstrates that the pstB2 strain accumulates nascent PtdSer in the Golgi apparatus and a novel light membrane fraction, consistent with a defect in lipid transport processes that control substrate access to PtdSer decarboxylase 2.

Alterations by perfluorooctanoic acid of glycerolipid metabolism in rat liver

The effects of perfluorooctanoic acid (PFOA) feeding on hepatic levels of glycerolipids and the underlying mechanism were investigated. Feeding of rats with 0.01% of PFOA in the diet for 1 week caused an increase in the contents of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and triglyceride (TG), which were 2.2, 2.4, 2.4, 1.6 and 5.2 times over control, respectively, on the basis of whole liver. The activities of glycerol-3-phosphate acyltransferase, diacylglycerol kinase and PtdSer decarboxylase were significantly increased upon PFOA feeding, whereas the activities of CTP:phosphoethanolamine cytidylyltransferase and PtdEtn N-methyltransferase were decreased. On the other hand, the activity of CTP:phosphocholine cytidylyltransferase was not increased by PFOA. Upon PFOA feeding, hepatic level of 16:0–18:1 PtdCho was markedly increased and, by contrast, the levels of molecular species of PtdCho which contain 18:2 were decreased, resulting in the reduced concentration of molecular species of serum PtdCho containing 18:2. The increase in the level of hepatic 16:0–18:1 PtdCho seemed to be due to 3-fold increase in the activities of both Δ9 desaturase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase. The mechanism by which PFOA causes the accumulation of glycerolipids in liver was discussed.

A Genetic Screen for Aminophospholipid Transport Mutants Identifies the Phosphatidylinositol 4-Kinase, Stt4p, as an Essential Component in Phosphatidylserine Metabolism

In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae. A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed. We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity. The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport. A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase. The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant. Creation of an STT4 null allele (stt4Delta::HIS3) demonstrates the gene is essential. These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.