Topogenesis and Homeostasis of Fatty Acyl-CoA Reductase 1

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.     

A Novel Pex14 Protein-interacting Site of Human Pex5 Is Critical for Matrix Protein Import into Peroxisomes

Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14. A systematic screening of a Pex5 peptide library by ligand blot analysis revealed a novel Pex5-Pex14 interaction site of Pex5. The novel motif composes the sequence LVAEF with the evolutionarily conserved consensus sequence LVXEF. Replacement of the amino acid LVAEF sequence by alanines strongly affects matrix protein import into peroxisomes in vivo. The NMR structure of a complex of Pex5-(57–71) with the Pex14-N-terminal domain showed that the novel motif binds in a similar α-helical orientation as the WXXX(F/Y) motif but that the tryptophan pocket is now occupied by a leucine residue. Surface plasmon resonance analyses revealed 33 times faster dissociation rates for the LVXEF ligand when compared with a WXXX(F/Y) motif. Surprisingly, substitution of the novel motif with the higher affinity WXXX(F/Y) motif impairs protein import into peroxisomes. These data indicate that the distinct kinetic properties of the novel Pex14-binding site in Pex5 are important for processing of the peroxisomal targeting signal 1 receptor at the peroxisomal membrane. The novel Pex14-binding site may represent the initial tethering site of Pex5 from which the cargo-loaded receptor is further processed in a sequential manner.     

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.     

Dual Functions of the Trans-2-Enoyl-CoA Reductase TER in the Sphingosine 1-Phosphate Metabolic Pathway and in Fatty Acid Elongation

The sphingolipid metabolite sphingosine 1-phosphate (S1P) functions as a lipid mediator and as a key intermediate of the sole sphingolipid to glycerophospholipid metabolic pathway (S1P metabolic pathway). In this pathway, S1P is converted to palmitoyl-CoA through 4 reactions, then incorporated mainly into glycerophospholipids. Although most of the genes responsible for the S1P metabolic pathway have been identified, the gene encoding the trans-2-enoyl-CoA reductase, responsible for the saturation step (conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA) remains unidentified. In the present study, we show that TER is the missing gene in mammals using analyses involving yeast cells, deleting the TER homolog TSC13, and TER-knockdown HeLa cells. TER is known to be involved in the production of very long-chain fatty acids (VLCFAs). A significant proportion of the saturated and monounsaturated VLCFAs are used for sphingolipid synthesis. Therefore, TER is involved in both the production of VLCFAs used in the fatty acid moiety of sphingolipids as well as in the degradation of the sphingosine moiety of sphingolipids via S1P.     

The TOR Complex 1 Is Distributed in Endosomes and in Retrograde Vesicles That Form from the Vacuole Membrane and Plays an Important Role in the Vacuole Import and Degradation Pathway

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.     

The TGL2 Gene of Saccharomyces cerevisiae Encodes an Active Acylglycerol Lipase Located in the Mitochondria

The Saccharomyces cerevisiae Tgl2 protein shows sequence homology to Pseudomonas triacylglycerol (TAG) lipases, but its role in the yeast lipid metabolism is not known. Using hemagglutinin-tagged Tgl2p purified from yeast, we report that this protein carries a significant lipolytic activity toward long-chain TAG. Importantly, mutant hemagglutinin-Tgl2p^S144A, which contains alanine 144 in place of serine 144 in the lipase consensus sequence (G/A)XSXG exhibits no such activity. Although cellular TAG hydrolysis is reduced in the tgl2 deletion mutant, overproduction of Tgl2p in this mutant leads to an increase in TAG degradation in the presence of fatty acid synthesis inhibitor cerulenin, but that of Tgl2p^S144A does not. This result demonstrates the lipolytic function of Tgl2p in yeast. Although other yeast TAG lipases are localized to lipid particles, Tgl2p is enriched in the mitochondria. The mitochondrial fraction purified from the TGL2-overexpressing yeast shows a strong lipolytic activity, which was absent in the tgl2 deletion mutant. Therefore, we conclude that Tgl2p is a functional lipase of the yeast mitochondria. By analyzing phenotypic effects of TGL2-deficient yeast, we also find that lipolysis-competent Tgl2p is required for the viability of cells treated with antimitotic drug. The addition of oleic acid, the product of Tgl2p-catalyzed lipolysis, fully complements the antimitotic drug sensitivity of the tgl2 null mutation. Thus, we propose that the mitochondrial Tgl2p-dependent lipolysis is crucial for the survival of cells under antimitotic drug treatment.     

Regulation of Phagocytosis in Macrophages by Neuraminidase 1

The differentiation of monocytes into macrophages and dendritic cells is accompanied by induction of cell-surface neuraminidase 1 (Neu1) and cathepsin A (CathA), the latter forming a complex with and activating Neu1. To clarify the biological importance of this phenomenon we have developed the gene-targeted mouse models of a CathA deficiency (CathA^S190A) and a double CathA/Neu1 deficiency (CathA^S190A-Neo). Macrophages of CathA^S190A-Neo mice and their immature dendritic cells showed a significantly reduced capacity to engulf Gram-positive and Gram-negative bacteria and positively and negatively charged polymer beads as well as IgG-opsonized beads and erythrocytes. Properties of the cells derived from CathA^S190A mice were indistinguishable from those of wild-type controls, suggesting that the absence of Neu1, which results in the increased sialylation of the cell surface proteins, probably affects multiple receptors for phagocytosis. Indeed, treatment of the cells with purified mouse Neu1 reduced surface sialylation and restored phagocytosis. Because Neu1-deficient cells showed reduced internalization of IgG-opsonized sheep erythrocytes whereas binding of the erythrocytes to the cells at 4 °C persisted, we speculate that the absence of Neu1 in particular affected transduction of signals from the Fc receptors for immunoglobulin G (FcγR). Indeed the macrophages from the Neu1-deficient mice showed increased sialylation and impaired phosphorylation of FcγR as well as markedly reduced phosphorylation of Syk kinase in response to treatment with IgG-opsonized beads. Altogether our data suggest that the cell surface Neu1 activates the phagocytosis in macrophages and dendritic cells through desialylation of surface receptors, thus, contributing to their functional integrity.     

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD.     

Filling the Gap, Evolutionarily Conserved Omp85 in Plastids of Chromalveolates

Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.     

Substrate Specificity, Membrane Topology, and Activity Regulation of Human Alkaline Ceramidase 2 (ACER2)

Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Δypc1Δydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca²⁺ for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2ΔN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2ΔN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2ΔN13, but not ACER2ΔN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2ΔN13 or ACER2ΔN36 inhibited the glycosylation of integrin β1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca²⁺ for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.     

The Conformational Transition Pathway of ATP Binding Cassette Transporter MsbA Revealed by Atomistic Simulations

ATP binding cassette transporters are integral membrane proteins that use the energy released from ATP hydrolysis at the two nucleotide binding domains (NBDs) to translocate a wide variety of substrates through a channel at the two transmembrane domains (TMDs) across the cell membranes. MsbA from Gram-negative bacteria is a lipid and multidrug resistance ATP binding cassette exporter that can undergo large scale conformational changes between the outward-facing and the inward-facing conformations revealed by crystal structures in different states. Here, we use targeted molecular dynamics simulation methods to explore the atomic details of the conformational transition from the outward-facing to the inward-facing states of MsbA. The molecular dynamics trajectories revealed a clear spatiotemporal order of the conformational movements. The disruption of the nucleotide binding sites at the NBD dimer interface is the very first event that initiates the following conformational changes, verifying the assumption that the conformational conversion is triggered by ATP hydrolysis. The conserved x-loops of the NBDs were identified to participate in the interaction network that stabilizes the cytoplasmic tetrahelix bundle of the TMDs and play an important role in mediating the cross-talk between the NBD and TMD. The movement of the NBD dimer is transmitted through x-loops to break the tetrahelix bundle, inducing the packing rearrangements of the transmembrane helices at the cytoplasmic side and the periplasmic side sequentially. The packing rearrangement within each periplasmic wing of TMD that results in exposure of the substrate binding sites occurred at the end stage of the trajectory, preventing the wrong timing of the binding site accessibility.     

Isoform-specific and Protein Kinase C-mediated Regulation of CTP:Phosphoethanolamine Cytidylyltransferase Phosphorylation

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -α and -β; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50–90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI, and PKCβII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -β-phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation.     

N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol

Cholera toxin travels from the plasma membrane to the endoplasmic reticulum of host cells, where a portion of the toxin, the A1-chain, is unfolded and targeted to a protein-conducting channel for retrotranslocation to the cytosol. Unlike most retrotranslocation substrates, the A1-chain escapes degradation by the proteasome and refolds in the cytosol to induce disease. How this occurs remains poorly understood. Here, we show that an unstructured peptide appended to the N terminus of the A1-chain renders the toxin functionally inactive. Cleavage of the peptide extension prior to cell entry rescues toxin half-life and function. The loss of toxicity is explained by rapid degradation by the proteasome after retrotranslocation to the cytosol. Degradation of the mutant toxin does not follow the N-end rule but depends on the two Lys residues at positions 4 and 17 of the native A1-chain, consistent with polyubiquitination at these sites. Thus, retrotranslocation and refolding of the wild-type A1-chain must proceed in a way that protects these Lys residues from attack by E3 ligases.     

An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins: APPLICATION TO CLASS I MYOSINS, PAKS, AND CARMIL

Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro.     

Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation

Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and by-products of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels.     

LPS impairs phospholipid synthesis by triggering beta-transducin repeat-containing protein (beta-TrCP)-mediated polyubiquitination and degradation of the surfactant enzyme acyl-CoA:lysophosphatidylcholine acyltransferase I (LPCAT1)

Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, β-transducin repeat-containing protein (β-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser(178) within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3β (GSK-3β) phosphorylation that recruits β-TrCP docking within the enzyme. β-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys(221)), as substitution of Lys(221) with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to β-TrCP and GSK-3β or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3β-mediated phosphorylation within a canonical phosphodegron for β-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for β-TrCP, and the results suggest that modulation of the GSK-3β-SCFβ(TrCP) E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis.     

Protein Kinase A-mediated Phosphorylation of Pah1p Phosphatidate Phosphatase Functions in Conjunction with the Pho85p-Pho80p and Cdc28p-Cyclin B Kinases to Regulate Lipid Synthesis in Yeast

Pah1p, which functions as phosphatidate phosphatase (PAP) in the yeast Saccharomyces cerevisiae, plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The diacylglycerol produced by PAP is used for the synthesis of triacylglycerol as well as for the synthesis of phospholipids via the Kennedy pathway. Pah1p is a highly phosphorylated protein in vivo and has been previously shown to be phosphorylated by the protein kinases Pho85p-Pho80p and Cdc28p-cyclin B. In this work, we showed that Pah1p was a bona fide substrate for protein kinase A, and we identified by mass spectrometry and mutagenesis that Ser-10, Ser-677, Ser-773, Ser-774, and Ser-788 were the target sites of phosphorylation. Protein kinase A-mediated phosphorylation of Pah1p inhibited its PAP activity by decreasing catalytic efficiency, and the inhibitory effect was primarily conferred by phosphorylation at Ser-10. Analysis of the S10A and S10D mutations (mimicking dephosphorylation and phosphorylation, respectively), alone or in combination with the seven alanine (7A) mutations of the sites phosphorylated by Pho85p-Pho80p and Cdc28p-cyclin B, indicated that phosphorylation at Ser-10 stabilized Pah1p abundance and inhibited its association with membranes, PAP activity, and triacylglycerol synthesis. The S10A mutation enhanced the physiological effects imparted by the 7A mutations, whereas the S10D mutations attenuated the effects of the 7A mutations. These data indicated that the protein kinase A-mediated phosphorylation of Ser-10 functions in conjunction with the phosphorylations mediated by Pho85p-Pho80p and Cdc28p-cyclin B and that phospho-Ser-10 should be dephosphorylated for proper PAP function.     

The Bifunctional Protein TtFARAT from Tetrahymena thermophila Catalyzes the Formation of both Precursors Required to Initiate Ether Lipid Biosynthesis

The biosynthesis of ether lipids and wax esters requires as precursors fatty alcohols, which are synthesized by fatty acyl reductases (FARs). The presence of ether glycerolipids as well as branched wax esters has been reported in several free-living ciliate protozoa. In the genome of Tetrahymena thermophila, the only ORF sharing similarities with FARs is fused to an acyltransferase-like domain, whereas, in most other organisms, FARs are monofunctional proteins of similar size and domain structure. Here, we used heterologous expression in plant and yeast to functionally characterize the activities catalyzed by this protozoan protein. Transient expression in tobacco epidermis of a truncated form fused to the green fluorescence protein followed by confocal microscopy analysis suggested peroxisomal localization. In vivo approaches conducted in yeast indicated that the N-terminal FAR-like domain produced both 16:0 and 18:0 fatty alcohols, whereas the C-terminal acyltransferase-like domain was able to rescue the lethal phenotype of the yeast double mutant gat1Δ gat2Δ. Using in vitro approaches, we further demonstrated that this domain is a dihydroxyacetone phosphate acyltransferase that uses preferentially 16:0-coenzyme A as an acyl donor. Finally, coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway. Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis.