New perspectives on the regulation of intermembrane glycerophospholipid traffic

In eukaryotes, phosphatidylserine (PtdSer) can serve as a precursor of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho), which are the major cellular phospholipids. PtdSer synthesis originates in the endoplasmic reticulum (ER) and its subdomain named the mitochondria-associated membrane (MAM). PtdSer is transported to the mitochondria in mammalian cells and yeast, and decarboxylated by PtdSer decarboxylase 1 (Psd1p) to form PtdEtn. A second decarboxylase, Psd2p, is also found in yeast in the Golgi-vacuole. PtdEtn produced by Psd1p and Psd2p can be transported to the ER, where it is methylated to form PtdCho. Organelle-specific metabolism of the aminoglycerophospholipids is a powerful tool for experimentally following lipid traffic that is now enabling identification of new proteins involved in the regulation of this process. Genetic and biochemical experiments demonstrate that transport of PtdSer between the MAM and mitochondria is regulated by protein ubiquitination, which affects events at both membranes. Similar analyses of PtdSer transport to the locus of Psd2p now indicate that a membrane-bound phosphatidylinositol transfer protein and the C2 domain of Psd2p are both required on the acceptor membrane for efficient transport of PtdSer. Collectively, these recent findings indicate that novel multiprotein assemblies on both donor and acceptor membranes participate in interorganelle phospholipid transport.     

Interorganelle transport of aminoglycerophospholipids

The aminoglycerophospholipids of eukaryotic cells, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), can be synthesized by multiple pathways. The PtdSer pathway encompasses the synthesis of PtdSer, its decarboxylation to PtdEtn and subsequent methylation reactions to form PtdCho. The Kennedy pathways consist of the synthesis of PtdEtn and PtdCho from Etn and Cho precursors via CDP-Etn and CDP-Cho intermediates. The reactions along the PtdSer pathway are spatially segregated with PtdSer synthesis occurring in the endoplasmic reticulum or mitochondria-associated membrane (MAM), PtdEtn formation occurring in the mitochondria and Golgi/vacuole compartments and PtdCho formation occurring in the endoplasmic reticulum or MAM. The organelle-specific metabolism of the different lipids in the PtdSer pathway has provided a convenient biochemical means for defining events in the interorganelle transport of the aminoglycerophospholipids in intact cells, isolated organelles and permeabilized cells. Studies with both mammalian cells and yeast demonstrate many significant similarities in lipid transport processes between the two systems. Genetic experiments in yeast now provide the tools to create new strains with mutations along the PtdSer pathway that can be conditionally rescued by the Kennedy pathway reactions. The genetic studies in yeast indicate that it is now possible to begin to define genes that participate in the interorganelle transport of the aminoglycerophospholipids.     

Molecular cloning of canine bullous pemphigoid antigen 2 cDNA and immunomapping of NC16A domain by canine bullous pemphigoid autoantibodies

The aminoglycerophospholipids of eukaryotic cells, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), can be synthesized by multiple pathways. The PtdSer pathway encompasses the synthesis of PtdSer, its decarboxylation to PtdEtn and subsequent methylation reactions to form PtdCho. The Kennedy pathways consist of the synthesis of PtdEtn and PtdCho from Etn and Cho precursors via CDP-Etn and CDP-Cho intermediates. The reactions along the PtdSer pathway are spatially segregated with PtdSer synthesis occurring in the endoplasmic reticulum or mitochondria-associated membrane (MAM), PtdEtn formation occurring in the mitochondria and Golgi/vacuole compartments and PtdCho formation occurring in the endoplasmic reticulum or MAM. The organelle-specific metabolism of the different lipids in the PtdSer pathway has provided a convenient biochemical means for defining events in the interorganelle transport of the aminoglycerophospholipids in intact cells, isolated organelles and permeabilized cells. Studies with both mammalian cells and yeast demonstrate many significant similarities in lipid transport processes between the two systems. Genetic experiments in yeast now provide the tools to create new strains with mutations along the PtdSer pathway that can be conditionally rescued by the Kennedy pathway reactions. The genetic studies in yeast indicate that it is now possible to begin to define genes that participate in the interorganelle transport of the aminoglycerophospholipids.     

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.     

Transport of Phosphatidylserine from the Endoplasmic Reticulum to the Site of Phosphatidylserine Decarboxylase2 in Yeast

Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non‐vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport‐dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p‐specific transport pathway is one in which the enzyme and its non‐catalytic N‐terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes.      

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.     

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.     

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.     

Phosphatidylethanolamine synthesized by three different pathways is supplied to peroxisomes of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae three pathways lead to the formation of phosphatidylethanolamine (PE), namely decarboxylation of phosphatidylserine (PS) (i) by Psd1p in mitochondria, and (ii) by Psd2p in a Golgi/vacuolar compartment; and (iii) synthesis via CDP–ethanolamine pathway in the endoplasmic reticulum. To determine the contribution of these pathways to the supply of PE to peroxisomes, we subjected mutants bearing defects in the respective metabolic routes to biochemical and cell biological analysis. Despite these defects in PE formation mutants were able to grow on oleic acid indicating induction of peroxisome proliferation. Biochemical analysis revealed that PE formed through all three pathways was supplied to peroxisomes. These analyses also demonstrated that selective as well as equilibrium interorganelle flux of PE appear to be equally important for cellular homeostasis of this phospholipid. Electron microscopic inspection confirmed that defects in PE synthesis still allowed formation of peroxisomes, although these organelles from strains lacking PSD1 were significantly smaller than wild type. The fact that peroxisomes were always found in close vicinity to mitochondria, ER and lipid particles supported the view that membrane contact may play a role in lipid traffic between these organelles.     

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[³H(G)]serine and [¹⁴C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of “correct” PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.     

Contribution of different biosynthetic pathways to species selectivity of aminoglycerophospholipids assembled into mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, three pathways lead to the formation of cellular phosphatidylethanolamine (PtdEtn), namely the mitochondrial conversion of phosphatidylserine (PtdSer) to PtdEtn catalyzed by phosphatidylserine decarboxylase 1 (Psd1p), the equivalent reaction catalyzed by phosphatidylserine decarboxylase 2 (Psd2p) in the Golgi, and the CDP-ethanolamine branch of the so-called Kennedy pathway which is located to the microsomal fraction. To investigate the contributions of these three pathways to the cellular pattern of PtdEtn species (fatty acid composition) we subjected lipids of wild-type and yeast mutant strains with distinct defects in the respective pathways to mass spectrometric analysis. We also analyzed species of PtdSer and phosphatidylcholine (PtdCho) of these strains because formation of the three aminoglycerophospholipids is linked through their biosynthetic route. We demonstrate that all three pathways involved in PtdEtn synthesis exhibit a preference for the formation of C34:2 and C32:2 species resulting in a high degree of unsaturation in total cellular PtdEtn. In PtdSer, the ratio of unsaturated to saturated fatty acids is much lower than in PtdEtn, suggesting a high species selectivity of PtdSer decarboxylases. Finally, PtdCho is characterized by its higher ratio of C16 to C18 fatty acids compared to PtdSer and PtdEtn. In contrast to biosynthetic steps, import of all three aminoglycerophospholipids into mitochondria of wild-type and mutant cells is not highly specific with respect to species transported. Thus, the species pattern of aminoglycerophospholipids in mitochondria is mainly the result of enzyme specificities, but not of translocation processes involved. Our results support a model that suggests equilibrium transport of aminoglycerophospholipids between mitochondria and microsomes based on membrane contact between the two compartments.     

Biosynthetic regulation and intracellular transport of phosphatidylserine in mammalian cells

In mammalian cells, phosphatidylserine (PtdSer) is synthesized through the action of the endoplasmic reticulum enzymes, PtdSer synthase 1 and 2, and the decarboxylation of PtdSer accounts for the majority of phosphatidylethanolamine (PtdEtn) synthesis. PtdSer decarboxylation for PtdEtn formation occurs in the mitochondria. In addition, the transport of PtdSer from the endoplasmic reticulum to the mitochondria is probably a rate limiting step for PtdEtn synthesis through the decarboxylation pathway. Therefore, the regulation of PtdSer synthesis and its intracellular transport appear to be essential events for the maintenance of normal cellular PtdSer and PtdEtn levels. Here we describe the current understanding of the regulation of PtdSer biosynthesis and the transport of PtdSer from the ER to the mitochondria in mammalian cells.     

Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in Saccharomyces cerevisiae

To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.     

The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function

Phosphatidylserine decarboxylase 2 (Psd2p) is currently being used to study lipid trafficking processes in intact and permeabilized yeast cells. The Psd2p contains a C2 homology domain and a putative Golgi retention/localization (GR) domain. C2 domains play important functions in membrane binding and docking reactions involving phospholipids and proteins. We constructed a C2 domain deletion variant (C2Delta) and a GR deletion variant (GRDelta) of Psd2p and examined their effects on in vivo function and catalysis. Immunoblotting confirmed that the predicted immature and mature forms of Psd2(C2Delta)p, Psd2(GRDelta)p, and wild type Psd2p were produced in vivo and that the proteins localized normally. Enzymology revealed that the Psd2(C2Delta)p and Psd2(GRDelta)p were catalytically active and could readily be expressed at levels 10-fold higher than endogenous Psd2p. Both Psd2p and Psd2(GRDelta)p expression complemented the growth defect of psd1Deltapsd2Delta strains and resulted in normal aminoglycerophospholipid metabolism. In contrast, the Psd2(C2Delta)p failed to complement psd1Deltapsd2Delta strains, and [(3)H]serine labeling revealed a severe defect in the formation of PtdEtn in both intact and permeabilized cells, indicative of disruption of lipid trafficking. These findings identify an essential, non-catalytic function of the C2 domain of Psd2p and raise the possibility that it plays a direct role in membrane docking and/or PtdSer transport to the enzyme.     

Contribution of different pathways to the supply of phosphatidylethanolamine and phosphatidylcholine to mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast, three biosynthetic pathways lead to the formation of phosphatidylethanolamine (PtdEtn): (i) decarboxylation of phosphatidylserine (PtdSer) by phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria; (ii) decarboxylation of PtdSer by Psd2p in a Golgi/vacuolar compartment; and (iii) the CDP-ethanolamine (CDP-Etn) branch of the Kennedy pathway. The major phospholipid of the yeast, phosphatidylcholine (PtdCho), is formed either by methylation of PtdEtn or via the CDP-choline branch of the Kennedy pathway. To study the contribution of these pathways to the supply of PtdEtn and PtdCho to mitochondrial membranes, labeling experiments in vivo with [(3)H]serine and [(14)C]ethanolamine, or with [(3)H]serine and [(14)C]choline, respectively, and subsequent cell fractionation were performed with psd1Delta and psd2Delta mutants. As shown by comparison of the labeling patterns of the different strains, the major source of cellular and mitochondrial PtdEtn is Psd1p. PtdEtn formed by Psd2p or the CDP-Etn pathway, however, can be imported into mitochondria, although with moderate efficiency. In contrast to mitochondria, microsomal PtdEtn is mainly derived from the CDP-Etn pathway. PtdEtn formed by Psd2p is the preferred substrate for PtdCho synthesis. PtdCho derived from the different pathways appears to be supplied to subcellular membranes from a single PtdCho pool. Thus, the different pathways of PtdEtn biosynthesis play different roles in the assembly of PtdEtn into cellular membranes.     

Deficiency in ethanolamine plasmalogen leads to altered cholesterol transport

Plasmalogens are a major sub-class of ethanolamine and choline phospholipids in which the sn-1 position has a long chain fatty alcohol attached through a vinyl ether bond. These phospholipids are proposed to play a role in membrane fusion-mediated events. In this study, we investigated the role of the ethanolamine plasmalogen plasmenylethanolamine (PlsEtn) in intracellular cholesterol transport in Chinese hamster ovary cell mutants NRel-4 and NZel-1, which have single gene defects in PlsEtn biosynthesis. We found that PlsEtn was essential for specific cholesterol transport pathways, those from the cell surface or endocytic compartments to acyl-CoA/cholesterol acyltransferase in the endoplasmic reticulum. The movement of cholesterol from the endoplasmic reticulum or endocytic compartments to the cell surface was normal in PlsEtn-deficient cells. Also, vesicle trafficking was normal in PlsEtn-deficient cells, as measured by fluid phase endocytosis and exocytosis, as was the movement of newly-synthesized proteins to the cell surface. The mutant cholesterol transport phenotype was due to the lack of PlsEtn, since it was corrected when NRel-4 cells were transfected with a cDNA encoding the missing enzyme or supplied with a metabolic intermediate that enters the PlsEtn biosynthetic pathway downstream of the defect. Future work must determine the precise role that plasmalogens have on cholesterol transport to the endoplasmic reticulum.     

Newly made phosphatidylserine and phosphatidylethanolamine are preferentially translocated between rat liver mitochondria and endoplasmic reticulum

The translocation of: (i) phosphatidylserine (PtdSer) from its site of synthesis on microsomal membranes to its site decarboxylation in mitochondrial membranes and (ii) phosphatidylethanolamine (PtdEtn) from the mitochondria to its site of methylation to phosphatidylcholine on microsomal membranes has been reconstituted in cell-free systems consisting of rat liver mitochondria and microsomes. Two types of systems have been reconstituted. In one, the translocation of newly made PtdSer or PtdEtn was examined by incubation of microsomes and mitochondria with [3-3H]serine. In the other, membranes were prelabeled with radioactive PtdSer or PtdEtn, and the transfer of these two lipids between mitochondria and microsomes was monitored. For the transfer of both PtdSer from microsomes to mitochondria and PtdEtn from mitochondria to microsomes, newly made phospholipids were translocated much more readily than pre-existing phospholipids. The data suggest that with respect to their translocation between these two organelles, the pools of newly synthesized PtdSer and PtdEtn were distinct from the pools of "older" phospholipids pre-existing in the membranes. Transfer of neither phospholipid in vitro depended on the presence of cytosolic proteins (i.e. soluble phospholipid transfer proteins) or on the hydrolysis of ATP, although there was some stimulation of PtdSer transfer by ATP and several other nucleoside mono-, di-, and triphosphates. The data are consistent with a collision-based mechanism in which the endoplasmic reticulum and mitochondria come into contact with one another, thereby effecting the transfer of phospholipids. The proposal that there is contact between the endoplasmic reticulum and mitochondria is supported by the recent isolation of a membrane fraction having many, but not all, of the properties of the endoplasmic reticulum, but which was isolated in association with mitochondria (Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256).     

Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris

Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris , which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae . Using P. pastoris psd1 Δ and psd2 Δ mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)–ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris , which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.     

Phosphatidylethanolamine Biosynthesis in Mitochondria: PHOSPHATIDYLSERINE (PS) TRAFFICKING IS INDEPENDENT OF A PS DECARBOXYLASE AND INTERMEMBRANE SPACE PROTEINS UPS1P AND UPS2P*

Phosphatidylethanolamine (PE) plays important roles for the structure and function of mitochondria and other intracellular organelles. In yeast, the majority of PE is produced from phosphatidylserine (PS) by a mitochondrion-located PS decarboxylase, Psd1p. Because PS is synthesized in the endoplasmic reticulum (ER), PS is transported from the ER to mitochondria and converted to PE. After its synthesis, a portion of PE moves back to the ER. Two mitochondrial proteins located in the intermembrane space, Ups1p and Ups2p, have been shown to regulate PE metabolism by controlling the export of PE. It remains to be determined where PS is decarboxylated in mitochondria and whether decarboxylation is coupled to trafficking of PS. Here, using fluorescent PS as a substrate in an in vitro assay for Psd1p-dependent PE production in isolated mitochondria, we show that PS is transferred from the mitochondrial outer membrane to the inner membrane independently of Psd1p, Ups1p, and Ups2p and decarboxylated to PE by Psd1p in the inner membrane. Interestingly, Ups1p is required for the maintenance of Psd1p and therefore PE production. Restoration of Psd1p levels rescued PE production defects in ups1Δ mitochondria. Our data provide novel mechanistic insight into PE biogenesis in mitochondria.     

The ADP-ribosylation factor GTPase-activating protein Glo3p is involved in ER retrieval.

Retrograde transport of proteins from the Golgi to the endoplasmic reticulum (ER) has been the subject of some interest in the recent past. Here a new thermosensitive yeast mutant defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum was characterized. The ret4-1 mutant also exhibited a selective defect in forward ER-to-Golgi transport of some secreted proteins at the non-permissive temperature. The corresponding RET4 gene was found to encode Glo3p, a GTPase-activating protein (GAP) specific for ADP-ribosylation factor (ARF). In vitro, the Glo3 thermosensitive mutant showed a reduced ARF1-GAP activity. The Glo3 protein belongs to a family of zinc finger proteins that may include additional ARF-GAPs. Gene deletion experiments of other family members showed that only GLO3 deletion resulted in impaired retrieval of dilysine-tagged proteins back to the ER. These results demonstrate that Glo3p is the main ARF-GAP specifically involved in ER retrieval.