The wide world of non-mammalian phospholipase D enzymes.

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

Phospholipid metabolism and membrane dynamics.

Genetic and biochemical approaches are shedding new light on the distinct physiological functions of specific phospholipid metabolic pathways and the mechanisms by which phospholipids are mobilized between intracellular compartments. In particular, phosphatidylinositol-transfer proteins have recently been revealed to play fascinating and unanticipated roles in the coordination of phospholipid metabolism with vesicle-trafficking and signal-transducing reactions.

Mammalian phospholipase D: Function, and therapeutics.

Despite being discovered over 60 years ago, the precise role of phospholipase D (PLD) is still being elucidated. PLD enzymes catalyze the hydrolysis of the phosphodiester bond of glycerophospholipids producing phosphatidic acid and the free headgroup. PLD family members are found in organisms ranging from viruses, and bacteria to plants, and mammals. They display a range of substrate specificities, are regulated by a diverse range of molecules, and have been implicated in a broad range of cellular processes including receptor signaling, cytoskeletal regulation and membrane trafficking. Recent technological advances including: the development of PLD knockout mice, isoform-specific antibodies, and specific inhibitors are finally permitting a thorough analysis of the in vivo role of mammalian PLDs. These studies are facilitating increased recognition of PLD's role in disease states including cancers and Alzheimer's disease, offering potential as a target for therapeutic intervention.

Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation.

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro-alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.

Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function.

The budding mode of Saccharomyces cerevisiae cell growth demands that a high degree of secretory polarity be established and directed toward the emerging bud. We report here our demonstration that mutations in SAC1, a gene identified by virtue of its allele-specific genetic interactions with yeast actin defects, were also capable of suppressing sec14 lethalities associated with yeast Golgi defects. Moreover, these sac1 suppressor properties also extended to sec6 and sec9 secretory vesicle defects. The genetic data are consistent with the notion that SAC1p modulates both secretory pathway and actin cytoskeleton function. On this basis, we suggest that SAC1p may represent one aspect of the mechanism whereby secretory and cytoskeletal activities are coordinated, so that proper spatial regulation of secretion might be achieved.

The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex.

We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.

SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast.

Mutations in the SAC1 gene exhibit allele-specific genetic interactions with yeast actin structural gene defects and effect a bypass of the cellular requirement for the yeast phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p), a protein whose function is essential for sustained Golgi secretory function. We report that SAC1p is an integral membrane protein that localizes to the yeast Golgi complex and to the yeast ER, but does not exhibit a detectable association with the bulk of the yeast F-actin cytoskeleton. The data also indicate that the profound in vivo effects on Golgi secretory function and the organization of the actin cytoskeleton observed in sac1 mutants result from loss of SAC1p function. This cosuppression of actin and SEC14p defects is a unique feature of sac1 alleles as mutations in other SAC genes that result in a suppression of actin defects do not result in phenotypic suppression of SEC14p defects. Finally, we report that sac1 mutants also exhibit a specific inositol auxotrophy that is not exhibited by the other sac mutant strains. This sac1-associated inositol auxotrophy is not manifested by measurable defects in de novo inositol biosynthesis, nor is it the result of some obvious defect in the ability of sac1 mutants to utilize inositol for phosphatidylinositol biosynthesis. Thus, sac1 mutants represent a novel class of inositol auxotroph in that these mutants appear to require elevated levels of inositol for growth. On the basis of the collective data, we suggest that SAC1p dysfunction exerts its pleiotropic effects on yeast Golgi function, the organization of the actin cytoskeleton, and the cellular requirement for inositol, through altered metabolism of inositol glycerophospholipids.

The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation.

The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPalpha, a classical PI transfer protein, for RdgB's PITP domain (PITPalpha or PITPalpha-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPalpha-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

The SEC14SC gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14SC gene product (SEC14pSC) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14YL, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14YL encodes a primary translation product (SEC14YL) that is predicted to be a 497-residue polypeptide of which the amino-terminal 300 residues are highly homologous to the entire SEC14pSC, and the carboxyl-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14pSC, SEC14pYL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14pYL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14pYL for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y. lipolytica as cell-free lysates prepared from delta sec14YL strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14pYL dysfunction results in the inability of Y. lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, delta sec14YL mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14pYL in promoting the differentiation of Y. lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms.

A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes.

SEC14p is required for protein transport from the yeast Golgi complex. We describe a quantitative analysis of yeast bulk membrane and Golgi membrane phospholipid composition under conditions where Golgi secretory function has been uncoupled from its usual SEC14p requirement. The data demonstrate that SEC14p specifically functions to maintain a reduced phosphatidylcholine content in Golgi membranes and indicate that overproduction of SEC14p markedly reduces the apparent rate of phosphatidylcholine biosynthesis via the CDP-choline pathway in vivo. We suggest that SEC14p serves as a sensor of Golgi membrane phospholipid composition through which the activity of the CDP-choline pathway in Golgi membranes is regulated such that a phosphatidylcholine content that is compatible with the essential secretory function of these membranes is maintained.

Mechanistic insights relevant to protein secretion in yeast.

During the past year, a powerful combination of genetic and biochemical approaches has yielded fascinating information with respect to the question of how proteins cross membranes and subsequently traffic between intracellular compartments of the yeast secretory pathway. Fundamental advances have been made in two specific areas. These include experiments that have provided new perspectives with respect to the nature of the soluble machinery involved in facilitating protein traffic from the cytoplasm to the lumen of the endoplasmic reticulum, and work that has provided a biochemical description of what may in effect represent a membranous ligand-gated channel that is required for protein translocation into the endoplasmic reticulum lumen.

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

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

The Schizosaccharomyces pombe spo20(+) gene encoding a homologue of Saccharomyces cerevisiae Sec14 plays an important role in forespore membrane formation.

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

Pleiotropic alterations in lipid metabolism in yeast sac1 mutants: relationship to "bypass Sec14p" and inositol auxotrophy.

SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect "bypass Sec14p" will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene.

Phosphatidylinositol/phosphatidylcholine transfer proteins in yeast.

Phosphatidylinositol transfer proteins (PITPs) are now becoming widely recognized as intriguing proteins that participate in the coordination and coupling of phospholipid metabolism with vesicle trafficking, and in the regulation of important signaling cascades. Yet, only in one case is there a large body of evidence that speaks to the precise identities of PITP-dependent cellular reactions, and to the mechanisms by which PITPs execute function in eukaryotic cells. At present, yeast provide the most powerful system for analysis of the physiology of PITP function in vivo, and the mechanism by which this function is carried out. Here, we review the recent progress and remaining questions in the area of PITP function in yeast.

The contribution of lipids and lipid metabolism to cellular functions of the Golgi complex.

The history of the Golgi complex now reaches its 100 year anniversary. Over the past several decades, tremendous effort has gone into cataloguing Golgi resident proteins, measuring the lipid compositions of Golgi membranes, and in elucidating the pathways by which proteins and lipids traffic through this unique organelle. Only in the past 8 years or so has experimental scrutiny extended to the investigation of how lipids and proteins cooperate to endow the Golgi with its various capabilities regarding protein/lipid transport and sorting. In this chapter we review some of the most recent advances in deciphering the functional interfaces between lipids and proteins of the Golgi complex.

Phospholipid transfer proteins and physiological functions.

Issues of how cells generate and maintain unique lipid compositions in distinct intracellular membrane systems remain the subject of much study. A ubiquitous class of soluble proteins capable of transporting phospholipid monomers from membrane to membrane across an aqueous milieu has been thought to define part of the mechanism by which lipids are sorted in cells. Progress in the study of these phospholipid transfer proteins (PLTPs) raises questions regarding their physiological functions in cells and the mechanisms by which these proteins execute them. It is now clear that across the eukaryotic kingdom, members of this protein family exert essential roles in the regulation of phospholipid metabolism and central aspects of phospholipid-mediated signaling. Indeed, it is now known that dysfunction of specific PLTPs defines the basis of inherited diseases in mammals, and this list is expected to grow. Phospholipid transfer proteins, their biochemical properties, and the emerging clues regarding their physiological functions are reviewed.

The yeast BSD2-1 mutation influences both the requirement for phosphatidylinositol transfer protein function and derepression of phospholipid biosynthetic gene expression in yeast.

The BSD2-1 allele renders Saccharomyces cerevisiae independent of its normally essential requirement for phosphatidylinositol transfer protein (Sec14p) in the stimulation of Golgi secretory function and cell viability. We now report that BSD2-1 yeast mutants also exhibit yet another phenotype, an inositol auxotrophy. We demonstrate that the basis for this Ino- phenotype is the inability of BSD2-1 strains to derepress transcription of INO1, the structural gene for the enzyme that catalyzes the committed step in de novo inositol biosynthesis in yeast. This constitutive repression of INO1 expression is mediated through specific inactivation of Ino2p, a factor required for trans-activation of INO1 transcription, and we show that these transcriptional regulatory defects can be uncoupled from the "bypass Sec14p" phenotype of BSD2-1 strains. Finally, we present evidence that newly synthesized phosphatidylinositol is subject to accelerated turnover in BSD2-1 mutants and that prevention of this accelerated phosphatidyl-inositol turnover in turn negates suppression of Sec14p defects by BSD2-1. We propose that, in BSD2-1 strains, a product(s) generated by phosphatidylinositol turnover coordinately modulates the activities of both the Sec14p/Golgi pathway and the pathway through which transcription of phospholipid biosynthetic genes is derepressed.

Secretory pathway function in Saccharomyces cerevisiae.

A genetic analysis of secretory pathway function in yeast was initiated some 12 years ago in the laboratory of Randy Schekman. These mutants held great promise in terms of providing an experimental system with which molecular participants of secretory pathway function could be investigated. This early promise has not failed. For the last five years, analysis of yeast secretory pathway function has been at the cutting edge of our understanding of the mechanisms by which proteins travel between intracellular compartments. In some cases, Sacch. cerevisiae has provided a valuable in vivo corroboration of the concepts derived from biochemical studies of mammalian intercompartmental protein transport in vitro. In other cases, studies conducted in the yeast system have defined previously unanticipated involvements for known catalytic activities in the secretory process. It is clear that yeast will continue to play a major role in setting the pace of research directed towards a detailed molecular understanding of protein secretion. Since it is now apparent that the basic strategies that underlie secretory pathway function have been conserved among eukaryotes, further exploitation of the powerful and complementary yeast and mammalian experimental systems guarantees that the next decade will see even greater progress towards our understanding of protein secretion in eukaryotic cells than did the first.