SPO14 separation-of-function mutations define unique roles for phospholipase D in secretion and cellular differentiation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid. SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion). We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation. Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct. Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis. Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein. Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth. These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis.

The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks.

The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination.

Preparation of bacterial plasmid DNA.

The protocols in this unit describe methods for preparing bacterial plasmid DNA free from chromosomal DNA. The first is an alkaline lysis miniprep suitable for screening a moderate number of bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. The second is the first step to producing large amounts (milligrams) of plasmid DNA and is also based on alkaline lysis of the bacterial cells. The crude lysate generated in this protocol can be further purified by centrifugation using CsCl/ethidium bromide (CsCl/EtBr) equilibrium density gradients. Three support protocols provide information on how to grow overnight and larger cultures of bacteria, and how to monitor bacterial growth with a spectrophotometer. Other methods, some relying on commercially available ion-exchange columns, are discussed in the commentary.

Preparation of plasmid DNA.

This appendix presents basic procedures for alkaline lysis minipreps, both in tubes and in microtiter plates, and also provides an Alternate protocol for boiling lysis. A Support Protocol describes storage of plasmid DNA.

Preparation of bacterial plasmid DNA.

The protocols in this unit describe methods for preparing bacterial plasmid DNA free from chromosomal DNA. The first is an alkaline lysis miniprep suitable for screening a moderate number of bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. The second is the first step to producing large amounts (milligrams) of plasmid DNA and is also based on alkaline lysis of the bacterial cells. The crude lysate generated in this protocol can be further purified by centrifugation using CsCl/ethidium bromide (CsCl/EtBr) equilibrium density gradients. Three support protocols provide information on how to grow overnight and larger cultures of bacteria, and how to monitor bacterial growth with a spectrophotometer. Other methods, some relying on commercially available ion-exchange columns, are discussed in the commentary.

Minipreps of plasmid DNA.

Although there are a large number of protocols for the isolation of small quantities of plasmid DNA from bacterial cells (minipreps), this unit presents four procedures based on their speed and success: the alkaline lysis prep, a modification of the alkaline lysis prep that is performed in 1.5-ml tubes or 96-well microtiter dishes, the boiling method, and a lithium-based procedure. A support protocol provides information on storing plasmid DNA.

TEP1, the yeast homolog of the human tumor suppressor gene PTEN/MMAC1/TEP1, is linked to the phosphatidylinositol pathway and plays a role in the developmental process of sporulation.

PTEN/MMAC1/TEP1 (PTEN, phosphatase deleted on chromosome ten; MMAC1, mutated in multiple advanced cancers; TEP1, tensin-like phosphatase) is a major human tumor suppressor gene whose suppressive activity operates on the phosphatidylinositol pathway. A single homologue of this gene, TEP1 (YNL128w), exists in the budding yeast Saccharomyces cerevisiae. Yeast strains deleted for TEP1 exhibit essentially no phenotype in haploids; however, diploids exhibit resistance to the phosphatidylinositol-3-phosphate kinase inhibitor wortmannin and to lithium ions. Although rates of cancer increase with age, neither tep1 haploids nor diploids have altered life spans. TEP1 RNA is present throughout the cell cycle, and levels are dramatically up-regulated during meiotic development. Although homozygous tep1 mutants initiate the meiotic program and form spores with wild-type kinetics, analysis of the spores produced in tep1 mutants indicates a specific defect in the trafficking or deposition of dityrosine, a major component of yeast spore walls, to the surface. Introduction of a common PTEN mutation found in human tumors into the analogous position in Tep1p produces a nonfunctional protein based on in vivo activity. These studies implicate Tep1p in a specific developmental trafficking or deposition event and suggest that Tep1p, like its mammalian counterpart, impinges on the phosphatidylinositol pathway.

Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.

Phosphoinositides are both substrates for second messenger-generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking. Phosphatidylcholine-specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD-dependent cellular processes is not known. To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function. This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides. Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are inactive against cellular lipid substrates. Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides. The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo. Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.

Regulation and function of PLDs in yeast.

While yeast contain multiple phospholipase D activities, only one, encoded by SPO14, appears to be a member of the phosphatidylcholine-specific phospholipase D gene family. Genetic analyses have revealed a role for this enzyme in regulated membrane trafficking events.

Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects.

Yeast phosphatidylinositol transfer protein (Sec14p) function is essential for production of Golgi-derived secretory vesicles, and this requirement is bypassed by mutations in at least seven genes. Analyses of such 'bypass Sec14p' mutants suggest that Sec14p acts to maintain an essential Golgi membrane diacylglycerol (DAG) pool that somehow acts to promote Golgi secretory function. SPO14 encodes the sole yeast phosphatidylinositol-4,5-bisphosphate-activated phospholipase D (PLD). PLD function, while essential for meiosis, is dispensable for vegetative growth. Herein, we report specific physiological circumstances under which an unanticipated requirement for PLD activity in yeast vegetative Golgi secretory function is revealed. This PLD involvement is essential in 'bypass Sec14p' mutants where normally Sec14p-dependent Golgi secretory reactions are occurring in a Sec14p-independent manner. PLD catalytic activity is necessary but not sufficient for 'bypass Sec14p', and yeast operating under 'bypass Sec14p' conditions are ethanol-sensitive. These data suggest that PLD supports 'bypass Sec14p' by generating a phosphatidic acid pool that is somehow utilized in supporting yeast Golgi secretory function.

ADP-Ribosylation factors do not activate yeast phospholipase Ds but are required for sporulation.

ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2. The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate. arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores. The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation. Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated. This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants. Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD. These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities. The implications of these results are discussed with respect to current models of Arf signaling.

Yeast dom34 mutants are defective in multiple developmental pathways and exhibit decreased levels of polyribosomes.

The DOM34 gene of Saccharomyces cerevisiae is similar to genes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminisehd. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34delta growth defect. dom34delta mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34delata mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

Yeast meiotic mutants proficient for the induction of ectopic recombination.

A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination.

Relocalization of phospholipase D activity mediates membrane formation during meiosis.

Phospholipase D (PLD) enzymes catalyze the hydrolysis of phosphatidylcholine and are involved in membrane trafficking and cytoskeletal reorganization. The Saccharomyces cerevisiae SPO14 gene encodes a PLD that is essential for meiosis. We have analyzed the role of PLD in meiosis by examining two mutant proteins, one with a point mutation in a conserved residue (Spo14pK--> H) and one with an amino-terminal deletion (Spo14pDeltaN), neither of which can restore meiosis in a spo14 deletion strain. Spo14pK--> H is enzymatically inactive, indicating that PLD activity is required, whereas Spo14pDeltaN retains PLD catalytic activity in vitro, indicating that PLD activity is not sufficient for meiosis. To explore other aspects of Spo14 function, we followed the localization of the enzyme during meiosis. Spo14p is initially distributed throughout the cell, becomes concentrated at the spindle pole bodies after the meiosis I division, and at meiosis II localizes to the new spore membrane as it surrounds the nuclei and then expands to encapsulate the associated cytoplasm during the formation of spores. The catalytically inactive protein also undergoes relocalization during meiosis; however, in the absence of PLD activity, no membrane is formed. In contrast, Spo14pDeltaN does not relocalize properly, indicating that the failure of this protein to complement a spo14 mutant is due to its inability to localize its PLD activity. Furthermore, we find that Spo14p movement is correlated with phosphorylation of the protein. These experiments indicate that PLD participates in regulated membrane formation during meiosis, and that both its catalytic activity and subcellular redistribution are essential for this function.

Measurement of phospholipase D activity.

Phosphodiesteric cleavage of phosphatidylcholine by members of a growing family of phospholipases D produces choline and phosphatidic acid. These enzymes can also catalyse a transphosphatidylation reaction in which the aliphatic chain of a primary alcohol is transferred to the phosphatidyl moiety of the phosphatidic acid product. PLD enzymes are found in a variety of organisms including bacteria, yeast, plants, and vertebrates. In mammalian systems, biochemical and cell biological approaches have identified phosphatidic acid as a mediator (or progenitor of mediators) that play important roles in the transduction of extracellular signals. Phosphatidic acid or its metabolites may be regulators of key cellular processes such as the control of intracellular protein trafficking, secretion, and alterations in cell morphology and motility. This review discusses methods for the determination of PLD activity both in vitro and in intact cells.

Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity.

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family.

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.

Phospholipase D signaling is essential for meiosis.

Phospholipid metabolism plays an important role in cellular regulation by generating second messengers for signal transduction. Many stimuli activate a phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine, producing phosphatidic acid and choline. Here we report that the yeast SP014 gene, which is essential for meiosis [Honigberg, S. M., Conicella, C. & Esposito, R. E. (1992) Genetics 130, 703-716], encodes a phospholipase D. SP014 RNA and protein activity are induced during late meiotic prophase, and the enzyme has properties similar to mammalian phosphatidylinositol 4,5-bisphosphate-regulated phospholipase D. Characterization of an unusual allele of SP014 defines regions of the protein important for enzyme catalysis and regulation. These results implicate phospholipase D signaling in regulating cellular differentiation.