JoAnne Engebrecht

ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis

ZIP1 is a novel meiosis-specific gene required for chromosome synapsis and cell cycle progression in S. cerevisiae. zip1 strains undergo homologous chromosome pairing, but are defective in synaptonemal complex (SC) formation. The zip1 mutation confers a uniform arrest in meiosis prior to the first division. zip1 strains display nearly wild-type levels of commitment to meiotic recombination; however, mature reciprocal recombinants are not formed until cells are released from meiotic arrest by return to growth medium. DNA sequence analysis of ZIP1 reveals structural homology to a number of proteins containing coiled coils. Immunofluorescence experiments using anti-ZIP1 antibodies demonstrate that the ZIP1 protein localizes to synapsed meiotic chromosomes but not to unsynapsed axial elements. Taken together, these data suggest that ZIP1 is a component of the central region of the SC. We propose a model in which ZIP1 acts as a molecular zipper to bring homologous chromosomes in close apposition.

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.

Structure and regulation of phospholipase D

Phosphar:S~:~oline-specific phos>holipase D (PLDs) are phosphodi‘stemses that catalyse the hydrolysis If phosphatidylcholine (PC) to pmjute phosphatidic acid (PA) and :holine. Diverse stimuli mcreasePLD activity in many cells and PA, or a netabolite of this lipid, is probably he orimarv mediator of PLD-initia , ited cell signalltng. Investigations of PLD regulation irt oitro suggest that .hese enzymes may play roles in nhacellular protein trafficking and nitogenic signaIling processes conrolled by members of the ADPribosylatibn factor (ARFj anJ Rho :amilies of monomeric G uroteins. l’he identification of genes encoding veast and human PLD enzymes xomises to provide the molecular tools necessy this reaction concurrmtly generates Ivso-Dhomhatidic acid. which can e of receptor-mechated PLD activation has been presumed to be the plasma membrane there are convincing reports of PLD activities associated with the nucleus and Co& apparat&?

Meiosis-specific RNA splicing in yeast

Previous studies have suggested that the differentiated state of meiosis in yeast is regulated primarily at the transcriptional level. This study reports a case of posttranscriptional regulation of a gene whose product is essential for meiosis. The MER2 gene is transcribed in mitosis as well as meiosis; however, the transcript is spliced efficiently to generate a functional gene product only in meiosis. Meiotic levels of splicing depend on the MER1 gene product, which is also essential for meiosis and which is produced only in meiotic cells. Therefore, at least one of the functions of the MER1 protein is to mediate splicing of the MER2 transcript. Genetic data suggest that the MER1 gene may also be responsible for splicing the transcript of at least one other gene.

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.

Meiotic gene conversion and crossing over: Their relationship to each other and to chromosome synapsis and segregation

The yeast mer1 mutant produces inviable spores and is defective in both meiotic recombination and chromosome pairing. A gene called MER2 partially suppresses the mer1 phenotype when present in high copy number. Both gene conversion and chromosome pairing are completely restored in mer1 strains overexpressing MER2; however, reciprocal crossing over and spore viability are not restored. The data presented are consistent with a model in which chromosome pairing is a direct consequence of a homology search mediated through gene conversion. Analysis of random viable spores indicates that the crossovers that occur in mer1 strains overexpressing MER2 are more effective in ensuring meiosis I disjunction than those that occur in mer1 strains. One interpretation of this result is that only those crossovers that occur in the context of the synaptonemal complex lead to the establishment of functional chiasmata. The MER2 gene product is essential for meiosis.

Phospholipase D and the SNARE Sso1p are necessary for vesicle fusion during sporulation in yeast

Spore formation in Saccharomyces cerevisiae requires the de novo formation of prospore membranes. The coalescence of secretory vesicles into a membrane sheet occurs on the cytoplasmic surface of the spindle pole body. Spo14p, the major yeast phospholipase D, is necessary for prospore membrane formation; however, the specific function of Spo14p in this process has not been elucidated. We report that loss of Spo14p blocks vesicle fusion, leading to the accumulation of prospore membrane precursor vesicles docked on the spindle pole body. A similar phenotype was seen when the t-SNARE Sso1p, or the partially redundant t-SNAREs Sec9p and Spo20p were mutated. Although phosphatidic acid, the product of phospholipase D action, was necessary to recruit Spo20p to the precursor vesicles, independent targeting of Spo20p to the membrane was not sufficient to promote fusion in the absence of SPO14. These results demonstrate a role for phospholipase D in vesicle fusion and suggest that phospholipase D-generated phosphatidic acid plays multiple roles in the fusion process.

Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes.

Phospholipase D (PLD) generates lipid signals that coordinate membrane trafficking with cellular signaling. PLD activity in vitro and in vivo is dependent on phosphoinositides with a vicinal 4,5-phosphate pair. Yeast and mammalian PLDs contain an NH2-terminal pleckstrin homology (PH) domain that has been speculated to specify both subcellular localization and regulation of PLD activity through interaction with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2). We report that mutation of the PH domains of yeast and mammalian PLD enzymes generates catalytically active PI(4,5)P2-regulated enzymes with impaired biological functions. Disruption of the PH domain of mammalian PLD2 results in relocalization of the protein from the PI(4,5)P2-containing plasma membrane to endosomes. As a result of this mislocalization, mutations within the PH domain render the protein unresponsive to activation in vivo. Furthermore, the integrity of the PH domain is vital for yeast PLD function in both meiosis and secretion. Binding of PLD2 to model membranes is enhanced by acidic phospholipids. Studies with PLD2-derived peptides suggest that this binding involves a previously identified polybasic motif that mediates activation of the enzyme by PI(4,5)P2. By comparison, the PLD2 PH domain binds PI(4,5)P2 with lower affinity but sufficient selectivity to function in concert with the polybasic motif to target the protein to PI(4,5)P2-rich membranes. Phosphoinositides therefore have a dual role in PLD regulation: membrane targeting mediated by the PH domain and stimulation of catalysis mediated by the polybasic motif.

Genetic and morphological approaches for the analysis of meiotic chromosomes in yeast

Publisher Summary Meiosis is a specialized cell division in which the chromosome number is reduced to half, generating haploid gametes for sexual reproduction. Chromosome behavior during meiosis is complex, comprising a single round of replication followed by two successive rounds of segregation. The meiosis I division is unique in that homologous chromosomes disjoin from each other; at meiosis II, such as mitosis, sister chromatids segregate from one another. Although some details differ for different organisms, the fundamental process of meiotic chromosome segregation is conserved. Therefore, meiosis can be studied in the yeast Saccharomyces cerevisiae, taking advantage of its powerful genetics. During prophase of meiosis I, homologous chromosomes pair, synapse along the meiosis-specific organelle called “the synaptonemal complex (SC),” undergo recombination. These processes are essential for the correct alignment of the homologous chromosomes on the metaphase plate and subsequent segregation. Genetic, biochemical, and cytological analyses of meiosis in yeast led to the identification of key regulatory molecules and structural components of the SC and the meiotic recombination machinery.

SYP-3 restricts synaptonemal complex assembly to bridge paired chromosome axes during meiosis in Caenorhabditis elegans.

Synaptonemal complex (SC) formation must be regulated to occur only between aligned pairs of homologous chromosomes, ultimately ensuring proper chromosome segregation in meiosis. Here we identify SYP-3, a coiled-coil protein that is required for assembly of the central region of the SC and for restricting its loading to occur only in an appropriate context, forming structures that bridge the axes of paired meiotic chromosomes in Caenorhabditis elegans. We find that inappropriate loading of central region proteins interferes with homolog pairing, likely by triggering a premature change in chromosome configuration during early prophase that terminates the search for homologs. As a result, syp-3 mutants lack chiasmata and exhibit increased chromosome missegregation. Altogether, our studies lead us to propose that SYP-3 regulates synapsis along chromosomes, contributing to meiotic progression in early prophase.