Aaron M. Neiman

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

Perinuclear localization of chromatin facilitates transcriptional silencing

Transcriptional silencing in Saccharomyces cerevisiae at the HM mating-type loci and telomeres occurs through the formation of a heterochromatin-like structure. HM silencing is regulated by cis-acting elements, termed silencers, and by trans-acting factors that bind to the silencers. These factors attract the four SIR (silent information regulator) proteins, three of which (SIR2–4) spread from the silencers to alter chromatin, hence silencing nearby genes. We show here that an HMR locus with a defective silencer can be silenced by anchoring the locus to the nuclear periphery. This was accomplished by fusing integral membrane proteins to the GAL4 DNA-binding domain and overproducing the hybrid proteins, causing them to accumulate in the endoplasmic reticulum and the nuclear membrane. We expressed the hybrid proteins in a strain carrying an HMR silencer with GAL4-binding sites (UASG) replacing silencer elements, causing the silencer to become anchored to the nuclear periphery and leading to silencing of a nearby reporter gene. This silencing required the hybrids of the GAL4 DNA-binding domain with membrane proteins, the UASG sites and the SIR proteins. Our results indicate that perinuclear localization helps to establish transcriptionally silent chromatin.

Biological imaging by soft x-ray diffraction microscopy

We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cells major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffraction microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.

Sporulation in the Budding Yeast Saccharomyces cerevisiae

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.

Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo.

BACKGROUND Many signals are transduced from the cell surface to the nucleus through mitogen-activated protein (MAP) kinase cascades. Activation of MAP kinase requires phosphorylation by MEK, which in turn is controlled by Raf, Mos or a group of structurally related kinases termed MEKKs. It is not understood how MEKKs are regulated by extracellular signals. In yeast, the MEKK Ste11p functions in multiple MAP kinase cascades activated in response to pheromones, high osmolarity and nutrient starvation. Genetic evidence suggests that the p21-activated protein kinase (PAK) Ste20p functions upstream of Ste11p, and Ste20p has been shown to phosphorylate Ste11p in vitro. RESULTS Ste20p phosphorylated Ste11p on Ser302 and/or Ser306 and Thr307 in yeast, residues that are conserved in MEKKs of other organisms. Mutating these sites to non-phosphorylatable residues abolished Ste11p function, whereas changing them to aspartic acid to mimic the phosphorylated form constitutively activated Ste11p in vivo in a Ste20p-independent manner. The amino-terminal regulatory domain of Ste11p interacted with its catalytic domain, and overexpression of a small amino-terminal fragment of Ste11p was able to inhibit signaling in response to pheromones. Mutational analysis suggested that this interaction was regulated by phosphorylation and dependent on Thr596, which is located in the substrate cleft of the catalytic domain. CONCLUSIONS Our results suggest that, in response to multiple extracellular signals, phosphorylation of Ste11p by Ste20p removes an amino-terminal inhibitory domain, leading to activation of the Ste11 protein kinase. This mechanism may serve as a paradigm for the activation of mammalian MEKKs.

The Yeast Spore Wall Enables Spores to Survive Passage through the Digestive Tract of Drosophila

In nature, yeasts are subject to predation by flies of the genus Drosophila. In response to nutritional starvation Saccharomyces cerevisiae differentiates into a dormant cell type, termed a spore, which is resistant to many types of environmental stress. The stress resistance of the spore is due primarily to a spore wall that is more elaborate than the vegetative cell wall. We report here that S. cerevisiae spores survive passage through the gut of Drosophila melanogaster. Constituents of the spore wall that distinguish it from the vegetative cell wall are necessary for this resistance. Ascospores of the distantly related yeast Schizosaccharomyces pombe also display resistance to digestion by D. melanogaster. These results suggest that the primary function of the yeast ascospore is as a cell type specialized for dispersion by insect vectors.

ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASE CASCADES BY P21-ACTIVATED PROTEIN KINASES IN CELL-FREE EXTRACTS OF XENOPUS OOCYTES

In the evolutionarily distant yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, genetic evidence suggests that activation of pheromone-induced mitogen-activated protein kinase (MAPK) cascades involves the function of the p21-activated protein kinases (PAKs) Ste20 and Shk1, respectively. In this report, we show that purified Ste20 and Shk1 were each capable of inducing p42 activation in cell-free extracts of Xenopus laevis oocytes, while a mammalian Ste20/Shk1-related protein kinase, p65 (Pak1), did not induce activation of p42. In contrast to p42, activation of JNK/SAPK in Xenopus oocyte extracts was induced by both the yeast Ste20 and Shk1 kinases, as well as by mammalian Pak1. Our results demonstrate that MAPK cascades that are responsive to PAKs are conserved in higher eukaryotes and suggest that distinct PAKs may regulate distinct MAPK modules.

Morphogenetic Pathway of Spore Wall Assembly in Saccharomyces cerevisiae

ABSTRACT The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.

A Gip1p–Glc7p phosphatase complex regulates septin organization and spore wall formation

Sporulation of Saccharomyces cerevisiae is a developmental process in which a single cell is converted into four haploid spores. GIP1, encoding a developmentally regulated protein phosphatase 1 interacting protein, is required for spore formation. Here we show that GIP1 and the protein phosphatase 1 encoded by GLC7 play essential roles in spore development. The gip1Δ mutant undergoes meiosis and prospore membrane formation normally, but is specifically defective in spore wall synthesis. We demonstrate that in wild-type cells, distinct layers of the spore wall are deposited in a specific temporal order, and that gip1Δ cells display a discrete arrest at the onset of spore wall deposition. Localization studies revealed that Gip1p and Glc7p colocalize with the septins in structures underlying the growing prospore membranes. Interestingly, in the gip1Δ mutant, not only is Glc7p localization altered, but septins are also delocalized. Similar phenotypes were observed in a glc7–136 mutant, which expresses a Glc7p defective in interacting with Gip1p. These results indicate that a Gip1p–Glc7p phosphatase complex is required for proper septin organization and initiation of spore wall formation during sporulation.