An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

https://www.webdepot.umontreal.ca/Usagers/chartrpa/MonDepotPublic/Chartrand/Publications/PC199802.pdf

Localization of ASH1 mRNA Particles in Living Yeast

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4-64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4-64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4-64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4-64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.

/pdf/a-new-vital-stain-for-visualizing-vacuolar-membrane-dynamics-1e6ae5yogm.pdf

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

The plant hormone auxin is central in many aspects of plant development. Previous studies have implicated the ubiquitin-ligase SCFTIR1 and the AUX/IAA proteins in auxin response. Dominant mutations in several AUX/IAA genes confer pleiotropic auxin-related phenotypes, whereas recessive mutations affecting the function of SCFTIR1 decrease auxin response. Here we show that SCFTIR1 is required for AUX/IAA degradation. We demonstrate that SCFTIR1 interacts with AXR2/IAA7 and AXR3/IAA17, and that domain II of these proteins is necessary and sufficient for this interaction. Further, auxin stimulates binding of SCFTIR1 to the AUX/IAA proteins, and their degradation. Because domain II is conserved in nearly all AUX/IAA proteins in Arabidopsis, we propose that auxin promotes the degradation of this large family of transcriptional regulators, leading to diverse downstream effects.

/pdf/auxin-regulates-scf-tir1-dependent-degradation-of-aux-iaa-3skr2yolsp.pdf

Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins.

Differentiation is a continuously regulated process and interactions between the cell and its environment play a major role in maintaining stable expression of differentiation specific genes (Blau and Baltimore, 1991). An important component of the cellular environment is the extracel-lular matrix (ECM), which is composed of glycoproteins, proteoglycans and glycosaminoglycans that are secreted and assembled locally into an organised network to which cells adhere (Hay, 1981). An ECM is present within mam-malian embryos from the two-cell stage and is a component of the environment of all cell types, although the composition of the ECM and the spatial relationships between cells and ECM differ between tissues. Cells may be completely surrounded by ECM, as is the case for chondrocytes, or may contact the ECM only at one surface, as exemplified by epithelial and endothelial cells. In some tissues only a proportion of the cells are exposed to ECM: for example , in stratified epithelia. The ECM offers structural support for cells, and can also act as a physical barrier or selective filter to soluble molecules. It has been clear for many years (Grobstein, 1954; Bis-sell et al., 1982) that the ECM plays a role in regulating the differentiated phenotype of cells (reviewed by Watt, 1986), but the mechanisms involved remained largely mysterious until recently, when cell-binding sites within individual ECM glycoproteins and specific ECM receptors were identified. The cell-binding sites were mapped by using pro-teolytic fragments and synthetic peptides to define the minimal sequences responsible for adhesive activity. In the case of fibronectin, the primary determinant of cell-binding activity for many cell types resides in the sequence GRGDSP, which occurs in one of the type III repeats that form the central domain of the molecule (Ruoslahti and Pierschbacher, 1987). Subsequently, RGD-containing sequences have been found in other matrix proteins, and additional short linear adhesive sequence motifs have been defined, although it is clear that the three-dimensional structure of matrix proteins is also an important determinant of adhesive activity (reviewed by Humphries, 1990). Affinity chromatography techniques, together with adhesion-perturbing antibodies that recognise specific plasma membrane glycoproteins, allowed the identification of ECM receptors, many of which belong to the integrin family of α/β het-erodimers (Ruoslahti and Pierschbacher, 1987; Hynes, 1987). In this review, we will summarise some of the evidence that ECM components regulate differentiation and development , describe the regulatory mechanisms involved and, finally, discuss the intracellular events that may transduce signals …

Regulation of development and differentiation by the extracellular matrix

Publisher Summary This chapter provides protocols for the application of immunofluorescence procedures to yeast. It should perhaps be stressed that immunofluorescence and other light microscopic techniques play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light microscopy. The potential advantages of light microscopic approaches derive from various facts: (1) they can be applied to lightly processed or (in some cases) living cells, (2) Much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) Some structures (for example, the cytoplasmic microtubules) have simply been easier to see by light microscopy than by electron microscopy.

Immunofluorescence Methods for Yeast

Publisher Summary This chapter reviews and provides detailed protocols for the application of immunofluorescence and other fluorescence-microscopic procedures to yeast. These procedures play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light (including fluorescence) microscopy. The potential advantages of light-microscopic approaches derive from the facts (1) that they can be applied to lightly processed or living cells, (2) that much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) that some structures have simply been easier to see by light microscopy than by electron microscopy. The methods are also effective with other yeasts such as Schizosaccharomyces pombe and Candida albicans .

Fluorescence microscopy methods for yeast.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDC10, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. Biol. 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normal-looking ring structures at the bases of the first zygotic buds.

/pdf/cellular-morphogenesis-in-the-saccharomyces-cerevisiae-cell-3pe4hxurz6.pdf

Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the "preimmune" sera of all rabbits tested.

Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck.

We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S. cerevisiae (Magdolen, V., U. Oechsner, G. Muller, and W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin. Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin. Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells. In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.

/pdf/purification-of-profilin-from-saccharomyces-cerevisiae-and-2vflp5d6s8.pdf

Brian Haarer

Papers

Immunofluorescence Methods for Yeast

Fluorescence microscopy methods for yeast.

Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site.

Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck.

Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells.