Cell wall assembly in Saccharomyces cerevisiae.

An extracellular matrix composed of a layered meshwork of beta-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.     

Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae

Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed. The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000–500,000 (mean of about 200,000) at early exponential phase to 250,000–350,000 (mean of about 300,000) at late exponential phase. These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000–100,000 at all growth stages and the level of O-glycosylation changed only slightly. After, incubation of the purified walls with concanavalin A-ferritin and subsequent analysis by electron microscopy, labelling was localized at the external and internal faces of the walls. The middle space of these was labelled after digestion of the glucan network with Zymolyase, which demonstrate the presence of mannoproteins in close contact with the structural glucan molecules throughout the wall.Abbreviations BSA bovine serum albumin - Con A concanavalin A - SDS sodium dodecyl sulphate     

Prediction of Saccharomyces cerevisiae replication origins

Background  

Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible.     

Cell Wall Assembly in Saccharomyces cerevisiae

An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.     

Saccharomyces cerevisiae structural cell wall mannoprotein

A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion. The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate. The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids. Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme. Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of [3H]proline into this fraction. In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel. Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material.     

Nuclear mitochondrial DNA activates replication in Saccharomyces cerevisiae

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in.     

酿酒酵母DNA复制的精确时空控制

DNA复制是最基本的生命活动之一。DNA复制本身的错误及其过程控制的异常是细胞内基因组不稳定的主要来源,会导致细胞生长异常、衰老、癌变乃至死亡。为了保证基因组DNA能够精确且完整的复制,DNA复制受到严格的调控。在G1期,DNA复制解旋酶的核心组分Mcm2-7复合体被招募到复制起点,获得复制许可资格。进入S期后,在两个周期性蛋白激酶及多个支架蛋白的作用下,复制解旋酶的激活因子Cdc45和GINS复合体被招募至Mcm2-7,形成解旋酶全酶Cdc45-Mcm2-7-GINS (CMG)复合体。随后,众多复制相关蛋白在精准的时空控制下被招募至CMG平台并组装成复制机器,起始DNA双向复制。当相向而行的两个复制叉相遇,复制机器会从DNA链上解离下来,从而完成DNA复制。关于DNA复制过程的研究在近十年来取得了跨越式的突破。本文以酿酒酵母为例,围绕所有真核生物中都高度保守的DNA复制控制开关——CMG解旋酶,对真核生物DNA复制的最新进展进行综述。     

Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae

Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of D-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.     

Dynamics of cell wall structure in Saccharomyces cerevisiae

The cell wall of Saccharomyces cerevisiae is an elastic structure that provides osmotic and physical protection and determines the shape of the cell. The inner layer of the wall is largely responsible for the mechanical strength of the wall and also provides the attachment sites for the proteins that form the outer layer of the wall. Here we find among others the sexual agglutinins and the flocculins. The outer protein layer also limits the permeability of the cell wall, thus shielding the plasma membrane from attack by foreign enzymes and membrane-perturbing compounds. The main features of the molecular organization of the yeast cell wall are now known. Importantly, the molecular composition and organization of the cell wall may vary considerably. For example, the incorporation of many cell wall proteins is temporally and spatially controlled and depends strongly on environmental conditions. Similarly, the formation of specific cell wall protein-polysaccharide complexes is strongly affected by external conditions. This points to a tight regulation of cell wall construction. Indeed, all five mitogen-activated protein kinase pathways in bakers' yeast affect the cell wall, and additional cell wall-related signaling routes have been identified. Finally, some potential targets for new antifungal compounds related to cell wall construction are discussed.     

Cell wall mutants of Saccharomyces cerevisiae with increased digestibility by cell wall lytic enzymes and protein extractability

Yeast cell wall mutants were obtained by mutagenesis of Saccharomyces cerevisiae X2180-1A, a haplid strain, with N-methyl-N′-nitro-N-nitrosoguanidine. The two S. cerevisiae mutants showed considerable morphological changes and digestibilities by lytic enzymes. Sequential extractions of proteins and polysaccharides from the mutant and wild type cells indicate that the mutants have high protein extractability and lack some wall proteins as well as some polysaccharide fractions.     

Cell polarity and morphogenesis in Saccharomyces cerevisiae

Polarized cell growth and division are fundamental to cellular differentiation and tissue formation in eukaryotes. Analysis of cell polarity in the budding yeast Saccharomyces cerevisiae has allowed the identification of many regulatory, secretory and cytoskeletal components involved in these processes, as well as the elucidation of various steps in these events. Many of these components and processes may be similar in other eukaryotes.     

Defective break-induced replication leads to half-crossovers in Saccharomyces cerevisiae

Break-induced replication (BIR) is an important process of DNA metabolism that has been implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities, including loss of heterozygosity, translocations, and alternative telomere lengthening. Therefore, knowledge of how BIR is carried out and regulated is important for better understanding the maintenance of genomic stability in eukaryotes. Here we present a new yeast experimental system that enables the genetic control of BIR to be investigated. Analysis of mutations selected on the basis of their sensitivity to various DNA-damaging agents demonstrated that deletion of POL32, which encodes a third, nonessential subunit of polymerase delta, significantly reduced the efficiency of BIR, although some POL32-independent BIR was still observed. Importantly, the BIR defect in pol32Delta cells was associated with the formation of half-crossovers. We propose that these half-crossovers resulted from aberrant processing of BIR intermediates. Furthermore, we suggest that the half-crossovers observed in our system are analogous to nonreciprocal translocations (NRTs) described in mammalian tumor cells and, thus, our system could represent an opportunity to further study the NRT mechanism in yeast.     

Cell wall permeability and export of proteins into the culture broth of Saccharomyces cerevisiae

The upper limit of Saccharomyces cerevisiae cell wall permeability is such that only molecules with a mass not greater than 2-3 kDa can pass across the cell wall. However, proteins with a much higher molecular mass are exported into the cultural broth when the organism is cultivated in a liquid medium with peptone. Under certain conditions, exogenous proteins interact with the cytoplasmic membrane. These as well as some other findings imply that the cell wall may have microplots with an anomalous permeability, via which the regulated exchange of macromolecules is realized between the intracellular and outer media.