Marina Vai

The Gas1 glycoprotein, a putative wall polymer cross-linker

The yeast cell wall, which for years has been regarded as a static cellular component, has been revealed to be dynamic in its structure and composition and complex in its enzymatic activity. The S. cerevisiae cell wall is composed of beta-1,3/beta-1,6-glucans, mannoproteins, and chitin, which are assembled into an extracellular matrix essential for maintenance of cell integrity. Gas1p, a glycoprotein anchored to the outer leaflet of the plasma membrane through a glycosylphosphatidylinositol, plays a key role in cell wall assembly. Loss of Gas1p leads to several morphogenetic defects and to a decrease in the amount of cross-links between the cell wall glucans. These defects in turn trigger a compensatory response that guarantees cell viability. Several Gas1p homologs have been isolated from Candida species and S. pombe. The Gas1p family also includes two plant proteins with endo-beta-1,3-glucanase activity. Sequence comparisons reveal that Gas1p family proteins have a modular organization of domains. The genetic and molecular analyses reviewed here suggest that Gas1p could play a role as a polymer cross-linker, presumably by catalyzing a transglycosylation reaction.

Deletion of GEL2 encoding for a β(1-3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus

The first fungal glycosylphosphatidylinositol an‐chored β(1–3)glucanosyltranferase (Gel1p) has been described in Aspergillus fumigatus and its encoding gene GEL1 identified. Glycosylphosphatidylinositol‐anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. We characterize here GEL2, a homologue of GEL1. Both homologues share common characteristics: (i) GEL1 and GEL2 are constitutively expressed during over a range of growth conditions; (ii) Gel2p is also a putative GPI‐anchored protein and shares the same β(1–3)glucanosyltransferase activity as Gel1p and (iii) GEL2, like GEL1, is able to complement the Δgas1 deletion in Saccharomyces cerevisiae confirming that Gelp and Gasp have the same enzymatic activity. However, disruption of GEL1 did not result in a phenotype whereas a Δgel2 mutant and the double mutant Δgel1Δgel2 exhibit slower growth, abnormal conidiogenesis, and an altered cell wall composition. In addition, the Δgel2 and the Δgel1Δgel2 mutant have reduced virulence in a murine model of invasive aspergillosis. These data suggest for the first time that β(1–3)glucanosyltransferase activity is required for both morphogenesis and virulence in A. fumigatus.

Chitin synthesis in a gas1 mutant of Saccharomyces cerevisiae

The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas1 delta. CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the CHS1 gene. However, deletion of the CHS1 gene did not affect the phenotype of the gas1 delta mutant and only slightly reduced the chitin content. Interestingly, in chs1 gas1 double mutants the lysed-bud phenotype, typical of chs1 null mutant, was suppressed, although in gas1 cells there was no reduction in chitinase activity. CHS3 expression was not affected in the gas1 mutant. Deletion of the CHS3 gene severely compromised the phenotype of gas1 cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change. Furthermore, in chs3 gas1 cells the chitin level was about 10% that of gas1 cells. Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress. However, the level of enzyme or the in vitro CSIII activity does not change. This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin.

Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.

The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.

Deletion or Overexpression of Mitochondrial NAD+ Carriers in Saccharomyces cerevisiae Alters Cellular NAD and ATP Contents and Affects Mitochondrial Metabolism and the Rate of Glycolysis

ABSTRACT The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.

Improved Secretion of Native Human Insulin-Like Growth Factor 1 from gas1 Mutant Saccharomyces cerevisiae Cells

ABSTRACT We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1gene was fused to the mating factor α prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.

SFP1 is involved in cell size modulation in respiro-fermentative growth conditions

Saccharomyces cerevisiae grows fast on glucose, while growth slows down on ethanol as cells move from glucose fermentation to oxidative metabolism. The type of carbon source influences both the specific growth rate and cell cycle progression, as well as cell size. Yeast cells grown on glucose have a larger size than cells grown on ethanol. Here, we analysed the behaviour of a sfp1 null mutant during balanced and transitory states of growth in batch in response to changes in the growth medium carbon sources. In a screening for mutants affected in cell size at Start, SFP1 has been identified as a gene whose deletion caused one of the smallest whi phenotype. Findings presented in this work indicate that in the sfp1 null mutant the reduction in cell size is not only a consequence of the reduced growth rate but it is tightly linked to the cellular metabolism. The SFP1 gene product is required to sustain the increase of both rRNA and protein content that in wild‐type cells takes place in respiro‐fermentative growth conditions, while it seems dispensable for growth on non‐fermentable carbon sources. It follows that sfp1 cells growing on ethanol have a larger size than cells growing on glucose and, noticeably, the former enter the S phase with a critical cell size higher than the latter. These features, combined with the role of Sfp1p as a transcriptional factor, suggest that Sfp1p could be an important element in the control of the cell size modulated by nutrients. Copyright

Effect of tunicamycin on cell cycle progression in budding yeast

Tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharides, prevents bud formation and growth in Saccharomyces cerevisiae cells that are either growing exponentially or recovering from different cell cycle arrests at start. Analysis of tunicamycin-treated cells by flow microfluorometry clearly shows that cells have a postsynthetic DNA content, but there is no evidence of an increase in binucleate cells. Therefore tunicamycin affects bud emergence and initiation of DNA synthesis, two events correlated under physiological conditions, in different ways. A bulk glycoprotein synthesis is shown to be required for bud emergence and localized chitin deposition, probably to sustain directional secretory vesicle transport, which allows polar growth of the bud. No evidence for a glycoprotein requirement for entrance into the S phase is obtained from the present experiments.

Immunological cross-reactivity of fungal and yeast plasma membrane H+-ATPase.

The plasma membrane H+‐ATPases from fungi and yeasts have similar catalytic and molecular properties. A structural comparison has been performed using immunoblot analysis with polyclonal antibodies directed toward the 102 kDa polypeptide of the plasma membrane H+‐ATPase from Neurospora crassa. A strong cross‐reactivity is observed between the fungal H+‐ATPase and the enzyme from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Structural homologies are indicated also by the analysis of the cross‐reactive peptides originated by proteolytic digestion of Neurospora and S.cerevisiae purified enzymes. Neither enzyme from these two sources appears to be glycosylated by a highly sensitive concanavalin A affinity assay on blotted proteins. A glycoprotein of M r 115000 and pI 4.8–5, which comigrates with a cell cycle‐modulated protein on 2D gel, is present in partially purified preparations of plasma membrane H+‐ATPase of S.cerevisiae and it is shown to be structurally unrelated to H+‐ATPase.