Absence of Gup1p inSaccharomyces cerevisiaeresults in defective cell wall composition, assembly, stability and morphology

Abstract

Saccharomyces cerevisiae Gup1p and its homologue Gup2p, members of the superfamily of membrane-bound O-acyl transferases, were previously associated with glycerol-mediated salt-stress recovery and glycerol symporter activity. Several other phenotypes suggested Gup1p involvement in processes connected with cell structure organization and biogenesis. The gup1Delta mutant is also thermosensitive and exhibits an altered plasma membrane lipid composition. The present work shows that the thermosensitivity is independent of glycerol production and retention. Furthermore, the mutant grows poorly on salt, ethanol and weak carboxylic acids, suggestive of a malfunctioning membrane potential. Additionally, gup1Delta is sensitive to cell wall-perturbing agents, such as Calcofluor white, Zymolyase, lyticase and sodium dodecyl sulphate and exhibits a sedimentation/aggregation phenotype. Quantitative analysis of cell wall components yielded increased contents of chitin and beta-1,3-glucans and lower amounts of mannoproteins. Consistently, scanning electron microscopy showed a strikingly rough surface morphology of the mutant cells. These results suggest that the gup1Delta is affected in cell wall assembly and stability, although the Slt2p/MAP kinase from the PKC pathway was phosphorylated during hypo-osmotic shock to a normal extent. Results emphasize the pleiotropic nature of gup1Delta, and are consistent with a role of Gulp1p in connection with several pathways for cell maintenance and construction/remodelling.

Fungal Chitin Induces Trained Immunity in Human Monocytes during Cross-talk of the Host with Saccharomyces cerevisiae

“…Cell Wall Characterization and Chitin Purification-Cell wall characterization has been performed as described by Ferreira et al (23) with modifications. The cells were cultured in YPD liquid medium at 28°C and collected at late exponential phase.…”

“…Cell walls were harvested by centrifugation for 30 min at 4200 ϫ g and dried in a SpeedVac concentrator. To extract chitin, cell wall dried biomass was subjected to alkaline extraction, followed by acid extraction according to the protocol described by Ferreira et al (23). Chitin was finally obtained by dialysis of the extract and lyophilized to determine the dry weight.…”

Methodologies to generate, extract, purify and fractionate yeast ECM for analytical use in proteomics and glycomics

“…In yeasts, proteins found using the present procedures may derive from the outer layers of the cell wall as previously suggested (reviewed by [27]). Their location in vivo has though to be secured by loose attachment, since the present procedures preclude the extraction of covalently linked cell wall materials that require harsh methods to be extracted [29,30]. The identification of all the proteins present in yECM, will be possible analysing all the spots present in the 2DE, or using a high throughput technique, as liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).…”

Mutations in the RAM network confer resistance to the thiol oxidant 4,4′-dipyridyl disulfide

“…Therefore, we thought that a post-mitotic cell separation defect per se could be advantageous for resistance to DPS. However, mutants like Dgup1 (lacking a putative membrane-bound O-acyltransferase) and Dgas1 (lacking a cell wall-bound 1.3-b-glucanosyltransferase) show cell separation defects related to abnormal cell wall built-up (Popolo et al 1993;Ferreira et al 2006), but are not resistant to DPS. By contrast, these mutants are sensitive to DPS (Fig.…”