0000000000218937
AUTHOR
Ana I. Martínez
Analysis of the proteins involved in the structure and synthesis of the cell wall of Ustilago maydis
Abstract A study of the proteins involved in the synthesis and structure of the cell wall of Ustilago maydis was made by in silico analysis of the fungal genome, with reference to supporting experimental evidence. The composition of the cell wall of U. maydis shows similarities with the structural composition of the walls of Ascomycetes, but also shows important differential features. Accordingly, the enzymes involved in the synthesis of the U. maydis wall polysaccharides chitin and β-1,6 glucans displayed some differential characteristics. The most salient difference in protein composition was the predicted absence of Pir proteins, an important class of proteins present in the Ascomycetes.…
Identification and study of a Candida albicans protein homologous to Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein
After screening of aCandida albicansgenome database, the product of an ORF (IPF 3054) that has 62 % homology withSaccharomyces cerevisiaeSsr1p, an internal cell-wall protein, was identified and named CaSsr1p. The deduced amino acid sequence shows that CaSsr1p contains an N-terminal hydrophobic signal peptide, is rich in Ser and Thr amino acids and has a potential glycosylphosphatidylinositol-attachment signal. CaSsr1p is released following degradation of isolated cell walls by zymolyase (mainly a 1,3-β-glucanase) and therefore seems to be covalently linked to theβ-glucan of the cell walls. Both disruption and overexpression of theCaSSR1gene caused an increased sensitivity to calcofluor whit…
Determination of the stability of protein pools from the cell wall of fungi.
Stability of the protein populations present in the cell wall of three ascomycetous fungi Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica was investigated. Cell wall proteins were either labeled with biotin or radiolabeled with amino acids, and chased for a period of time representing several generations. Proteins linked by non-covalent or covalent bonds were separated and their turnover was analyzed. No significant turnover took place during the chase period, and in fact radioactive proteins were accumulated in the wall during the period possibly by transfer through the secretory pathway. This transfer did not involve de novo protein synthesis; it was inhibited by azide,…
Genomic response programs of Saccharomyces cerevisiae following protoplasting and regeneration.
Abstract Global transcription profiling during regeneration of Saccharomyces cerevisiae protoplasts was explored. DNA microarrays measured the expression of 6388 genes and wall removal resulted initially in over-expression of 861 genes that decayed later on, a behaviour expected from a transient stress response. Kinetics of expression divided the genes into 25 clusters. Transcription of the genes from clusters 14–25 was initially up-regulated, suggesting that the grouped genes permitted cell adaptation to the removal of the wall. Clustering of genes involved in “wall structure and biosynthesis” showed that most of them had initially low levels of expression that increased along the process.…
Functional analysis of the cysteine residues and the repetitive sequence ofSaccharomyces cerevisiaePir4/Cis3: the repetitive sequence is needed for binding to the cell wall β-1,3-glucan
Identification of PIR/CIS3 gene was carried out by amino-terminal sequencing of a protein band released by β-mercaptoethanol (β-ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with β-1,3-glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys214-12aa-Cys227-COOH), reduced but did not eliminate incorporation of Pir4 (protein with internal repeats) by disulphide bridges. Similarly, site-directed mutation of two other cysteine amino acids (Cys130Ser or Cys197Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2-unprocessed Pir4. Therefore, it seems th…
Anchorage of Candida albicans Ssr1 to the cell wall, and transcript profiling of the null mutant.
Incorporation into the wall of Candida albicans Ssr1, a GPI-dependent protein, was investigated by construction of different truncated genes for which the three potential omega sites (S199, S215 and G216) and the corresponding omega+1 and omega+2 were eliminated or modified. Cells of the C. albicans ssr1Delta mutant were transformed with pADH-pl harboring the truncated versions of CaSSR1, pADH-DeltaCaSSR1t(217-234) (lacking a C-terminal hydrophobic stretch of 18 aa including the putative omega+2 and omega+1, omega+2 of S215 and G216) or pADH-DeltaCaSSR1t(199-201) (lacking three serine residues), and their walls were analyzed for the protein. Results suggested that the three serine residues …
Role of Pir1 in the construction of the Candida albicans cell wall
Searches in a Candida albicans database (http://genolist.pasteur.fr/CandidaDB/) identified two Individual Protein Files (IPF 15363 and 19968) whose deduced amino acid sequences showed 42 % and 45 % homology with Saccharomyces cerevisiae Pir4. The two DNA sequences are alleles of the same gene (CaPIR1) but IPF 19968 has a deletion of 117 bases. IPF 19968 encodes a putative polypeptide of 364 aa, which is highly O-glycosylated and has an N-mannosylated chain, four cysteine residues and seven repeats. Both alleles are expressed under different growth conditions and during wall construction by regenerating protoplasts. The heterozygous mutant cells are elongated, form clumps of several cells an…
A study of the Candida albicans cell wall proteome
Considering the importance of proteins in the structure and function of the cell wall of Candida albicans, we analyzed the cell wall subproteome of this important human pathogen by LC coupled to MS (LC-MS) using different protein extraction procedures. The analyzed samples included material extracted by hydrogen fluoride-pyridine (HF-pyridine), and whole SDS-extracted cell walls. The use of this latter innovative procedure gave similar data as compared to the analysis of HF-pyridine extracted proteins. A total of 21 cell wall proteins predicted to contain a signal peptide were identified, together with a high content of potentially glycosylated Ser/Thr residues, and the presence of a GPI mo…
Genomic response programs of Candida albicans following protoplasting and regeneration
Transcription profiling of Candida albicans cells responding to the elimination of the wall (protoplasts) and posterior regeneration was explored. DNA microarrays were used to measure changes in the expression of 6039 genes, and the upregulated genes during regeneration at 28 degrees C were assigned to fourteen categories. A total of 407 genes were upregulated during the process, of which 144 reached a maximum after 1 h. MKC1, a gene encoding a member of the regulatory pathway involved in cell wall integrity was overexpressed. Time-dependent expression divided the genes into 40 clusters. Clusters 1-19 were highly expressed initially (time 0) and downregulated following incubation, whereas t…