A.F.J. Ram

In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae

Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N‐terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl‐phosphatidylinositol (GPI)‐attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI‐attachment sites, it was found that in yeast the most probable amino acids for GPI‐attachment are asparagine and glycine.

Identification of two cell cycle regulated genes affecting the β1,3-glucan content of cell walls in Saccharomyces cerevisiae

The Calcofluor white‐hypersensitive mutants cwh52 and cwh53 are severely reduced in β1,3‐glucan. CWH52 was equivalent to GAS1. CWH53 represented a new gene, located on the right arm of chromosone XII, and predicted to encode a 215 kDa protein with multiple transmembrane domains. The transcription of CWH53 was cell cycle‐dependent and, similar to GAS1/CWH52, increased in late G1, indicating that the formation of β‐glucan is cell cycle‐regulated. Further, in some mutant alleles of both gas1/cwh52 and cwh53 lethal concentrations of Calcofluor induced growth arrest at a specific phase of the cell cycle.

Identification of SPT14/CWH6 as the yeast homologue of hPIG-A, a gene involved in the biosynthesis of GPI anchors

Cwh6 is a temperature-sensitive cell wall mutant of Saccharomyces cerevisiae. CWH6 was found to be identical to SPT14, a gene that is highly homologous to both human PIG-A and to RFAK from Salmonella typhimurium. PIG-A and RFAK are involved in transferring N-acetylglucosamine to, respectively, a GPI anchor precursor and to lipopolysaccharides. Because cell walls of cwh6 are greatly reduced in mannose, and because some cell wall proteins are known to be incorporated into the cell wall through a GPI-anchor dependent mechanism, we propose that Spt14p/Cwh6p is involved in transferring N-acetylglucosamine to a precursor of GPI anchors. We further propose that the majority of cell wall proteins are incorporated into the cell wall through a GPI anchor.

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway.

The Saccharomyces cerevisiae cwh43‐2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1–MPK1 pathway, including a growth defect and increased release of β‐1,6‐glucan and β‐glucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14–16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N‐terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2‐ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster. The C‐terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43–GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43‐2 mutant. This allele‐specific phenotype appears to be due to a G–R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high‐copy‐number expression of genes encoding downstream proteins of the PKC1 pathway However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G1, no growth defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1‐dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes. Copyright

Posttranslational modifications of secretory proteins

Publisher Summary This chapter focuses on post-translational modifications of secretory proteins. Proteins that traverse the secretory pathway and reach the plasma membrane may be targeted to various locations. One final destination is the plasma membrane itself. The plasma membrane contains not only many transmembrane proteins, but also a special group of proteins, which are C-terminally linked to the outer leaflet of the plasma membrane through a so-called “glycosylphosphatidylinositol” (GPI) anchor. Between the plasma membrane and the cell wall, periplasmic proteins, such as invertase and acid phosphatase, may accumulate. A limited number of proteins, such as chitinase, and several heat shock proteins are secreted into the medium. Secretory proteins are modified in various ways during their passage through the secretory pathway. The chapter discusses techniques to identify and characterize the post-translational modifications of secretory proteins. It is important to realize that these techniques, although primarily developed for Succhuromyces cereuisiae , are in many cases also valid for other Ascomycetes, including the filamentous fungi belonging to this taxonomic group. The chapter discusses the glycosylation of secretory proteins and the detection of N - and O -glycosylation in secretory proteins.

The protein kinase Kic1 affects 1,6-beta-glucan levels in the cell wall of Saccharomyces cerevisiae.

KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity. Both over- and underexpressing conditions of KIC1 affected cell wall composition. Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-beta-glucan. When Kic1-deficient cells were crossed with various kre mutants, which also have less 1,6-beta-glucan in their wall, the double mutants displayed synthetic growth defects. However, when crossed with the 1,3-beta-glucan-deficient strain fks1delta, no synthetic growth defect was observed, supporting a specific role for KIC1 in regulating 1,6-beta-glucan levels. Kic1-deficient cells also became highly resistant to the cell wall-degrading enzyme mixture Zymolyase, and exhibited higher transcript levels of the cell wall protein-encoding genes CWP2 and SED1. Conversely, overexpression of KIC1 resulted in increased sensitivity to Zymolyase and in a higher level of 1,6-beta-glucan. Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3. Consistent with this, expression levels of RHO3 correlated with 1,6-beta-glucan levels in the cell wall. Interestingly, expression levels of KIC1 and the MAP kinase kinase PBS2 had opposite effects on Zymolyase sensitivity of the cells and on cell wall 1,6-beta-glucan levels in the wall. It is proposed that Kic1 affects cell wall construction in multiple ways and in particular in regulating 1,6-beta-glucan levels in the wall.