The sporulation-specific GAS2 and GAS4 genes are essential for the proper assembly of spore wall in Saccharomyces cerevisiae

The GAS family comprehends 5 paralogs encoding GPI-anchored proteins endowed with glucanosyltransferase activity. GAS1 is expressed during vegetative growth and plays a crucial role in cell wall assembly. In order to study the role of GAS2 and GAS4, we analysed the expression profiles during the entire sporulation process by use of Quantitative RT-PCR. GAS2 and GAS4 expression was periodic and overlapping. A peak occurred at 7 h from induction of sporulation and at 10 h the mRNA levels were already decreased. Immunoblot analysis using polyclonal antibodies raised against the purified Gas4p revealed that Gas4p is a GPI-anchored 60 kDa-membrane protein that was absent at the moment of induction of sporulation and reached a maximum at 11 h from transfer of the cells to sporulation medium. The double null mutant gas2 gas4/gas2 gas4 showed a severe reduction of the efficiency of sporulation whereas the single null mutants, gas2/gas2 and gas4/gas4 were similar to the isogenic strain. Only 4% of mature asci were present in the double mutant after 24 h of sporulation. These results indicate that GAS2 and GAS4 are redundant genes which play partially overlapping functions in spore morphogenesis. To determine if meiosis was affected in the double null mutant, we monitored the kinetics of nuclei formation. Meiotic progression occurred normally when Gas2 and Gas4 proteins were absent. A detailed analysis of the spore wall defects was performed using different kinds of assays. Spores of the double null mutant resulted to be highly permeable to Calcofluor. The spores released by a zymolyase treatment of the asci were unviable. Detailed analysis by electron microscopy of the spore walls of the double mutant indicated that the glucan and chitosan layers were dissociated and an abnormal accumulation of spore wall material occurred. These results suggests that Gas2 and Gas4 proteins play a crucial role in spore morphogenesis by directing the cross-linking between the inner and outer spore wall layers to each other