GAS2 and GAS4 : a pair of developmentally regulated genes involved in spore wall assembly in yeast

The GAS multigene family comprehends 5 paralogs encoding GPI-anchored proteins endowed with glucanosyltransferase activity. Gas1p, the best characterized protein of the family, is expressed during vegetative growth and plays a crucial role in cell wall assembly. In order to study the role of GAS2 and GAS4 genes, that were previously classified as middle meiotic genes, we analysed their expression profiles during the entire sporulation process by Quantitative RT-PCR in a SK1 strain. GAS2 and GAS4 expression profiles were 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 is absent at the moment of induction of sporulation and reaches 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. 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 morphologically aberrant, less viable, highly permeable to Zymolyase and Calcofluor. TEM and SEM analysis of the spores of the double mutant indicated that the glucan and chitosan layers are dissociated and an abnormal accumulation of spore wall material occurs. These results suggests that Gas2 and Gas4 proteins play a crucial role in spore morphogenesis by direecting the attachment of the inner and outer spore wall layers to each other .