Mechanisms for targeting of the Saccharomyces cerevisiaeGPI-anchored cell wall protein Crh2p to polarised growth sites

The cell wall is an essential structure that preserves the osmotic integrity of fungal cells and determines cellular morphology during developmental programs. The high number of different wall components demands a variety of processes to deliver precursors and synthetic proteins to the proper location at the right time for wall development and modification. Here,the specificity of the mechanisms that regulate the temporal and spatial localisation of cell wall proteins to sites of polarised growth in Saccharomyces cerevisiae is investigated. For this purpose, the localisation of Crh2p, a cell wall glycosylphosphatidylinositol (GPI)-anchored mannoprotein that we have recently described as involved in cell wall construction and localised to polarised growth sites, was followed using a Crh2p-GFP fusion protein. Crh2p distribution was studied in several genetic backgrounds affected in different steps of the cell polarity establishment machinery or/and bud morphogenesis. Crh2p is localised at the mother-bud neck in bud1 cells following the random budding pattern characteristic of this mutant. The Crh2p distribution was greatly altered in a cdc42-1mutant, indicating complete dependence on an organised actin cytoskeleton for polarised Crh2p distribution. The usual deposition of Crh2p in a ring at the base of growing buds was lacking in cdc10-11 cells growing under restrictive temperature conditions, whereas Crh2p deposition at the septum region was absent in both cdc10-11 and cdc15-lyt1 cells. These results point to the dependence of Crh2p localisation at the bud-neck on both septins and septum integrity. Furthermore, in the absence of Bni4p, a scaffold protein involved in the targeting of the chitin synthase III complex to the bud neck, Crh2p was not longer found at the neck in large-budded cells undergoing cytokinesis. Finally, Crh2p was not properly localised in cells deleted in CHS5, which encodes a protein involved in the transport of Chs3p, and was completely mislocalised in sbe2/sbe22 mutants,suggesting that the transport systems for Chs3p and Crh2p are to a certain extent coincident. The transport of other GPI-cell wall proteins, such as Cwp1p, however, does not depend on these systems as the localisation of the latter protein was not affected in either of these mutants.