A protein interaction map for cell polarity development

Many genes required for cell polarity development in budding yeast have bee n identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in di verse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms tha t coordinate the numerous events involved, we carried out a large-scale two -hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins as sociated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express similar to 90% of the predicted Saccharomyces cerevisiae open readi ng frames as Gal4 activation domain fusions. 191 protein-protein interactio ns were detected, of which 128 had not been described previously. 44 intera ctions implicated 20 previously uncharacterized proteins in cell polarity d evelopment. Further insights into possible roles of 13 of these proteins we re revealed by their multiple two-hybrid interactions and by subcellular lo calization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization , actin assembly, microtubule organization, autophagy, cytokinesis, and cel l wall synthesis. Other interactions suggested direct connections between R ho1- and Cdc42-regulated pathways; the secretory apparatus and regulators o f polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactio ns that provide an integrated response of signaling proteins, the cytoskele ton, and organelles to the spatial cues that direct polarity development wa s revealed.