TARGETING OF CHITIN SYNTHASE-3 TO POLARIZED GROWTH SITES IN YEAST REQUIRES CHS5P AND MYO2P

Chitin is an essential structural component of the yeast cell wall who se deposition is regulated throughout the yeast life cycle. The tempor al and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localizati on of the putative catalytic subunit of chitin synthase III, Chs3p, an d its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p rev ealed a novel localization pattern that is cell cycle-dependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the n eck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In larg e-budded cells undergoing cytokinesis, it reappears as a ring at the n eck. In cells responding to mating pheromone, Chs3p is found throughou t the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also fo und in cytoplasmic patches in cells at different stages of the cell cy cle. Epitope-tagged Chs3p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly al l Chs3p patches contain Chs5p, whereas some Chs5p patches lack detecta ble Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic pat ches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furt hermore, Chs5p localization is not affected either by temperature shif t or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p po larization to cortical sites in yeast is dependent on both Chs5p and t he actin cytoskeleton/Myo2p.