Abstract. Bud emergence, spindle pole body duplication and DNA replication are all dependent on the activation of the CDC28 protein kinase at the Start point in the G1 phase of the cell cycle. Bud emergence requires polarization of the cytoskeleton and secretory vesicles to a specific site on the cell surface. Cdc28p activated by Gl-cyclins triggers polarization of actin to the site of bud emergence and favors apical bud growth (Lew, D. J., and S. I. Reed. 1993. J. Cell Biol. 120:1305-1320 C ELL reproduction involves cell cycle-specific events occurring both in the nucleus and in the cytoplasm. In the budding cell cycle of Saccharomyces cerevisiae, a number of stage-specific events direct the position and the morphology of the bud. These events include the choice of a bud site, the localization of a specific group of proteins to the site from which the bud will emerge, and the polarization of cell surface growth to the new bud site. Assembly of the bud-site complex is required for the polarization of the yeast cytoskeleton and the targeting of secretory vesicles to the bud site (for reviews see references 9, 12, 32).The initial growth of the bud is highly polarized. Actin cortical patches (22), calmodulin (6), and the SPA2 protein (53, 54) are localized to the tips of the bud during this period of apical growth. Actin localization and bud growth are less polarized later in the cell cycle. The timing of this shift and the degree of delocalization determine bud shape, with isotropic growth favoring spherical forms (28,55).The entry into a new cell cycle involves the coordinated but independent pathways of bud initiation, spindle pole body duplication, and nuclear DNA synthesis (43). The triggering of these pathways all require the activation of the CDC28 protein kinase in the G1 phase of the ceil cycle (45). This regulatory point of the cell cycle has been baptized "Start" (18). The manner in which the CDC28 ldnase promotes bud formation at Start is not well defined. Lew and Reed (28) have shown that actin polarization can be triggered by the kinase activity of Cdc28p complexed with G1 cyclins, and they mapped the time at which actin polarization occurs in their synchronized cells to be soon after Start. Localization of Cdc3p (23) and Spa2p (54) to the presumptive bud site occurs substantially before bud emergence, but the precise timing of these events relative to CDC28 kinase activation at Start was not examined. However, cdc28 mutants at their restrictive temperature and wild-type cells treated with mating pheromone can polarize their growth to form a projection in the apparent absence of CDC28 kinase activity (28,31,54). These observations suggest that a pathway for polarizing growth independent of Cdc28p also exists in S. cerevisiae.We isolated a mutation of the SLT2 (MPKI) MAP kinase gene that augments the division defect of cells expressing a partially inactivated cdc28 mutant. We show that sit2 mutants exhibit defects in cell polarity and accumulate secretory vesicles. We suggest that Slt2p acts downst...