| Fus2p is a protein required for cell fusion during mating in Saccharomyces cerevisiae. In fus2 matings, intervening cell walls are not degraded and cells accumulate as prezygotes. Time-lapse microscopy of Fus2p-GFP revealed unexpected dynamic localization during mating. In pheromone-responding cells (shmoos), Fus2p showed four sites of localization: (1) nuclear, (2) mobile cytoplasmic, (3) stable, immobile cortical puncta, and (4) unstable cortical puncta. Time-lapse studies suggested that Fus2p was initially nuclear, but became concentrated at the shmoo tip. In wildtype pre-zygotes, Fus2p localized to the cell fusion zone, dilating as a ring during fusion.; Fus2p localization was dependent on both actin and Fus1p. Cells treated with latrunculin-A failed to show Fus2p movement, and Fus2p was localized more broadly as a cap at the shmoo tip. In a fus1Delta strain treated with latrunculin A, Fus2p neither moved nor localized. The results suggested that actin is required for Fus2p transport, whereas Fus1p is required for cortical anchoring.; Consistent with the dependence on actin, Fus2p localization was impaired in polarity mutants (bni1, pea2, and spa2). Fus2p was also mis-localized in rvs161 mutants specifically defective for binding to Fus2p. Nevertheless, in all of these mutant prezygotes, Fus2p localized to the cell fusion zone. These results, and the behavior of an epitope-tagged form of Fus2p, imply that a separate prezygote-specific signal for localization exists.; Dynamic localization of Fus2p also reflects critical regulatory events during mating. First, localization depends on pheromone concentration; Fus2p was exclusively nuclear at lower levels of pheromone, and in matings to partners producing inadequate levels. Second, Fus2p ectopically expressed in vegetative cells was nuclear, but localized to the shmoo tip in response to pheromone. Third, Fus2p remained nuclear in fus3 shmoos (FUS3 encodes the MAP kinase in the pheromone response pathway). Finally, after cell fusion, Fus2p returned to the nucleus. These results suggested that Fus2p is activated in the nucleus and that nuclear export occurs in response to pheromone. Relocalization to the nucleus after cell fusion may sequester excess Fus2p.; In sum, Fus2p, a critical component of the mating pathway, localizes dynamically in response to extracellular signaling and intracellular cell polarity determinants. |
