| Cell fusion is ubiquitous among eukaryotes, fulfilling essential roles in fertilization, development and tissue repair. In yeast, a polarized shmoo contacts its mating partner, forming a zone of cell fusion (ZCF). Fus2p, a key regulator of cell wall removal during fusion, interacts with Rvs161p, an amphiphysin, and the complex localizes to the shmoo tip. We found that Rvs161p facilitates cortical localization through membrane interaction, but Fus2p's C-terminus forms a novel Rvs161p-independent localization signal. Analysis of C-terminal Fus2p truncations suggested that an auto-inhibitory mechanism interferes with localization via an upstream region. Fus2p is known to be retained at the shmoo tip by both the pheromone-induced transmembrane protein, Fus1p, and actin. We find that the actin-dependent pathway requires the C-terminus, whereas the Fus1p pathway depends on more internal sequences.;To identify additional Fus2p-interacting proteins, we performed a high copy suppressor screen of the C-terminal mutations, and identified Kel1p. Kel1p, a kelch-domain protein, was implicated in cell fusion, though its function was unknown. Kel1p and its homologue, Kel2p, are required for Fus2p localization, and act through the actin-dependent pathway. Kel1p also functions in mating independent of Fus2p localization. To facilitate fusion, Fus2p interacts with Cdc42p, a Rho-like GTPase that promotes growth and morphogenesis. Through suppression analysis of Cdc42p mutations, we hypothesize that Fus2p, Cdc42p and Kel1p form a complex during fusion. In support, Kel1p interacts with two different domains of Fus2p, dependent on Cdc42p.;We next examined Cdc42p's function during cell wall degradation. In prezygotes, Cdc42p localized to a novel focus at the center of the ZCF, and remained associated with remnant cell walls after fusion. Cdc42p and Fus2p co-localized, and Cdc42p localization was dependent upon Fus2p. Earlier, during shmooing, Cdc42p localization was independent of Fus2p and the two proteins were spatially separate. Cdc42p focus formation was dependent on changing membrane curvature at the ZCF. Signaling and morphogenesis did not require a Cdc42p focus, but a cdc42 secretion-specific allele blocked fusion. As both fus2 and cdc42 mutant zygotes accumulate vesicles, these observations support a model where Fus2p localizes Cdc42p to the flat ZCF to promote exocytosis of hydrolases that remove the cell wall. |
