Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein.

Abstract

We characterized two genes, FUSJ and FUS2, which are required for fusion of Saccharomyces cerevisiae cells during conjugation. Mutations in these genes lead to an interruption of the mating process at a point just before cytoplasmic fusion; the partition dividing the mating pair remains undissolved several hours after the cells have initially formed a stable "prezygote." Fusion is only moderately impaired when the two parents together harbor one or two mutant fus genes, and it is severely compromised only when three or all four fus genes are inactivated. Cloning of FUSI and FUS2 revealed that they share some functional homology; FUSI on a high-copy number plasnmid can partially suppress a fus2 mutant, and vice versa. FUS) remains essentially unexpressed in vegetative cells, but is strongly induced by incubation of haploid cells with the appropriate mating pheromone. Immunofluorescence microscopy of a factor-induced a cells harboring afusl-LACZ fusion showed the fusion protein to be localized at the cell surface, concentrated at one end of the cell (the shmoo tip). FUSI maps near HIS4, and the intervening region (including BIKI, a gene required for nuclear fusion) was sequenced along with FUSI. The sequence of FUS] revealed the presence of three copies of a hexamer (TGAAAC) conserved in the 5' noncoding regions of other pheromone-inducible genes. The deduced FUS1 protein sequence exhibits a striking concentration of serines and threonines at the amino terminus (46%; 33 of 71), followed by a 25-amino acid hydrophobic stretch and a predominantly hydrophilic carboxy terminus, which contains several potential N-glycosylation sites (Asn-X-Ser/Thr). This sequence suggests that FUSI encodes a membrane-anchored glycoprotein with both N-and 0-linked sugars.Conjugation in the yeast Saccharomyces cerevisiae involves the fusion of haploid cells of opposite mating type followed by the fusion of nuclei to form a diploid. The zygote formed by this process buds off diploid cells capable of vegetative growth. Formation of the zygote requires the coordination of two processes-cell fusion and nuclear fusion. Both processes are initiated by mating pheromones: a cells produce a factor, to which a cells respond, and a cells produce a factor, which specifically acts on a cells (13,44).Cells stimulated by the appropriate mating pheromone produce surface agglutinins (resulting in extensive clumping of conjugating cultures) (33, 40), arrest their cell cycle at the Gl stage, and elongate to form a discernible tip (a process dubbed shmooing) (12, 44; for a review, see reference 41). When the appropriate partners have achieved contact, presumably at the shmoo tip, the cells rapidly fuse, a process that requires the degradation or reorganization (or both) of the cell wall and the fusion of the two plasma membranes (see Fig. 8). The nuclei subsequently fuse within this dumbbell-shap-ed zygote, and the resultant diploid nucleus begins a series of division cycles, each of which yields a new diploid nucletis that ehters an enierging bud. Recent e...

Hsp90 Is Required for Pheromone Signaling in Yeast

“…To test the Hsp90 requirement in the pheromone-dependent transactiva- tion of mating genes such as FUS1, we assessed the induction of a FUS1-LacZ reporter gene (Trueheart et al, 1987) upon treatment of cells with ␣-factor. The results shown in Figure 1C indicate that, similarly to what was observed in the case of the G 1 arrest, the activation of FUS1-LacZ in response to ␣-factor is strongly reduced in Hsp90 mutant strains compared with a strain with wild-type Hsp82.…”

“…The FUS1-LacZ reporter plasmid pSB234 was used to measure the transcriptional output of the pheromone pathway (Trueheart et al, 1987). Wild-type and mutant strains were grown to early logarithmic phase and exposed to 5 M ␣-factor for 2 h after addition of 10 mM Na-citrate, pH 4.3.…”

Interferon-γ Activation of a Mitogen-activated Protein Kinase, KFR1, in the Bloodstream Form of Trypanosoma brucei

“…pRS-FUS3 contains FUS3 gene in pRS314 under GAL1 promoter. pFus1-LacZ contains the Fus1-LacZ gene and the LEU2 gene (19,20). To clone KFR1 into pRS314 vector, BamHI and SalI sites were generated flanking the KFR1 (16) by polymerase chain reaction (PCR) using primer B (5Ј-GAAGGATCCATGGTGTCGTTCAGCATC) and primer S (5Ј-GGAG-GTCGACTATGGCTGAGCAACATTTC).…”

“…GAL1 promoter was activated by dextrose-free SC medium containing 2% galactose, 0.5% sucrose, 2% glycerol, 1% raffinose, and tryptophanfree amino acid mixture. After a 1-h induction by ␣-factor, LacZ expression was monitored as described (19).…”

CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.

“…Each of these haploid cell types secretes a specific peptide pheromone (a-factor and a-factor) to which the other cell type responds. In the presence of the appropriate pheromone, haploid cells arrest in Gl at START; induce transcription of particular genes such as FUS1 that are required for mating (Trueheart et al, 1987); and undergo a morphological change in which a projection is extended from one face of the cell, giving the cells an elongated shape (Levi, 1956). The signal transduction pathway that mediates this response has been well characterized genetically (Cross et al, 1988).…”