MOLECULAR AND CELLULAR BIOLOGY, Oct. 1986, p. 3490-3497 Vol. 6, No. 10 0270-7306/86/103490-08$02.00/0 Copyright C) 1986, American Society for Microbiology Saccharomyces cerevisiae Nuclear Fusion Requires Prior Activation by Alpha Factor MARK DAVID ROSE,t BONNIE R. PRICE, AND GERALD R. FINK* Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 Received 6 March 1986/Accepted 13 June 1986 We have developed a protocol for efficient fusion of spheroplasts of the same mating type. Nuclear fusion in this whole-cell system is also efficient and closely parallels nuclear fusion in heterosexual mating of intact cells. In the spheroplast fusion system, nuclear fusion is dependent on both the KARI gene and prior exposure to alpha factor. The major products of nuclear fusion in the spheroplast fusion assay were true diploids that were homozygous at the mating-type locus. An additional 10% of the products were cells of ploidy greater than diploid. The dependence of nuclear fusion on alpha factor treatment could not be replaced by synchronization in Gl by mutations in CDC28 and CDC35 or by prior arrest in stationary phase. These data suggest that nuclear fusion is not a constitutive function of the nucleus, but rather is specifically induced by mating hormone. In the yeast Saccharomyces cerevisiae, the nuclear enve- portion of the cell cycle, agglutination with cells of the lope remains intact throughout mitosis, meiosis, and conju- opposite mating type, morphological distortion (shmooing), gation (1). Consequently, formation of the diploid nucleus and selective removal of a portion of the cell wall to permit during conjugation (karyogamy) requires fusion of the nu- cytoplasmic fusion. A similar activation of the nucleus could clear membranes of the two haploid nuclei. Observation of be required for nuclear fusion. Alternatively, nuclear fusion conjugation by electron microscopy (2, 3) indicates that could be constitutive, occurring spontaneously once the two nuclear fusion (karyogamy) is mediated by extranuclear nuclei are present in the same cell. In this report we use a microtubules emanating from the spindle plaques embedded spheroplast fusion assay to fuse cells of the same mating within the nuclear envelope. These microtubules span the type. Our results show that nuclei, like cells, require activa- gap between the two haploid nuclei, which then move tion by conjugation-specific signals in order to fuse. together and fuse in the region of the spindle plaques. As a result, the newly formed diploid nucleus includes a novel MATERIALS AND METHODS zygotic spindle plaque. During conjugation, nuclear fusion occurs with high fidelity; usually more than 95% of the Strains and media. The S. cerevisiae strains used in this zygotes produce buds having strictly diploid nuclei. study are described in Table 1. Strains MY346 and MY348 Several mutations have been identified that disrupt nu- were transformed with a plasmid carrying the wildtype clear fusion without affecting cell fusion (4, 5, 7, 9, 21; J. H. KARI gene. The isolation of this plasmid will be described Thomas, doctoral thesis, Massachusetts Institute of Tech- elsewhere. nology, 1984). The first mutation to be associated with a Yeast strains were propagated nonselectively in liquid defect in nuclear fusion is karl-i (4, 9). In crosses with YPD medium (2% peptone, 1% yeast extract, and 2% karl-1, nuclear fusion fails in approximately 90 to 95% of all glucose) or on the surface of agar plates (YPD with 2% agar) matings. This defect is manifest to the same extent whether essentially as described in Sherman et al. (27). Selective karl-i is mated with the wild type or with karl-i strains. If medium was YNB (yeast nitrogen base [Difco Laboratories], both the cytoplasm and nuclei of the parents are appropri- 0.67%) supplemented with glucose to 2% and any essential ately marked, it can be shown that the buds from these zygotes contain one or more haploid nuclei and cytoplasmic TABLE 1. Strain list contributions from both These recombinant parents. prog- S. cererisiae Relevant genotype or phenotype eny are called cytoductants. Subsequent buds show no strain higher frequency of diploid nuclei than do the first buds (18), MY573 MATa his4-34 ura3-52 indicating that the nuclei within the zygote do not subse- MY577 MATa leul Ieu2 ura3-52 quently fuse. These features of nuclear fusion suggest that MY578 MATa karl-I his4-34 ura3-52 the process is not solely a passive fusion of the two mem- MY575 MATa karl-I leul leu2 ura3-52 branes, but is mediated by specific gene products. MY248 MA Ta his4-34 ura3-52 Eryr In the normal sequence of events in conjugation, cell MY346 MATa leul Ieu2 karl-i ura3-52 (pMR6) fusion is an obligatory prelude to nuclear fusion. Therefore, MY348 MATa his4-34 karl-i ura3-52 (pMR6) it is difficult to separate the functions required for cell MY415 MATa leu2-3 leu2-112 cdc28-1 ura3-52 contact and fusion from the functions required for nuclear MY416 MATa leu2-3 leu2-112 ura3-52 fusion. Yeast cells must be activated so that they can leave MY418 MATa his4-34 ura3-52 MY419 MATa his4-34 cdc28-1 ura3-52 the mitotic cycle and undergo the developmental events L455 MA To/ta lysl-l/lysl-l required for cell fusion. Activation leads to arrest in the Gl L94 MATot Ivs1-I 67-1D MATa his4-34 ura3-52 * 67-3C MA Ta his4-34 leu2-3 leu2-112 Corresponding author. 67-liB MA Ta his4-34 cdc35-1 ura3-52 t Present address: Department of Molecular Biology, Princeton MA Ta /his4-34 leu2-3 University, Princeton, NJ 08544. 67-12D cdc35-1 leu2-112 3490 VOL. 6, 1986 S. CEREVISIAE NUCLEAR FUSION 3491 nutritional requirement where appropriate as described in -ou STRiAIN A STRAIN B Sherman et al. (27). YNB medium was supplemented rou- tinely with a mixture of all amino acids plus adenine and uracil (except for those specific supplements for which t1 1t selection against auxotrophy was desired). Spheroplasts were regenerated in a layer of top agar SPHEROPLAST I NG (regeneration agar) composed of YNB medium with 1 M sorbitol and 3% agar as described by Hinnen et al. (15) except that no YPD medium was added. The regeneration ::.. agar also normally contained a mixture of all amino acids ::.. save histidine and leucine, except when the regeneration CELL FUSIO frequency was measured, in which case all amino acids were ....:.:::°:.:..° ...... added. Erythromycin resistance was assessed on YPGE medium .... .... .. ... (2% peptone, 1% yeast extract, 2% glycerol, 2% ethanol, and ... ... ... .. ..... .... NUCLEAR ... .... .. ..... .. ..... 2% agar) to which 4.0 mg of erythromycin per ml was FUSIa ON V~~~~-" ODEFECT... added. ... .. ...... Spheroplast fusion. Spheroplast fusion was performed essentially as described by van Solingen and van der Plaat ..... (29). Strains were grown to a density of 1 x 107 to 2 x 107 .... ~~~~~~~~~~~~~~~~~~~~~~~~.......... cells per ml in YPD medium at 30°C. Prior to alpha factor treatment, the cells were centrifuged briefly and suspended *: in 2 volumes of YPD medium that had been adjusted to pH .-........ 4.0 with hydrochloric acid. Synthetic alpha factor (Sigma Chemical Co.) dissolved in methanol was added to a final concentration of 5 ,uM, and the cells were incubated with shaking until more than 90% of the cells had arrested (2 h at 30°C) and begun to undergo the characteristic morphological change associated with mating factor arrest (shmooing). In experiments with temperature shifts, cultures were grown at PROTOTRC)PHI C HAPLOI D 24°C and then incubated at 34°C for 3 h, by which time the DIPLOI DCYTODUCTANTS majority of the cdc mutant cells had arrested with charac- FIG. 1. teristic morphology. Half of the culture was then incubated Spheroplast fusion assay. See text for details. with alpha factor for 2 h at 34°C, and half was incubated without alpha factor at 34°C for 2 h. Approximately 109 cells regenerant, where the regeneration frequency used is that of were harvested by centrifugation and suspended in 10 ml of the strain with the lower regeneration frequency. 1 M sorbitol. Glusulase (Endo Labs) was added to 2% (vol/vol), and the cells were incubated with gentle shaking at 30°C for 2 h until spheroplasts formed. The culture was RESULTS assumed to have been adequately converted to spheroplasts Spheroplast fusion assay. Cell fusion can have two different when more than 95% of the cells lysed after dilution into outcomes. If the haploid nuclei fuse, the resulting nucleus 0.1% sodium dodecyl sulfate. An additional indication that will be diploid and, if appropriately marked genetically, cells had been converted to spheroplasts was provided by prototrophic. Growth of the prototrophic diploid will lead to measurement of the difference in the viable titer of a culture the formation of a macroscopic colony on selective minimal that had been diluted in H20 before and after Glusulase medium. If nuclear fusion fails, the fused cells will be treatment. Viability after dilution in H20 was routinely transiently prototrophic heterokaryons that can divide on reduced about 100-fold by treatment with Glusulase. Cells the minimal medium until the individual nuclei segregate into were centrifuged and washed in 10 ml of 1 M sorbitol different daughter cells. The progeny of the transient supplemented with 10 mM Tris hydrochloride (pH 7.5) and heterokaryons should be a mixture of cells of both haploid 10 mM CaCl2 (STC). Spheroplasts were centrifuged again nuclear genotypes containing cytoplasmic elements from and suspended at 2 x 109 cells per ml in STC. The actual both parents. fusion was carried out by mixing together 0.1 ml of each Our standard spheroplast fusion assay is depicted in Fig. parent strain and then adding 2 ml of a solution of 40%o 1.