University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan. Received 1 April 1993/Accepted 30 June 1993. The buoyant densities of the yeast cells of defective ...
Vol. 175, No. 17
JOURNAL OF BACTERIOLOGY, Sept. 1993, p. 5714-5716
0021-9193/93/175714-03$02.00/0 Copyright C) 1993, American Society for Microbiology
Density Fluctuation during the Cell Cycle in the Defective Vacuolar Morphology Mutants of Saccharomyces cerevisiae MARIKO OHSUMI,l* KEIKO UCHIYAMA,2 AND YOSHINORI OHSUMI2
Department ofBioscience, Faculty of Science and Engineering The Nishi-Tokyo University, Uenohara, Kita-Tsuru-gun, Yamanashi 409-01, 1 and Department ofBiology, College of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan Received 1 April 1993/Accepted 30 June 1993
The buoyant densities of the yeast cells of defective vacuolar morphology mutants were examined by equilibrium sedimentation centrifugation in a Percoll density gradient. These vacuoleless mutants also show density fluctuation as wild-type cells during the cell cycle. This suggests that morphological changes of the vacuole are not related to cyclic density fluctuation in Saccharomyces cerevisiae.
KL197-1D (vamS); haploid cells, X2180-1A (a), YW10-5B (a,
Budding yeast cells of Saccharomyces cerevisiae show periodic fluctuation in their buoyant densities during the cell cycle (2). When logarithmically growing cells are subjected to equilibrium sedimentation centrifugation by using a Renografin-sucrose density gradient (2) or a Ficoll density gradient (1), cells distribute in a broad band. Minimum density occurs at the time of cell separation, whereas the maximum occurs between DNA replication and nuclear
vamS adel), YWHO42-1D (a, vam8), and the mostly iso-
genic parental strain ANY21 (a, VAM). Each strain was cultured in yeast extract-peptone-dextrose medium to 2.0 x 107 cells per ml. Strains were quickly collected by centrifugation and then were resuspended in yeast extract-peptonedextrose medium at 2.0 x 108 cells per ml. After a brief sonication, 1 ml of each of the cell suspensions was quickly layered onto a Percoll density gradient and was centrifuged at 4,000 x g for 10 min in a swinging bucket with a KUBOTA-05PR centrifuge. The Percoll density gradient used was prepared according to the method of Baldwin et al. (1) with a slight modification. After bands were fractionated, cells of each fraction were collected by centrifugation and their morphologies were examined under a phase-contrast
division.
These methods are simple and good procedures for obtaining a synchronous culture of yeast cells. So far, similar density fluctuations during the cell cycle have only been reported with Chlorella spp. (11) and Candida utilis (8). However, various cells, including Eschenichia coli (5), mammalian cells (7), and the fission yeast Schizosaccharomyces pombe (6), were shown to have a constant density during their cell cycles. The reason for density fluctuation of budding yeast cells is not yet known. The largest intracellular compartment, the vacuole, has been previously shown to exhibit dynamic morphological changes during the cell cycle (9, 13, 14). Wiemken et al. (14) made freeze-fracture preparations of synchronously growing cells and observed that just prior to bud initiation, the few large vacuoles present in the mature mother cell began to shrink and fragment into the newly formed bud. Vacuolar sap contains mainly low-molecular-weight substances, such as amino acids, inorganic salts, and various hydrolytic enzymes. Since the concentration of protein in the vacuole is far lower than that of cytosol, it is possible that the density fluctuation is caused by a morphological change of the vacuolar compartment. Recently, Kitamoto et al. (3) and Wada et al. (12) isolated vacuolar morphology mutants. Four class I vam mutants, vaml, vamS, vam8, and vam9, certified as class C vps mutants (10), were shown to contain no apparent vacuolar compartments by biochemical and morphological analyses. These mutants may provide good experimental objects for studying the possibility discussed above. If the vacuole is relevant to cell buoyant density fluctuation, class I vam mutants may not change their buoyant densities during the cell cycle. The following strains were used in this work: diploid cells, X2180-1D (VAM) and the isogenic vamS homozygous diploid *
microscope. Both the top and the bottom portions were quickly resuspended in fresh, prewarmed yeast extract-peptone-dextrose medium, and their growth was monitored at 30°C. In order to see simply the density distribution of the cells, a 50-to-100% linear Percoll gradient was also used. The wild-type diploid cells X2180-1D gave a broad band in the Percoll gradient. The top portion contained mainly cells with a large bud or no bud (Fig. la), while cells in the bottom portion had a small bud (Fig. lc). These results confirmed observations by Hartwell (2) and Baldwin et al. (1). Cells from both the top and the bottom portions contained clear central vacuoles (Fig. lb and d), but the vacuoles of cells from the top of the band were apparently larger than those of cells from the bottom portion. KL197-1D, the vamS mutant, gave a similar broad band, but it was apparently at a position lower than that of X2180-1D in the Percoll gradient (data not shown). The buoyant densities of the wild-type cell and the vamS mutant of the peak fractions were 1.103 and 1.111 g/ml, respectively. This indicates that the vamS mutation makes the cell heavier. The top portion of the band of the mutant contained mostly cells with a large bud or unbudded cells (Fig. le), and the bottom portion contained cells with a small bud (Fig. lg). When both fractions were cultured in yeast extract-peptonedextrose medium, they gave good synchrony, at least in two cell cycles (data not shown). Careful examination of cells with a phase-contrast microscope (Fig. le and g) or a fluorescence microscope with quinacrine (Fig. lf and h) failed to show any vacuoles in the vam mutant. These results indicate that the vamS mutant also shows
Corresponding author. 5714
VOL. 175, 1993
NOTES
5715
FIG. 2. Isopycnic banding of exponentially growing haploid cells by Percoll density gradient centrifugation. Cells (1.0 ml) were layered onto a Percoll gradient and centrifuged at 4,000 x g for 10 min at room temperature with a swing rotor of KUBOTA-05PR. The strains used are X2180-1A (VAM) (1), YW10-2B (vamS) (2), YWH042-1D (vam8) (3), and ANY21 (VAM) (4).
fractions of X2180-1A (Fig. 3a and b) and YWH042-1D (Fig. 3c and d). Another class I mutant, the vam9 strain, also gave results similar to those by the vamS and vam8 mutants (data not shown).
During this work, we found that the buoyant density of S. cerevisiae also depends on its growth phase. Cells became dense when they reached the stationary phase. The extent of change during the growth phase was much greater than that of the change in one cell cycle. So we carefully compared the buoyant densities of all strains at the same growth stage in the middle-logarithmic phase.
FIG. 1. Phase-contrast (a, c, e, and g) and fluorescence (b, d, f, and h) photomicrographs of wild-type and vamS mutant cells. Cells of strains X2180-1D (a, b, c, and d) and KL197-1D (e, f, g, and h) were centrifuged in a Percoll density gradient. The top (a, b, e, and f) and the bottom (c, d, g, and h) portions of each band of cells were collected. Vacuolar morphologies were examined by quinacrine staining and fluorescence microscopy after staining with 1 mM quinacrine. Magnification, x 1,000.
buoyant density fluctuation as the wild type during the cell cycle. Next, we examined haploid cells of other class I vam mutants. As shown in Fig. 2, wild-type cells of X2180-1A or ANY21 gave as broad a band as diploid cells in the Percoll gradient. Phase-contrast microscopic observation and culture of the top and the bottom portions showed that the buoyant density of haploid cells fluctuates during the cell cycle in a manner similar to that of diploid cells (data not shown). Both YW10-2B (vamS) and YWHO42-1D (vam8) showed one broad band in the Percoll gradient that was lower than those of two wild-type strains (Fig. 2). Fractionation of these bands also proved the cell cycle-dependent fluctuation of buoyant density. Figure 3 shows the phasecontrast micrographs of cells from the top and the bottom
FIG. 3. Photomicrographs of haploid cells collected from the top (a and c) and the bottom (b and d) portions of bands obtained by Percoll density gradients. The strains used are X2180-1A (a and b) and YWHO42-1D (c and d). Magnification, x 1,000.
5716
J. BA=rRIOL.
NOTES
It is well-known that just before reaching the stationary phase, yeast cells accumulate storage polymers such as glycogen and polyphosphates (4). The periodic synthesis and breakdown of macromolecules in the cytosol may be the reason for density fluctuation during the cell cycle of S. cerevisiae. We are grateful to Yoh Wada of the University of Tokyo for kindly providing yeast strains of class T vam mutants.
REFERENCES 1. Baldwin, W. W., and H. E. Kubitschek 1984. Buoyant density variation during the cell cycle of Saccharomyces cerevisiae. J. Bacteriol. 158:701-704. 2. Hartwell, L. H. 1970. Periodic density fluctuation during the yeast cell cycle and the selection of synchronous culture. J. Bacteriol. 104:1280-1285. 3. Kitamoto, K., K. Yoshizawa, Y. Ohsumi, and Y. Anrakl. 1988. Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae. J. Bacteriol. 170:2683-2686. 4. Klionsky, D. J., P. K. Herman, and S. D. Emr. 1990. The fungal vacuole: composition, function, and biogenesis. Microbiol. Rev. 54:266-292. 5. Kubitschek, H. E., W. W. Baldwin, and R. Graetzer. 1983. Buoyant density constancy during the cell cycle of Escherichia coli. J. Bacteriol. 155:1027-1032. 6. Kubitschek, H. E., and R. A. Ward. 1985. Buoyant density
7. 8. 9.
10. 11.
12.
13. 14.
constancy of Schizosaccharomyces pombe cells. J. Bacteriol. 162:902-904. Loken, M. R., and H. E. Kubitschek 1984. Constancy of cell buoyant density for cultured murine cells. J. Cell. Physiol. 118:22-26. Nurse, P., and A. Wiemken. 1974. Amino acid pools and metabolism during the cell division cycle of arginine-grown Candida utilis. J. Bacteriol. 117:1108-1116. Raymond, C. K., P. J. O'Hara, G. Ellgr, J. H. Rothman, and T. H. Stevens. 1990. Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle. J. Cell Biol. 111:877892. Raymond, C. K., C. J. Roberts, K E. Moore, L Howard, and T. H. Stevens. 1992. Biogenesis of the vacuole in Saccharomyces cerevisiae. Int. Rev. Cytol. 139:59-120. Sitz, T. 0., A. B. Kent, H. A. Hopkins, and R R. Schmidt. 1970. Equilibrium density-gradient procedures for selection of synchronous cells from asynchronous cultures. Science 168:12311232. Wada, Y., Y. Ohsumi, and Y. Anrakn. 1992. Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. 1. Isolation and characterization of two classes of vam mutants. J. Biol. Chem. 267:18665-18670. Wlemken, A., H. K Hopkins, and P. H. Matgle. 1970. Properties of the vacuole in baker's yeast synchronized with a new method. Acta Fac. Med. Univ. Brun. 37:47-52. Wlemken, A., P. Matile, and H. Moor. 1970. Vacuolar dynamics in synchronously budding yeast. Arch. Mikrobiol. 70:89-103.
