Abstract. Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindies), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.A important class of questions about the cell division cycle concerns the dependency relationships or other coordinating mechanisms that ensure that cell-cycle events occur in an appropriate sequence. Such questions have been investigated in the yeast Saccharomyces cerevisiae by using mutations and inhibitors that block specific cellcycle events (Hartwell et al., 1974;Pringle, 1978;Pringle and Hartwell, 1981; Moir and Botstein, 1982;Wood and Hartwell, 1982;Jacobs et al., 1988; Hartwell and Weinert, 1989). In this context, temperature-sensitive (Ts-) ~ mutants defective in gene CDC24 have been of great interest. The observation that such mutants can continue DNA synthesis and nuclear division while bud emergence is blocked (Hartwell et al., 1973(Hartwell et al., , 1974 suggests that the nuclear cycle is not dependent on the cytoplasmic processes involved in budding. Conversely, experiments with a variety of other mutations and inhibitors suggest that bud emergence is not dependent on the nuclear cycle (Hartwell et al., 1974;Pringle and Hartwell, 1981). Thus, it appears that many of the events of the yeast cell cycle are organized into two parallel and indepen-A. E. M. Adams' present address is

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i .e ., increase in cell mass and cell volume) at restrictive temperature (36°C) but were unable to form buds . Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36°C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanateconjugated concanavalin A suggested that mannan incorporation was also delocali-Zed in mutant cells grown at 36°C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded cells as well as in unbudded cells.Growth of the mutant strains at temperatures <36°C revealed allele-specific differences in behavior . Two strains produced buds of abnormal shape during growth at 33°C. Moreover, these same strains displayed abnormal localization of budding sites when grown at 24°C (the normal permissive temperature for the mutants) ; in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses.Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape .Cellular morphogenesis is the process by which cellular form and spatial organization are generated . During the cell division cycle of the yeast Saccharomyces cerevisiae, cellular morphogenesis includes the following sequential events : (a) selection of a nonrandom site at which budding will occur (1,14,23,25, 41,46) ; (b) formation of a ring of chitin (the "bud scar") in the largely nonchitinous cell wall at that site (24, 34) ; (c) localization of new cell wall growth to the region bounded by the chitin ring, resulting in the appearance and selective growth of a bud (10,11,22,27,28); (d) localization of new cell wall growth to the tip of the growing bud (l0, 11, 27, 43); (e) cytokinesis and the formation of septal cell wall (6, 43) . In addition, it seems that periods of uniform growth of the bud cell wall precede and follow the period of tip growth (11) ; presumably, the relative amounts of tip growth and of uniform growth are adjusted to yield the normal ellipsoidal shape of the daughter cell (10, 11) .

In the budding yeast Saccharomyces cerevisiae, each bud appears within a ring of chitin formed in the cell wall of the mother cell. Temperature-sensitive mutants defective in gene cdc24 synthesize chitin at restrictive temperatures, but do not organize it into the discrete rings found in normal cells, nor do they form buds. The chitin ring or an annular precursor structure may play an essential role in reinforcing the region of the cell wall involved in budding.

Portions of this work were submitted in partial fulfillment of the requirements for the Ph.D. degree. The work was supported by funds from an NIH training grant in the Department of Zoology, The University of Michigan, (NIH 5T 01GM00989). 514Sloat & Allen: Lysosomal Acid Phosphatase MATERIALS AND METHODS Preparation of Liver Homogenates 575Normal tissue. Normal tissue used in quantitative and electrophoretic studies was obtained from 300 to 400-gram albino male rats. Animals were fed a diet of Purina Lab Chow and water ad libitum. After a starvation period of 12 to 18 hours, animals were sacrificed by cervical dislocation. The liver was removed immediately, weighed, and an initial 20% (w/v) homogenate prepared by bringing 20 g of tissue to a final volume of 100 ml with 0.25 M sucrose (Merck, reagent grade). Tissue was homogenized at 0°C in a smooth glass homogenizer with a Teflon pestle using 5 to 8 up-down strokes at 1200 revlmin.Regenerated tissue. Animals were partially hepatectomized under Nembutal anesthesia (Merck, 20 mg/ kg body weight) according to the method of Higgins and Anderson (1931). At the time of partial hepatectomy, the median and left lateral lobes were ligated and removed. This tissue was homogenized as above and utilized as control tissue. During the first 24 hours after the operation, animals were provided with 20% dextrose (Mallinckrodt analytical) ad libitum. After this period, food and water were given as usual. After the required regeneration period, animals were sacrificed and homogenates prepared as previously described. Difleren tial CentrifugationNormal and regenerated whole tissue homogenates were subjected to differential centrifugation at 4 O C according to one of the following schemes. Centrifugation times and g values are designated exclusive of acceleration and deceleration times and are based on average r-values. Scheme 1 :Nuclear fraction. Whole homogenates were centrifuged at 480 X g for 10 minutes followed by two washes ( 5 up-down strokes each), resuspensions, and recentrifugations at 270 X g for 10 minutes each. The washed pellet was resuspended to starting volume in 0.25 M sucrose to yield a final 20% fraction. All other pellets were resuspended to starting volume in the same manner.Mitochondrial-lysosomal fraction. The combined supernatants from the nuclear step were centrifuged at 22,000 X g for 30 minutes to sediment mitochondria and lysosomes.Microsomal-soluble fraction. The supernatant remaining after the mitochondrial-lysosomal centrifugation was designated the microsomal-soluble fraction. All centrifugations were carried out in a Servall RC 2 refrigerated centrifuge equipped with an SS-34 rotor. Scheme 2 :Nuclear fraction. Whole homogenates were centrifuged at 270 X g for 10 minutes. Two resuspensions (homogenized with 8 up-down strokes at 1,200 rev/ min) and recentrifugations followed at the same speed and time. The washed pellet was brought to starting volume with 0.25 M sucrose.Mitochondria1 fraction. The combined supernatants from the nuclear sedimentation wer...

Normal tissue. Normal tissue used in quantitative and electrophoretic studies was obtained from 300 to 400-gram albino male rats. Animals were fed a diet of Purina Lab Chow and water ad libitum. After a starvation period of 12 to 18 hours, animals were sacrificed by cervical dislocation. The liver was removed immediately, weighed, and an initial 20% (w/v) homogenate prepared by bringing 20 g of tissue to a final volume of 100 ml with 0.25 M sucrose (Merck, reagent grade). Tissue was homogenized at 0°C in a smooth glass homogenizer with a Teflon pestle using 5 to 8 up-down strokes at 1200 revlmin. Regenerated tissue. Animals were partially hepatectomized under Nembutal anesthesia (Merck, 20 mg/ kg body weight) according to the method of Higgins and Anderson (1931). At the time of partial hepatectomy, the median and left lateral lobes were ligated and removed. This tissue was homogenized as above and utilized as control tissue. During the first 24 hours after the operation, animals were provided with 20% dextrose (Mallinckrodt analytical) ad libitum. After this period, food and water were given as usual. After the required regeneration period, animals were sacrificed and homogenates prepared as previously described. Difleren tial Centrifugation Normal and regenerated whole tissue homogenates were subjected to differential centrifugation at 4 O C according to one of the following schemes. Centrifugation times and g values are designated exclusive of acceleration and deceleration times and are based on average r-values. Scheme 1 : Nuclear fraction. Whole homogenates were centrifuged at 480 X g for 10 minutes followed by two washes (5 up-down strokes each), resuspensions, and recentrifugations at 270 X g for 10 minutes each. The washed pellet was resuspended to starting volume in 0.25 M sucrose to yield a final 20% fraction. All other pellets were resuspended to starting volume in the same manner. Mitochondrial-lysosomal fraction. The combined supernatants from the nuclear step were centrifuged at 22,000 X g for 30 minutes to sediment mitochondria and lysosomes. Microsomal-soluble fraction. The supernatant remaining after the mitochondrial-lysosomal centrifugation was designated the microsomal-soluble fraction. All centrifugations were carried out in a Servall RC 2 refrigerated centrifuge equipped with an SS-34 rotor. Scheme 2 : Nuclear fraction. Whole homogenates were centrifuged at 270 X g for 10 minutes. Two resuspensions (homogenized with 8 up-down strokes at 1,200 rev/ min) and recentrifugations followed at the same speed and time. The washed pellet was brought to starting volume with 0.25 M sucrose. Mitochondria1 fraction. The combined supernatants from the nuclear sedimentation were centrifuged at 2,500 X g for 10 minutes. The pellet was resuspended, homogenized by hand, and recentrifuged at the same speed and time to yield the final mitochondria1 pellet. Resuspension to starting volume with 0.25 M sucrose followed.