The G₁ Cyclin Cln3p Controls Vacuolar Biogenesis in Saccharomyces cerevisiae

Corresponding author: Michael Polymenis, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128., polymenis{at}tamu.edu (E-mail)

Communicating editor: M. SACHS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

How organelle biogenesis and inheritance is linked to cell division is poorly understood. In the budding yeast Saccharomyces cerevisiae the G₁ cyclins Cln1,2,3p control initiation of cell division. Here we show that Cln3p controls vacuolar (lysosomal) biogenesis and segregation. First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar fragmentation. Although the vacuoles of cln3 cells were fragmented, together they occupied a large space, which accounted for a significant fraction of the overall cell size increase in cln3 cells. Second, cytosol prepared from cells lacking Cln3p had reduced vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was perturbed in cln3 cells. Our findings reveal a novel role for a eukaryotic G₁ cyclin in cytoplasmic organelle biogenesis and segregation.

OVERALL organelle morphology and copy number in proliferating cells remain constant, despite successive cell divisions. In yeast, as in animal cells, the enzymes that catalyze cell cycle transitions are complexes of a cyclin-dependent kinase (Cdk) and activating regulatory subunits called cyclins. In Saccharomyces cerevisiae, START is thought to represent a nodal point in late G₁, where various aspects of the cell's physiology are measured or monitored prior to initiation of DNA replication (PRINGLE and HARTWELL 1981 ). START is brought about by the activity of Cdc28p (a Cdk) in association with one of the G₁ cyclins, Cln1,2,3p (WITTENBERG and REED 1996 ). Cells lacking all three CLN genes are inviable and cannot complete START (RICHARDSON et al. 1989 ). During vegetative growth the only essential function of G₁ cyclins is to promote the phosphorylation and subsequent proteolysis of the B-type cyclin kinase inhibitor Sic1p (SCHNEIDER et al. 1996 ; TYERS 1996 ).

None of the CLN genes alone, however, is necessary for the cell's survival. This apparent redundancy has been challenged in the last few years, with Cln1,2p and Cln3p being functionally distinct. It is now thought that Cln3p functions upstream of Cln1,2p, activating the G₁/S transcription program (TYERS et al. 1993 ; DIRICK et al. 1995 ; STUART and WITTENBERG 1995 ; LEVINE et al. 1996 ), where >100 genes (CLN1,-2 among them) are transcribed in a temporal manner at the G₁/S transition (SPELLMAN et al. 1998 ). Cln1,2p/Cdc28p complexes may regulate polarized growth during budding (BENTON et al. 1993 ; CVRCKOVA and NASMYTH 1993 ). They may also serve as upstream activators of the protein kinase C (Pkc1p), which is involved in cell wall biosynthesis (HEINISCH et al. 1999 ). Thus, it seems that Cln3p controls the correct timing of G₁/S transcription, while Cln1,2p tethers G₁/S progression with the morphogenetic and biosynthetic aspects of making a bud.

The vacuole in S. cerevisiae is a large compartment, occupying a significant fraction (25%) of the total cellular volume (WIEMKEN and DURR 1974 ). Vacuoles serve as repositories of metabolites and low-molecular-weight compounds and they are analogous to the lysosomes of animal cells, containing numerous hydrolases (ROBERTS et al. 1991 ; JONES et al. 1997 ). In all eukaryotic cells the lysosomes or vacuoles play major cellular turnover roles, including autophagy where entire organelles are delivered to them for turnover (KLIONSKY and EMR 2000 ). These lysosomal or vacuolar functions are evident during responses to stress or nutrient limitation and also impact on developmental processes and human disease states (KLIONSKY and EMR 2000 ). Vacuoles, and many other organelles (e.g., the Golgi), are not usually synthesized de novo in daughter cells, but instead are inherited from mother cells. It is possible, however, for a daughter cell to slowly synthesize a new vacuole (the same is true for the Golgi) if it didn't inherit one (CATLETT and WEISMAN 2000 ). Since this is a slow and inefficient process, a vacuolar inheritance mechanism is believed to have evolved. Vacuolar morphology and inheritance is dynamic and is somehow coordinated with cell cycle progression (CATLETT and WEISMAN 2000 ). Yeast cells typically contain only one to three vacuoles, and their segregation to daughter cells follows an ordered pattern (WARREN and WICKNER 1996 ). Beginning at the G₁/S transition of the cell cycle, vesicles from the vacuole of the mother cell form a tubular structure and are transported into the newly formed bud, where they will eventually establish the vacuolar compartment of the daughter cell (BRYANT and STEVENS 1998 ; CATLETT and WEISMAN 2000 ). However, it is not known whether the molecular machinery that regulates cell cycle progression also affects vacuolar inheritance and vice versa.

Here we show that the G₁ cyclin Cln3p regulates vacuolar biogenesis and segregation. Our findings suggest an unexpected role for a G₁ cyclin that is specific to Cln3p and is not shared by other G₁ cyclins.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and media:
Cells were grown at 30° and were collected for further analysis while proliferating exponentially, unless otherwise indicated. Formulations for YPD, SC, and SD media were exactly as described elsewhere (KAISER et al. 1994 ). The cln3, cln2, pep3, and vac8 strains in the haploid BY4741 (MATa his3 leu2 met15 ura3) were generated by the yeast deletion project (WINZELER et al. 1999 ). All the homozygous diploid deletion strains in the BY4743 (MATa/ his3/his3 leu2/leu2 met15/met15 ura3/ura3) background were also generated by the yeast deletion project (WINZELER et al. 1999 ). The cdc28-1 strain from the Hartwell collection (in the A364A background, MATa ade1 ade2 ura1 his7 lys2 tyr1 gal1 SUC mal) was obtained from the Yeast Genetic Stock Center. The W303 strain (MATa ade2 trp1 leu2 his3 ura3 can1) and its otherwise isogenic derivatives (GT106, cln3::URA3; MT240, CLN2-3HA::URA3; and GT108, CLN3-3HA::URA3) were gifts from B. Futcher (TYERS et al. 1992 , TYERS et al. 1993 ). The strains used in the in vitro homotypic fusion assay lacking either alkaline phosphatase (DKY6281, MATa lys2 trp1 ura3 his3 leu2 suc2 pho8::TRP1) or the vacuolar proteases necessary for alkaline phosphatase maturation (BJ3505, MATa lys2 trp1 ura3 his3 gal2 can prb1 pep4::HIS3) have been described previously (EITZEN et al. 2000 ) and were a gift from W. Wickner. The YEp-VAC8 and YEp-CLN2 plasmids were gifts from L. Weisman (WANG et al. 2001 ) and B. Andrews (OGAS et al. 1991 ), respectively. The plasmid used to disrupt CLN3 in vac8 cells (BY4741 background) has been described previously (POLYMENIS and SCHMIDT 1997 ).

The CLN3-2 strains were generated by transformation of the corresponding wild-type strains with a URA3⁺ low-copy-number centromeric plasmid carrying the CLN3-2 allele (CROSS 1990 ). In the cln2 strain (BY4741 background), we disrupted CLN1 (cln1::URA3) by PCR-based single-step gene replacement (KAISER et al. 1994 ). The PCR product was generated by amplification of URA3 sequences with the following oligonucleotide primers: 5'-CCACCACTCCACTGCTCGTTAGCTATTTCTGTAAAATAAATAAAAAGATCATGTCGAAAGCTACATATAAGGAACG-3' and 5'-TAGTATTCCGTTATTAATTAAGTATATATGTAGGCTTGATGAGAAAATGGTCAGTTTTGCTGGCCGCATCTTCTC-3'.

All DNA manipulations, isolation of transformants, and verification of mutations were done as described previously (POLYMENIS and SCHMIDT 1997 ).

Microscopy and flow cytometry:
For microscopic examination of vacuolar membranes the cells were stained with FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide, as described by WANG et al. 1996 . Briefly, exponentially growing cells in rich YPD media were transferred in YPD growth medium containing 80 µM FM4-64 (Molecular Probes, Eugene, OR) for 1 hr, washed, resuspended in fresh medium, and allowed to grow for another 2 hr before they were examined microscopically. For the experiments shown in Fig 3 with the temperature-sensitive cdc28-1 strain, the cells were shifted to their nonpermissive temperature (37°) for 3 hr, stained with FM4-64 for 1 hr at 37°, cultured in dye-free medium at 37° for 2 hr, and then examined microscopically.

View larger version (45K): In this window In a new window Download PPT slide   Figure 1. Vacuolar fragmentation in cells lacking CLN3. Diploid cells of the indicated genotype (all in the BY4743 background) were exposed to FM4-64, a vital dye that stains the vacuolar membrane (see MATERIALS AND METHODS), and photographed through phase optics (left) and by fluorescence microscopy with a rhodamine filter (right).

View larger version (69K): In this window In a new window Download PPT slide   Figure 2. Electron micrographs of wild-type and cln3 cells. V indicates the vacuole.

View larger version (59K): In this window In a new window Download PPT slide   Figure 3. Vacuolar fragmentation in cdc28-1 cells. Wild-type and cdc28-1 cells (in the A364A background; see MATERIALS AND METHODS) were exposed to FM4-64 and photographed through phase optics (left) and by fluorescence microscopy with a rhodamine filter (right). The cells were photographed both during growth at room temperature and after they were shifted for 6 hr to 37°, the nonpermissive temperature of the cdc28-1 strain (see MATERIALS AND METHODS).

Cells and purified vacuoles were stained with the vital vacuolar stain 5-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA), as described previously (ROBERTS et al. 1991 ). CDCFDA was added at 10 µM in the culture media (with 50 mM sodium citrate, pH 5.0) for 20 min. The cells were then examined by either fluorescence microscopy or flow cytometry.

For samples analyzed by confocal microscopy, total cellular volume was evaluated by staining the exterior of the cells with rhodamine red, according to the manufacturer's instructions (Molecular Probes), while vacuoles were visualized using CDCFDA as we described above. Data for each strain were obtained from at least three different microscope fields, and we examined a minimum of 20 planes for each microscope field. The fluorescent area per cell was measured using Adobe Photoshop software. The sum of the areas corresponding to each cell was then used as an estimate of volume.

Electron microscopic analysis of ultrathin sections and acid phosphatase localization using cerium chloride as a capture agent to visualize the vacuole was carried out at the Texas A&M Microscopy and Imaging Center. Cells were fixed in a 2% acrolein and 0.1 M sodium cacodylate solution (pH 7.4) on ice for 30 min. The cells were then washed four times, 15 min each time, in 5% sucrose, 1% DMSO, 0.1 M sodium cacodylate solution (pH 7.4). The reaction mixture for acid phosphatase localization was 0.1 M sodium acetate (pH 5.0), 5% sucrose, 1 mM ß-glycerophosphate, 2 mM cerium chloride, and 0.01% Triton X-100. The cells were first incubated for 30 min at 30° in reaction mixture lacking the substrate (ß-glycerophosphate), followed by a 1-hr incubation in complete reaction medium at 30°. The reaction was stopped by two washes in ice-cold solution of 0.1 M sodium acetate (pH 5.0), 5% sucrose, followed by two washes in ice-cold 0.1 M sodium cacodylate solution (pH 7.4). The cells were then incubated overnight at 4° in a solution containing 1% OsO₄, 5% sucrose, and 0.1 M sodium cacodylate (pH 7.4). The samples were then dehydrated in a graded ethanol series, embedded in epoxy resin, sectioned, and examined without poststaining.

For flow cytometry for cellular DNA content (Fig 7), cells (1 x 10⁷ cells/ml) were fixed overnight in an ethanol-PBS solution (mixed at a 7:3 ratio). They were then resuspended in 50 mM sodium citrate buffer (pH 7.0). The sample was treated with RnaseA (0.25 mg/ml) overnight at 37°. Finally, the sample was resuspended in a 50 mM sodium citrate buffer (pH 7.0) containing 1 mM Sytox Green (Molecular Probes) before it was evaluated by flow cytometry. To generate the DNA content histograms (Fig 7), the same number of cells (30,000) was collected for any given strain.

View larger version (49K): In this window In a new window Download PPT slide   Figure 4. Cell and vacuole size in cells carrying different CLN3 alleles. All the strains shown were in the haploid BY4741 background. (A) Cells were photographed through phase optics (left) and by fluorescein fluorescence (right) to visualize the vacuole of exponentially growing cells in rich defined SC media (see MATERIALS AND METHODS). (B) Cell volume of the indicated strains was measured using a Channelyzer (left) or flow cytometry by forward angle scattering (FSC, middle). Vacuolar fluorescence was measured by flow cytometry (right). In every panel the measured parameter is shown on the x-axis, and the number of cells on the y-axis. The geometric mean is shown in each case.

View larger version (36K): In this window In a new window Download PPT slide   Figure 5. Cln3p regulates vacuole homotypic fusion in vitro. (A and B) Purified vacuoles were allowed to fuse in the presence of ATP and cytosol and stained with CDCFDA before they were photographed. (A) Vacuole fusion in the presence of cytosol prepared from the indicated strains, all in the BY4743 background. To visualize the vacuoles in the absence of ATP and cytosol (bottom right), we exposed for 30 sec, because shorter exposures were insufficient. In contrast, fused vacuoles, in the presence of ATP and cytosol, fluoresce much more intensely, so for wild-type cytosol (top left) we exposed for 6 sec, while for CLN3-2 cytosol (bottom left) the fluorescence was so intense that the exposure was only 4 sec. For cln3 cytosol (top right) or for the no cytosol control (middle right) we exposed for 10 sec. Aliquots of the vacuole fusion reactions shown were evaluated colorimetrically on the basis of the reconstitution of alkaline phosphatase activity. The average and standard deviations from at least seven independent experiments of relative alkaline phosphatase activity, normalized for background (no cytosol) and obtained from the indicated reactions, are shown. (B) Vacuole fusion in the presence of cytosol from untagged, CLN2-HA-, or CLN3-HA-tagged strains. All the cytosols were immunodepleted using an anti-HA monoclonal antibody (see MATERIALS AND METHODS) before they were used in the vacuole fusion reactions. The precipitated (P) and the supernatant (S) fractions were evaluated by immunobloting using the anti-HA antibody to measure the extent of cyclin depletion. An asterisk indicates nonspecific bands. Fusion was evaluated microscopically, and the exposure time was the same (10 sec) for all the photographs shown. Aliquots of the vacuole fusion reactions shown were also evaluated colorimetrically on the basis of the reconstitution of alkaline phosphatase activity. The average and standard deviations from three independent experiments of relative alkaline phosphatase activity, normalized for background (no cytosol) and obtained from the indicated reactions, are shown. (C) The intracellular steady-state levels of Pgk1p and the pro- and mature (m-) forms of CpY and ALP in cells of the indicated genotype were evaluated by immunobloting. The pep4, prb1 strain was BJ3505 (see MATERIALS AND METHODS), while all the others were in the BY4743 background. (D) Homotypic fusion reactions using cytosol from CLN3+ and cln3 cells (in the BY4743 background) transformed with an empty (vector) or a CLN2-containing (CLN2) high-copy plasmid were evaluated colorimetrically as described above. The average and standard deviations from four independent experiments of relative alkaline phosphatase activity, normalized for background (no cytosol) and obtained from the indicated reactions, are shown.

View larger version (19K): In this window In a new window Download PPT slide   Figure 6. Cells lacking CLN3 are defective in vacuolar segregation. Cells of the indicated genotype (all in the BY4743 background) were stained with FM4-64 to visualize vacuole membranes. (A) Random fields of cells were examined for vacuolar segregation 2 hr after vacuolar staining with FM4-64 (see MATERIALS AND METHODS). The number of cells examined is shown in parentheses. The scored cells were grouped into two groups on the basis of bud size, and within each group the percentage of cells with the indicated vacuolar morphology is shown. (B) Unequal vacuolar segregation in homozygous diploid cln3 cells, stained and photographed as described in the Fig 1 legend.

View larger version (57K): In this window In a new window Download PPT slide   Figure 7. CLN3 and VAC8 in vacuolar biogenesis and cell cycle progression. Cells of the indicated genotype (all in the BY4741 background) were exposed to FM4-64 and photographed through phase optics (left) and by fluorescence microscopy with a rhodamine filter (middle). Their cellular DNA content (right) was determined by fluorescence-activated cell sorter. For each strain the percentage of cells in G1, calculated by the ModFit software, is indicated. Cell numbers are plotted on the y-axis and the x-axis represents fluorescence.

Vacuole fusion:
For in vitro fusion assays cytosol and vacuoles were prepared as described in CONRADT et al. 1992 . Vacuole fusion was essentially performed according to HAAS and WICKNER (1996), using 0.34 mg/ml of vacuoles prepared from strains DKY6281 (pho8) and BJ3505 (pep4, prb1). Cytosolic extracts from the indicated strain in each case were added at the same concentration on the basis of their protein content. The in vitro fusion reaction was carried out for 2 hr. Before colorimetric measurement of alkaline phosphatase activity from in vitro fused vacuoles, cytosolic activity was removed according to HAAS and WICKNER (1996). For the immunodepletion experiments, cytosolic extracts were incubated for 1 hr at 4° with 60 µg of 12CA5 monoclonal anti-hemagglutinin (anti-HA) antibody prepared from ascites fluid, followed by a 1-hr incubation at 4° with 50 µl of a protein G-agarose bead solution (Pierce, Rockford, IL). The beads were then removed and the cytosolic extracts were used in vacuole fusion reactions as described above. Mock-depleted cytosols were prepared from the same batches of extracts but with the same volume of PBS instead of the anti-HA antibody. The effects of each immunodepleted Cln (Cln2p or Cln3p) on the fusion activity were evaluated by comparing it to its mock-depleted counterpart.

Other techniques:
For the immunoblots shown in Fig 5, cytosolic extracts were prepared as described above, while total cellular extracts were prepared as described previously (KAISER et al. 1994 ), separated by SDS-PAGE on a 10% acrylamide gel, and transferred onto nitrocellulose. The blots were blocked in PBS containing 5% (w/v) dry nonfat milk and 0.1% (v/v) Tween-20. Between incubations the blots were washed three times 10 min each in PBS. All the antibodies were added in blocking solution. The 12CA5 monoclonal antibody against HA was used at a 1:1000 dilution. The primary antibodies against yeast Prc1p (CpY), Pho8p (alkaline phosphatase, or ALP), and Pgk1p were from Molecular Probes, and they were used according to their instructions. Secondary antibody horseradish peroxidase conjugates were from Pierce and used at 1:5000 dilution. The blots were developed with chemiluminescent peroxidase reagent from Sigma (St. Louis), according to their instructions.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Vacuolar morphology in cln3 cells:
To examine vacuolar morphology we first visualized the vacuoles with the vital amphiphilic styryl dye FM4-64 (see MATERIALS AND METHODS), which stains the vacuolar membrane (HILL et al. 1996 ). The vacuolar compartment in 60–70% of cln3 cells had a fragmented and multilobular appearance (Fig 1). This was not the case, however, for cells lacking Cln1p and Cln2p (Fig 1). The extent of vacuolar fragmentation was comparable to that of vac8 cells (Fig 1), which are defective in vacuolar inheritance (CATLETT and WEISMAN 2000 ), and they display extensive vacuolar fragmentation (WANG et al. 2001 ). We also examined cells carrying the CLN3-2 allele, which effectively overexpress Cln3p because they produce a truncated but stable form of the protein (CROSS 1988 ), but the vacuolar morphology of these cells was indistinguishable from wild-type cells (data not shown). Next, we evaluated wild-type and cln3 cells by electron microscopy (Fig 2). Consistent with the fluorescence data, cells lacking Cln3p had more but smaller vacuoles (Fig 2). We also observed extensive vacuolar fragmentation of cdc28-1 cells shifted to their nonpermissive temperature (Fig 3). This is consistent with the known role of Cln3p as a regulatory subunit in a complex with Cdc28p, which is important for the G₁/S transition. These observations suggest that Cln3p, but not Cln1,2p, may be necessary for the maintenance of vacuolar morphology.

The vacuolar compartment in cln3 cells occupied a significant portion of the cell (Fig 1 and Fig 2), and we decided to address this issue in more detail. We examined living cells carrying a wild-type (CLN3⁺), a null (cln3), or a dominant (CLN3-2) CLN3 allele. The cells were stained with a vacuolar fluorescent probe, CDCFDA. CDCFDA is localized in the vacuole by diffusion, where it is hydrolyzed into an impermeant fluorescent anionic derivative (PRESTON et al. 1989 ). The cells were then examined by either fluorescence microscopy (Fig 4A) or flow cytometry (Fig 4B). CDCFDA fluorescence intensity, representing vacuolar size of live cells, was quantified by flow cytometry (Fig 4B) from the same samples that were used to obtain the forward angle scattering cell size estimates, to simultaneously obtain both parameters. Note that this analysis confirmed the expected small vacuolar compartment of pep3 cells (Table 1), which are known to have small vacuolar vesicles (PRESTON et al. 1991 ). In Table 1, we summarize the values of cell size and vacuolar size in CLN3⁺, cln3, and CLN3-2 cells in the BY4741 background. Importantly, we noted disparities in vacuolar size that did not correlate with cell size differences. For example, cln3 cells are 30% larger overall than CLN3⁺ cells, but their vacuole is 80% larger. This disproportional enlargement of the vacuolar compartment in cln3 cells was evident (P < 0.05, based on a Student's t-test) in all strains tested, irrespective of ploidy, haploid BY4741 vs. diploid BY4743, and genetic background, strains BY4741 vs. W303 (data not shown). Note that the above differences in vacuolar volume were not due to intravacuolar pH differences, which may alter the fluorescence of the vacuolar probe we used, for two reasons: First, both cln3 and CLN3-2 cells had a vacuolar pH in the same range as wild-type cells (5.98 and 6.14; data not shown). Second, the vacuolar size differences were evident even after the intravacuolar pH was equilibrated to that of external buffers (data not shown).

We also estimated cell size and vacuolar size using confocal microscopy. For CLN3⁺, cln3, and CLN3-2 haploid cells in the BY4741 strain background, cultured in defined SC media, the relative cell volume values were 1 ± 0.24 (n = 20), 1.35 ± 0.35 (n = 23), and 0.71 ± 0.19 (n = 20), respectively. In contrast, the vacuolar size of CLN3⁺, cln3, and CLN3-2 cells, relative to the overall cell size of wild-type cells, was 0.25 ± 0.13 (n = 40), 0.55 ± 0.17 (n = 50), and 0.14 ± 0.07 (n = 22), respectively. Our results are in very good agreement with earlier estimates of vacuolar size (25% of total cellular volume; WIEMKEN and DURR 1974 ) and with our own data regarding the relative vacuolar and cell size parameters in cln3 cells that we reported above (Table 1).

However, the vacuolar size in cells that lack another G₁ cyclin, Cln2p, was not significantly affected (Table 1). Even in cells that lacked both CLN1 and CLN2 and were quite large overall, the vacuole was not disproportionately enlarged. Instead, we noted a decrease in vacuolar size in cln1,2 cells (Table 1). Thus, on the basis of our results with the cln1,2 strain, we conclude that a decrease in vacuolar size is not necessarily accompanied by an overall cell size decrease. Specifically in the case of cln3 cells, however, loss of Cln3p clearly increases the size of the vacuolar compartment to a greater extent than what was predicted from cell size differences.

Cells that lack Cln3p are defective in vacuole homotypic fusion:
Given the fragmented vacuolar morphology of cln3 cells, we decided to test Cln3p's effects in an in vitro vacuole homotypic fusion assay developed by Wickner and colleagues (WICKNER and HAAS 2000 ). The process of vacuole vesicle fusion is crucial in determining the overall vacuole copy number and vacuolar biogenesis in general (WICKNER and HAAS 2000 ). Vacuoles were purified and then mixed in the presence of cytosol and ATP. To evaluate vacuole fusion microscopically (Fig 5A), the fused vacuoles were stained with CDCFDA. Cytosol from cells lacking Cln3p had significantly reduced fusion activity (Fig 5A). The extent of vacuole fusion can also be evaluated colorimetrically, because the purified vacuoles in this assay were prepared from two different strains, each lacking the ability to produce active alkaline phosphatase (encoded by PHO8). One of the strains lacks PHO8, while the other lacks vacuolar proteases necessary for Pho8p maturation. In this assay, processed vacuolar alkaline phosphatase can be produced only if the vacuolar constituents from the two strains mix after fusion (WICKNER and HAAS 2000 ). Although the enzymatic assay was not as sensitive as the direct microscopic observation, it was still clear that alkaline phosphatase activity, which reflects vacuole fusion, was lower (P < 0.05, based on a Student's t-test) in the presence of cytosol from cln3 cells (Fig 5A).

To more directly test the role of Cln3p in vacuolar homotypic fusion, we prepared cytosol from wild-type cells and from cells carrying epitope-tagged versions of Cln2p (CLN2-HA) and Cln3p (CLN3-HA). These epitope-tagged Cln2p and Cln3p are fully functional (TYERS et al. 1992 , TYERS et al. 1993 ). Cytosol from the untagged and tagged strain was either incubated with an antibody against the epitope for immunodepletion or mock depleted with PBS, and we then carried out immunoprecipitations to deplete the HA epitope-carrying polypeptides from the cytosolic extracts. The extent of immunodepletion was significant (>90%), as shown on the immunoblot below the micrographs in Fig 5B. The immunodepleted or mock-depleted cytosolic extracts were then evaluated for vacuole homotypic fusion activity (Fig 5B). It is clear that depletion of Cln3p, but not Cln2p, reduced homotypic fusion activity (Fig 5B). These results further strengthen the notion that Cln3p is required for homotypic fusion activity.

To test whether CLN3 deletion or overexpression might somehow affect secretory pathways of vacuolar protein sorting (JONES et al. 1997 ), we evaluated the maturation of two vacuolar enzymes, carboxypeptidase Y (CpY) and vacuolar membrane ALP, in CLN3⁺, cln3, and CLN3-2 cells (all in the BY4743 background). The CpY precursor, Prc1p, traffics through the endoplasmic reticulum, Golgi, and prevacuolar compartments before it is sorted to the vacuole (JONES et al. 1997 ). ALP bypasses the prevacuolar compartment. We found that steady-state levels of mature CpY and ALP were similar among wild-type, cln3, and CLN3-2 cells (Fig 5C). Thus, it does not appear that the secretory processes involved in CpY and ALP maturation are grossly affected in CLN3-2 or cln3 cells.

Finally, increasing the dosage of another G₁ cyclin, CLN2, could not suppress the defect in homotypic fusion observed in cln3 cells (Fig 5D). We also examined vacuolar morphology of these cells after staining with the vacuolar membrane stain FM4-64, and we found that the vacuolar fragmentation of cln3 cells that we described above (Fig 1) was not rescued by introducing CLN2 on a high-copy plasmid (data not shown).

Cln3p and vacuolar segregation:
After examining photographs of budded cln3 cells, we noted that in several cases the signal associated with the vacuolar compartment was not equally distributed between the bud and the mother cell (Fig 6A). This is different from wild-type cells, which distribute their vacuoles between the mother and the bud. In the known vacuolar inheritance mutants the bud fails to receive vacuoles, as was evident in vac8 cells (Fig 6A). Note that the vacuoles stained by FM4-64 do not represent all the vacuoles in the cell, because between the time of staining and the time of observation, new vacuoles, which are not stained, are synthesized in the cell (see MATERIALS AND METHODS). We were particularly surprised to find that there was also a fraction of cln3 cells that distributed their vacuoles almost exclusively in the bud and not in the mother (Fig 6A and Fig B). This has not been observed in known vac mutants.

VAC8 and CLN3:
We next examined vacuolar morphology and cell cycle progression of cells mutant for VAC8 and CLN3 (Fig 7). Combined loss of CLN3 and VAC8 does not lead to an apparent additive effect and the cells still have visible but fragmented vacuoles. However, overexpression of CLN3 in vac8 cells and vice versa do not suppress the vacuolar fragmentation defects of the singly mutant strains also (Fig 7). Thus, it does not appear that CLN3 and VAC8 function in a simple linear pathway.

We also examined cell cycle progression of these strains by flow cytometry (Fig 7, right). Note that vac8 cells proliferated at the same rate as wild-type or cln3 cells (data not shown). Interestingly, in vac8 cells there was an increase in the percentage of cells in the G₁ phase of the cell cycle. Thus, as is the case for cln3 cells (CROSS 1988 ; NASH et al. 1988 ), it appears that vac8 cells stay longer in G₁ but there is a compensatory shortening of subsequent cell cycle phases, resulting in no net change in doubling time. Overexpression of Vac8p had no effect on cell cycle progression (data not shown). Importantly, however, Cln3p overexpression in vac8 cells accelerated completion of START without suppressing the vacuolar fragmentation of these cells (Fig 7), suggesting that, at least in this case, vacuolar fragmentation is not necessarily linked to the timing of START. Taken together, our results indicate that Cln3p's role in vacuolar biogenesis is distinct from that of Vac8p and also separate from Cln3p's established function in G₁/S progression.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Besides the chromosomes in the nucleus, the size and copy number of all organelles must also be maintained during cell proliferation. This work documents a novel role for a eukaryotic G₁ cyclin in vacuolar (lysosomal) homeostasis, suggesting that the same cell cycle machinery that initiates cell division may also perform a separate function in the control of vacuolar biogenesis and segregation.

There are usually one to three vacuoles per cell (WARREN and WICKNER 1996 ). In cln3 (but not in cln1 and/or cln2) cells, the vacuolar compartment was fragmented (Fig 1 and Fig 2). Interestingly, the overall vacuolar compartment of cln3 cells was disproportionately enlarged (Fig 4). Even though vacuole size does not always dictate overall cell size (for example, cln3 and cln1,-2 cells are both large but the size of their vacuolar compartment differs by three- to fourfold; see Table 1), this result underscores the complexity of cell size regulation. Organelle contribution to overall cell size is not usually taken into consideration in studies of cell size control. How could Cln3p regulate vacuolar biogenesis? We provide evidence that Cln3p controls vacuolar homotypic fusion activity in vitro (Fig 5). These findings raise the issue of Cln3p's subcellular localization. Cln3p is certainly found in the nucleus (MILLER and CROSS 2000 ), but a more recent report clearly showed that Cln3p is also found in the cytoplasm (GARI et al. 2001 ). Note also that cytosolic extracts immunodepleted for Cln3p could not support homotypic fusion (Fig 5), arguing for a post-translational mechanism of regulation of homotypic fusion by Cln3p. Whether Cln3p needs to exit the nucleus or simply acts on a nuclear factor, which then exits the nucleus, is unclear at present, since our cytosolic preparations are not devoid of soluble nuclear proteins. Since forcing Cln3p in the cytoplasm leads to cell size enlargement (EDGINGTON and FUTCHER 2001 ), and since we show here (Fig 4) that the large size of cln3 cells is largely due to vacuolar enlargement, it is perhaps likely that Cln3p still functions in the nucleus where it perhaps modifies a factor that in turn exits into the cytoplasm and affects vacuolar biogenesis. In any case, it is clear that Cln3p's function in vacuolar biogenesis is separate from its established role in activating the G₁/S transcriptional program.

Self- (homotypic) fusion of organelle vesicles is essential for organelle homeostasis (WICKNER and HAAS 2000 ). In animal cells, inhibition of Golgi homotypic fusion in mitosis results in extensive fragmentation of the Golgi. This is important for Golgi inheritance because the resulting Golgi vesicles stochastically disperse in equal numbers in both daughter cells where, after completion of mitosis, they will fuse to regenerate the Golgi (WARREN and WICKNER 1996 ). Warren and colleagues have shown that mitotic inhibition of Golgi homotypic fusion is mediated by the cyclin-dependent kinase Cdc2 (LOWE et al. 1998 ; NELSON 2000 ). Is it possible that aspects of this established paradigm also operate in Cln3p's role during vacuolar biogenesis? Perhaps. Note that while the Cln3p/Cdc28p complex may be necessary for high vacuolar homotypic fusion activity, the mammalian Cdc2/cyclinB complex does the opposite, because it inhibits Golgi vesicle fusion. Overall, however, we think it is intriguing that, although in each case different organelles are affected at different cell cycle points, in both cases organelle homotypic fusion may be sensitive to changes in the activity of cyclin/Cdk complexes.

Loss of Cln3p not only affects vacuolar morphology, but also impacts on vacuolar segregation (Fig 6). To our knowledge, this is the first evidence linking a cell cycle regulator with vacuolar segregation. Overall, how do the vacuolar phenotypes of cln3 cells compare to those of other vacuolar mutants? We think that although cln3 cells share some characteristics with other vacuolar mutants, the combination of these characteristics makes them unique. For example, vacuolar enlargement and a concomitant overall cell enlargement are evident in fab1 mutants (GARY et al. 1998 ; JORGENSEN et al. 2002 ; ZHANG et al. 2002 ). But, in contrast to cln3 cells, the vacuole of fab1 cells is not fragmented, fab1 cells loose vacuolar acidity, and vacuolar protein sorting is impaired as well (GARY et al. 1998 ). During the course of this work, the fragmented vacuole of cln3 cells was also mentioned in a genome-wide study of vacuolar morphology (SEELEY et al. 2002 ). The fragmented vacuolar morphology of cln3 cells corresponds to class B vacuolar protein sorting mutants and is apparently similar to vac8 cells (SEELEY et al. 2002 ). Furthermore, both Vac8p (WANG et al. 2001 ) and Cln3p (Fig 5) appear to be required in vacuole fusion. However, on the basis of our results we think there are important differences between vac8 and cln3 cells. For example, note that in vac8 cells vacuolar fragmentation is not accompanied by overall vacuolar and cellular enlargement (see Fig 1 and Fig 6). Furthermore, our analysis of double CLN3 and VAC8 mutants argues against a simple linear relationship of the two gene products in vacuolar biogenesis (Fig 7). In known vac mutants (including vac8 cells) it is the daughter cells that do not receive enough vacuoles (CATLETT and WEISMAN 2000 ). However, in cln3 cells it appears that at least in some cases the opposite is true (Fig 6). Although known vac mutants are not defective in vacuolar retention, retention in the mother cell can play an important role during organelle partition, and it is an established aspect of mitochondria segregation in yeast (YANG et al. 1999 ). Finally, loss of Vac8p apparently delays the G₁/S transition (Fig 7), but it is not clear at this point whether vacuolar biogenesis can causally alter cell cycle progression. Note that vacuolar fragmentation per se in vac8 cells is not blocking acceleration of START in the context of the dominant CLN3-2 allele (Fig 7).

On the basis of the results we report here, it appears that the seemingly separate events of the nuclear cell division cycle and the cytoplasmic processes that control organelle segregation might be controlled by separate functions of the same machinery. Our finding that Cln3p is involved in vacuolar biogenesis at least provides a beginning toward a more detailed understanding of these phenomena in yeast. Since regulatory mechanisms of cell division and organelle biogenesis are highly conserved between yeast and humans, findings from yeast studies should be relevant to these processes in other organisms.

	ACKNOWLEDGMENTS

We thank F. R. Cross, B. Futcher, L. Weisman, B. Andrews, and W. Wickner for plasmids and strains; Ann Ellis and the Microscopy Imaging Center at Texas A&M University for electron microscopy; J. Miller for flow cytometry; R. Barhoumi and the Image Analysis Laboratory at the Texas A&M Veterinary Medical Center for confocal fluorescence microscopy; and J. C. Hu, A. LiWang, P. LiWang, and I. Hariharan for discussions. We are indebted to L. Bogomolnaya and other members of the Polymenis lab for their help and support. This work was supported by grants from the National Institutes of Health (GM-058770 to R.A. and GM-062377 to M.P.) and the American Heart Association—Texas Affiliate (0060115Y to M.P.).

Manuscript received March 4, 2003; Accepted for publication June 5, 2003.

	LITERATURE CITED

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