Animals, fungi and other eukaryotes have cells that are divided into sub-compartments that are called organelles. Each type of organelle serves a specific purpose that is essential for the life of the cell. Yeast cells have a large organelle called a vacuole; the inside of the vacuole is acidic and contains enzymes that can break down other molecules.

Previous studies have shown that when a budding yeast cell buds to produce a new daughter cell, a process ensures that some of the mother's vacuole is transferred to its daughter. However, yeast mutants that fail to inherit some of their mother's vacuole can still survive. This is because an ‘alternative’ mechanism allows the newly forming daughter to generate its own vacuole from scratch.

Jin and Weisman now unexpectedly show that a new daughter cell cannot become a mother cell until its new vacuole is formed. The experiments made use of yeast mutants that were defective in the ‘inheritance’ mechanism, and double mutants that were defective in both the inheritance and alternative mechanisms. The experiments also revealed that a signal from the vacuole is required before the yeast cell's nucleus can start the cycle of events that lead to the cell dividing. Jin and Weisman suggest that this newly identified communication between the vacuole and the nucleus may help to ensure that critical organelles are present in all cells.

Though it remains unclear why the yeast vacuole is critical for a cell to divide, these findings suggest that the mammalian lysosome (which is similar to the yeast vacuole) may perform a similar critical role in mammals. If this is the case, then understanding how these organelles communicate with the nucleus may provide new insights into how to prevent the uncontrolled growth of tumors and cancer.

Organelles are essential for cellular functions, and organelle inheritance is likely a major pathway that ensures the presence of organelles in all cells. A requirement for the inheritance of the mammalian Golgi is well established due in part to the elaborate changes in architecture that occur during interphase vs mitosis (Shorter and Warren, 2002). For other organelles, which are constitutively dispersed, organized mechanisms for their inheritance remain less clear. The budding yeast Saccharomyces cerevisiae provides an excellent model to study the spatial and temporal control of organelle inheritance, in part because its cell division is asymmetric. This asymmetric division requires active organelle transport in each cell-cycle. In budding yeast, most of the organelles are transmitted from mother to daughter cells (Fagarasanu and Rachubinski, 2007). These include the vacuole/lysosome, mitochondria, the endoplasmic reticulum, peroxisomes, secretory vesicles and late-Golgi elements. Transport of these organelles starts in G1 phase and occurs in coordination with the cell-cycle. However, it is unclear whether there are mechanisms that guarantee the presence of organelles prior to the next round of cell division. Here we present the unexpected finding that the presence of the vacuole is ensured because the vacuole plays an essential role in the initiation of the cell-cycle.

During cell division in budding yeast, the daughter cell inherits a vacuole from the mother cell (Weisman et al., 1987). The vacuole is transported by a vacuole transport complex, composed of the myosin V motor Myo2, the vacuole membrane anchored protein Vac8, and an adaptor protein Vac17 that links Myo2 and Vac8 (Catlett and Weisman, 1998; Wang et al., 1998; Ishikawa et al., 2003; Tang et al., 2003). Vacuole inheritance is initiated in G1 phase via Cdk1/Cdc28, which regulates the formation of the vacuole transport complex (Peng and Weisman, 2008). After formation of the complex, Myo2 moves the vacuole to the daughter cell along actin cables (Hill et al., 1996). At the end of the cell-cycle, vacuole transport is terminated by ubiquitylation of Vac17, which is then degraded by the 26S proteasome (Yau et al., 2014). Notably, Myo2 also delivers other cargoes including mitochondria, peroxisomes, secretory vesicles, late-Golgi elements, and astral microtubules. Myo2 binds to each cargo via cargo specific adaptors, which attach to the globular tail domain of Myo2 (Yin et al., 2000; Itoh et al., 2002; Boldogh et al., 2004; Itoh et al., 2004; Fagarasanu et al., 2006; Arai et al., 2008; Lipatova et al., 2008; Jin et al., 2011; Santiago-Tirado et al., 2011; Eves et al., 2012; Chernyakov et al., 2013). Moreover, some of the regulatory pathways for vacuole transport are also utilized by other Myo2 cargoes (Moore and Miller, 2007; Peng and Weisman, 2008; Fagarasanu et al., 2009; Jin et al., 2009; Yau et al., 2014).

Many of the proteins involved in vacuole inheritance are conserved among several species, which suggests that vacuole inheritance confers a selective advantage (Mast et al., 2012). These observations suggest that the vacuole plays essential roles. Surprisingly, mutations that block vacuole inheritance do not have a notable impact on cell viability (Catlett and Weisman, 1998; Ishikawa et al., 2003). Indeed, previous studies suggest that new vacuole synthesis occurs in the absence of vacuole inheritance (Weisman et al., 1990; Gomes De Mesquita et al., 1997), however at the time of those studies, there were no suitable methods to distinguish an old vacuole from newly formed vacuoles. Moreover the origin of the new vacuole was unknown. Importantly it was not clear how many pathways would need to be blocked in order to prevent vacuole biogenesis. Note that vacuole biogenesis utilizes at least three direct transport pathways: autophagy/Cvt (from the cytoplasm), AP-3/ALP (from the Golgi), and CPY (from the MVB/endosome) pathways (Bryant and Stevens, 1998; Hecht et al., 2014).

To test when and where a new vacuole is generated in the absence of vacuole inheritance, we monitored for the presence of a vacuole using two markers, Vph1 and FM4-64. Vacuoles were detected using GFP fused to the integral vacuole membrane protein Vph1, a V₀ subunit of the vacuolar ATPase (Manolson et al., 1992). The presence of inherited vacuoles or old vacuoles were specifically assessed via pulse chase experiments with the vital fluorophore FM4-64 (Vida and Emr, 1995). Exogenously added FM4-64 binds to the plasma membrane, is internalized by endocytosis and delivered to the vacuole. After a chase of one doubling time, all of the FM4-64 is trapped on the vacuole membrane. In wild-type cells, the vacuole is inherited and FM4-64 is distributed between the mother and daughter vacuole (Wang et al., 1996), and Vph1-GFP and FM4-64 always colocalize (Figure 1A, top panels). In contrast, in the vacuole inheritance mutant vac17Δ, FM4-64 is retained in the mother cell (Ishikawa et al., 2003). Interestingly in vac17Δ, buds contained small Vph1-GFP positive vacuoles that lack FM4-64 (Figure 1A; open white allow heads, see also [Anand et al., 2009]). This observation indicates that there is new vacuole synthesis in the absence of vacuole inheritance, and that the new synthesis initiates in the bud.

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The vacuole is required for cell growth

We observed that all vac17Δ mother cells and unbudded cells have a vacuole as defined by the presence of Vph1-GFP. This strongly suggests that the vacuole is required for cell growth and viability. If this were true, then a combination of a vacuole inheritance defect with an additional defect in the synthesis of a new vacuole would render the cell inviable. Similarly, an additional defect in vacuole function(s) that are required for bud emergence would result in non-viable cells (Figure 2A). Indeed, a high-throughput screen suggested that over twenty genes might be synthetically lethal with the vac17Δ mutant (Costanzo et al., 2010). We individually tested double mutants of vac17Δ with each of the previously proposed candidates that are not essential genes, and found that the double mutants, vac17Δ pep12Δ and vac17Δ vps45Δ displayed synthetic growth defects (Figure 2B,C). Importantly pep12Δ and vps45Δ were also synthetically lethal with additional mutants defective in vacuole inheritance, vac8Δ and myo2-N1304D mutants (Figure 2—figure supplement 1A–D). The corresponding wild-type genes, PEP12 and VPS45, likely play a critical role in the generation of a new vacuole.

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PEP12 encodes a t-SNARE, and VPS45 encodes a Sec1/Munc18 protein. These proteins function together in the vacuole-protein-sorting pathway from endosomes to the vacuole (Becherer et al., 1996; Burd et al., 1997). To test whether the growth defects of the vac17Δ pep12Δ double mutant are due to a defect in forming new vacuoles, we monitored vacuoles using two markers, Vph1-GFP and CMAC, a small molecule that is taken into the lumen of the vacuole (Stefan and Blumer, 1999). Wild-type and vac17Δ cells showed normal localization of Vph1-GFP and CMAC (Figure 2D,E). However, single pep12Δ cells, which have a defect in protein sorting to the vacuole, and a partial defect in vacuole inheritance (Raymond et al., 1992), showed an abnormal distribution of Vph1 (Piper et al., 1997), but not CMAC (Figure 2D,E). The vac17Δ pep12Δ double mutant cells showed defects in the localization of Vph1 and CMAC (Figure 2D,E). These findings suggest that the double mutant does not generate normal vacuoles.

PEP12 localization is consistent with its role in the synthesis of a new vacuole. GFP-Pep12 and Vph1-CFP were co-expressed in a vac17Δ mutant labeled with FM4-64. In this strain, 63% of vac17Δ cells had GFP-Pep12 on the newly synthesized vacuole in the bud, which was Vph1-CFP positive but lacked FM4-64 (Figure 2F,G). In 17% of daughter cells, GFP-Pep12 was present in buds without a vacuole, as indicated by the absence of Vph1-CFP (vacuole). This suggests that a Pep12-positive endosome appears first, and subsequently a Vph1-positive vacuole matures from the Pep12-positive endosome. Note that in only 1.6% of cells, Vph1-CFP was present without GFP-Pep12.

The vacuole is required for cell-cycle progression from early G1

That the vacuole in the mother cell must reach a specific size prior to bud emergence (Figure 1B) and that a vacuole is required for cell growth (Figure 2), raised the possibility that the vacuole is required for cell-cycle progression. Thus, we tested whether the vac17 pep12 double mutant arrests at a specific point in the cell-cycle. To perform this analysis, we used the pep12-60^tsf mutant, which is temperature sensitive for function (tsf). At elevated temperatures PEP12 function is acutely ablated, but the cells remain viable (Burd et al., 1997). Importantly, the vac17Δ pep12-60^tsf double mutant, but not pep12-60^tsf single mutant, showed a severe growth defect at 37°C (Figure 3A).

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To test cell-cycle progression, we labeled DNA with propidium iodide (PI), and measured DNA content via FACS analysis. Wild-type, vac17Δ, pep12-60^tsf, and vac17Δ pep12-60^tsf cells were incubated overnight at 24°C, then shifted to 37°C. At 24°C (0 hr of 37°C), the vac17Δ pep12-60^tsf double mutant had a normal cell-cycle profile (Figure 3B and Figure 3—figure supplement 1A). After incubation at 37°C for 24 hr, wild-type, vac17Δ and pep12-60^tsf showed a similar percent of G1 phase cells (1N DNA); 46(±2)%, 48(±2)%, and 43(±3)%, respectively (Figure 3B and Figure 3—figure supplement 1A). In contrast, after incubation at the restrictive temperature for 24 hr, 80(±3)% of the vac17Δ pep12-60^tsf double mutant cells arrested at G1 phase (1N DNA). Importantly, at 8, 12 and 24 hr after the shift to 37°C, the differences between the vac17Δ pep12-60^tsf double mutant and the other strains were statistically significant (all p-values < 1 × 10⁻³). Consistent with these findings, both the vac17Δ pep12Δ and vac17Δ vps45Δ double mutants exhibited an arrest in G1 phase (Figure 3—figure supplements 1B,C). These results strongly suggest that a functional vacuole is important for cell-cycle progression from G1 phase.

The finding that the vac17Δ pep12-60^tsf double mutant arrests in G1 phase, suggested that this might be the primary reason for the growth arrest (Figure 3A). To further test this hypothesis, we monitored cell viability at several time points following a shift to 37°C. Cells were counted with a hemocytometer, and the ability of these cells to form colonies at 24°C was assessed (Figure 3B). There was a measurable increase in the lethality of the vac17Δ pep12-60^tsf mutant. However the difference between the lethality of the pep12-60^tsf single mutant and the vac17Δ pep12-60^tsf double mutant was not statistically significant (p = 0.082) (Figure 3B). Together, these observations strongly suggest that cells without a functional vacuole arrest at G1 phase, and that this arrest is not an artifact of general cell death.

Additional evidence for a specific arrest in G1 phase, came from the finding that when the vac17Δ pep12-60^tsf mutant was incubated at the restrictive temperature, there was a striking increase of cells with Whi5 in the nucleus (Figure 3C and Figure 3—figure supplement 1D). Whi5 is a transcriptional repressor of the SBF complex (Swi4-Swi6), and is localized in the nucleus in early G1 phase (Costanzo et al., 2004; de Bruin et al., 2004). In wild-type cells, Whi5 nuclear localization is transient and released by Cdc28-Cln3 activity, which enables progression to early G1 phase.

Further evidence that this is a bona fide G1 arrest, came from the finding that the vac17Δ pep12-60^tsf mutant with 1N DNA content, arrests as unbudded cells. We collected 1N DNA cells by flow cytometry, and determined their morphology by microscopy. After incubation at 37°C for 24 hr, 99% of the G1 cells were unbudded, in both the wild-type and vac17Δ pep12-60^tsf mutant (Figure 3D). Together these results indicate that the vacuole is required for early G1 progression.

TORC1-SCH9 signaling from the new vacuole is required for cell-cycle progression

The above findings predict that regulation of the cell-cycle requires signaling from the vacuole. Evidence for a candidate signaling pathway came from studies which showed that deletion of Target Of Rapamycin 1 (TOR1) showed synthetic growth defects with vac17Δ and vac8Δ (Zurita-Martinez et al., 2007; Costanzo et al., 2010) (Figure 4—fiugre supplements 1A,B). Similarly, another vacuole inheritance mutant, myo2-N1304D, was synthetic lethal with tor1Δ (Figure 4—figure supplement 1C). Notably, the vac17Δ tor1Δ double mutant showed an increase in cells arrested at G1 phase (Figure 4A,B). This arrest in G1 phase was similar to that observed for the vac17Δ pep12-60^tsf and vac17Δ pep12Δ mutants (Figure 3—figure supplements 1A–C). This suggests that TORC1 signaling from the vacuole may account at least in part for the G1 arrest observed in the vac17Δ pep12-60^tsf and vac17Δ pep12Δ mutants. Interestingly, the vac17Δ tor1Δ mutants generated a new vacuole in the daughter cells (Figure 4C). This suggests that TOR1 functions after the synthesis of the new vacuole.

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TOR1 encodes a PIK-related protein kinase (Alarcon et al., 1999). In yeast, Tor1 functions in the TORC1 complex, which is composed of Tor1/2, Kog1, Lst8, and Tco89 (Loewith et al., 2002). In yeast, TORC1 localizes on the vacuole membrane (Reinke et al., 2004; Araki et al., 2005; Urban et al., 2007; Sturgill et al., 2008; Binda et al., 2009; Jin et al., 2014), and is a key determinant of nutrient status (Di Como and Arndt, 1996). We found that the kinase activity of Tor1 was required for growth of vac17Δ (Figure 4D), and that the TORC1 specific subunit KOG1 was also required for growth of vac17Δ (Figure 4—figure supplement 1D). In addition, the lst8-15 temperature sensitive mutant is also synthetically lethal with vac17Δ (Costanzo et al., 2010). These results suggest that the kinase activity of the TORC1 complex is required for normal growth of vacuole inheritance mutants.

One critical target of TORC1 is the Sch9 kinase, which shares overlapping functions with metazoan S6 kinase (Ballou et al., 1991; Oldham et al., 2000; Christie et al., 2002). TORC1 directly phosphorylates Sch9 on several serines and threonines, and the phospho-mimetic sch9-2D3E, but not wild-type SCH9 or the Ala-substituted sch9-5A mutant bypasses the TORC1 inhibitor, rapamycin (Loewith et al., 2002; Urban et al., 2007). Notably, the phospho-mimetic sch9-2D3E mutant partially suppressed the growth defect of the vac17Δ tor1Δ mutant (Figure 4E), suggesting that the arrest of this mutant is due in part to defects in TORC1 mediated signaling via SCH9.

It was previously shown that Sch9-2D3E localizes on the vacuole membrane (Urban et al., 2007). To test whether bypass of Tor1 by sch9-2D3E requires a functional vacuole, we tested whether sch9-2D3E can suppress the vac17Δ pep12Δ double mutant. Notably sch9-2D3E did not suppress the vac17Δ pep12Δ double mutant (Figure 4F). This suggests that a functional vacuole is required for the roles of the TORC1-SCH9 pathway in cell-cycle progression from G1 phase.

While previous studies showed that TORC1 signals from the vacuole/lysosome (Sancak et al., 2010), and that Sch9 is activated in that location (Urban et al., 2007), it was assumed that once Sch9 is activated, it no longer requires the vacuole for its further downstream functions. However our findings strongly suggest that the vacuole is required for Sch9 function(s) after Sch9 is phosphorylated by TORC1. One possible role of the vacuole in Sch9 function, is that target protein(s) of the Sch9 kinase must be present on the vacuole membrane. Alternatively or in addition, the full kinase activity of Sch9 may require other proteins that are on the vacuole membrane.

The newly synthesized vacuoles initially lack Sch9 and Fab1

If a cell does not receive a vacuole from the mother cell, the daughter cell generates a new vacuole. The observation that these new vacuoles grow to a specific size prior to generation of a bud, and that TORC1 signaling is also required, raised the possibility that that there are functional differences between newly synthesized vacuoles and inherited vacuoles. As a first approach, we tested the localization of several proteins that are involved in the TORC1 pathway, Tor1, Kog1 and Sch9. New vacuoles were defined as Vph1-CFP positive structures that failed to inherit FM4-64. Notably, in vac17Δ cells, GFP-Sch9 was defective in its localization to the new vacuoles, while the localization of Tor1 and Kog1 were unaffected (Figure 5A,B). To directly address whether Sch9 is eventually recruited to the newly formed vacuole and when this occurs, we correlated the presence of fluorescent signals for Vph1-CFP, Tor1-3xGFP and GFP-Sch9 (Figure 5C,D). These analyses show that Sch9 recruitment to the newly formed vacuole is slower than the recruitment of Vph1 and Tor1. Specifically, small budded cells did not have fluorescent signals for any of the proteins, which indicates that these small buds do not have a vacuole (Figure 5C–F). As the bud increases in size, in most cases, Vph1-CFP and Tor1-3xGFP appeared simultaneously (Figure 5C). This indicates that Tor1-3xGFP is immediately recruited to the newly formed vacuoles. Moreover in some small budded cells, Tor1-3xGFP was present without Vph1-CFP, which suggests that Tor1 may be present at endosomes in these small budded cells.

Figure 5

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In the vac17Δ mutant, although Tor1-3xGFP and Vph1-CFP are both present on the new vacuoles in medium buds (approximately 0.6 daughter size/mother size) (Figure 5C), GFP-Sch9 was generally not present until the bud size was larger (approximately 0.8) (Figure 5D). Thus Sch9 recruitment is delayed compared to Vph1 and Tor1, but eventually occurs. Notably the recruitment of the Sch9-2D3E mutant was similar to the recruitment of wild-type Sch9 (Figure 5E), indicating that the growth suppression of the vac17Δ tor1Δ mutant by sch9-2D3E is not due to a faster recruitment to the vacuole.

PI(3,5)P₂, an inositol lipid which is present at the vacuole membrane, is required for vacuole membrane targeting of Sch9 (Jin et al., 2014). To test whether the delay in Sch9 recruitment might be due to a delay in the generation of PI(3,5)P₂ on newly formed vacuoles, we tested the timing of the recruitment of Fab1, the sole lipid kinase that generates PI(3,5)P₂ (Gary et al., 1998). Notably the timing of the recruitment of Fab1 was similar to that observed for Sch9. In a vac17Δ mutant which co-expressed Vph1-CFP and Fab1-3xGFP, only Vph1-CFP is present in medium buds (approximately 0.6 daughter size/mother size) (Figure 5F). The average bud size where both Vph1 and Fab1 are present was 0.76(±0.10). Thus Fab1 recruitment is delayed compared to Tor1, but is similar to that observed for Sch9. These observations suggest that there are differences between new vacuoles and inherited vacuoles, and that the newly synthesized vacuoles are missing specific components of mature vacuoles that are essential for cell-cycle progression.

Together, these observations demonstrate that a functional vacuole is crucial for cell-cycle progression at G1 phase, and that the TORC1-SCH9 pathway is part of this critical function (Figure 4G). TORC1-SCH9 signaling from the vacuole may be involved in G1 progression through its known functions in a ribosome biogenesis and translation (Barbet et al., 1996; Jorgensen et al., 2004; Urban et al., 2007). Alternatively, the TORC1-SCH9 pathway may signal the cell-cycle machinery that a functional vacuole is present and hence the cell is ready to progress from G1. Previous studies showed that Sch9 activity is critical for cell size (Jorgensen et al., 2004; Urban et al., 2007), and that cell size correlates with vacuole size (Chan and Marshall, 2014). These observations together with the current study suggest that TORC1-SCH9 localization and signaling from the vacuole is critical for the regulation of cell size. It is tempting to speculate that an analogous regulation of the cell-cycle occurs from endo-lysosomal membranes in other organisms. In addition, these findings lead to the hypothesis that cells possess novel checkpoint mechanisms that prevent cell-cycle progression at G1 phase in the absence of essential organelles.

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Author details

1. Yui Jin

   Life Sciences Institute, Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

   YJ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Lois S Weisman

   Life Sciences Institute, Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

   LSW, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   lweisman@umich.edu

   Competing interests

   The authors declare that no competing interests exist.

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