Zn2+-treatment temporarily arrested cell growth.
Zinc, like any ion, is cytotoxic when its levels are sufficiently raised. Curiously, in the case of yeast, the sensitivity of cells to this metal proved to be dependent on the composition of the medium used. BY4741 grew well on a rich medium, YPD, supplemented with 13 mM ZnSO4 (). However, on standard minimal medium with glucose (SD), colony-forming ability decreased sharply when Zn2+ levels rose from 10 to 13 mM. As shows, even adding uracil to SD medium slightly increased cell tolerance for zinc.
Figure 1 Zn2+ disrupts cell metabolism and division. (A) The sensitivity of the parental strain, BY4741, transformed with the plasmid, YEp, was assessed using cultures grown in SD medium to mid-exponential phase. A sample was serially diluted by factors of 10, (more ...)
Further studies revealed that in liquid SD medium, cell division ceased within the first 2 h of exposure to 13 mM Zn2+
arresting the population with a multibudded phenotype () looking like mutants missing Cdc42.18
This period of stasis lasted for approximately 6 h (). By this time, 54.0 ± 1.5% of the Zn2+
-treated cells had accumulated large, refractile aggregates in their vacuoles (arrow, ), whereas untreated cells had none. Although we have not performed definitive chemical analysis on these inclusions, we have determined that they stained with DAPI (Fig. S1
), like polyphosphate inclusions,19
and were less abundant (6.3 ± 1.5%) in cells with a deletion of VTC4
, a gene needed for polyphosphate accumulation in the vacuole.20
When BY4741 cultures were diluted into medium without supplemental zinc, the number of cells harboring these inclusions dropped to 32.6 ± 1.9% within 2 h and continued falling thereafter. Unbudded cells began to appear some time later. On the other hand, cells that remained under zinc treatment gradually became permeable to propidium-iodide (PI) and lost viability (), dropping to 50% of the starting population by 22 h (data not shown).
Apoptotic processes played little role in the death of Zn2+-treated cells.
Nargand et al.21
found that Cd2+
, an ion chemically related to Zn2+
, induces metacaspase-dependent apoptotic death. Zn2+
, however, did not. First, if zinc-treated cells died apoptotically, the loss of the metacaspase gene, YCA1
or of other apoptosis-associated genes such as AIF1
would have decreased zinc sensitivity. Instead, these mutants proved to be as sensitive as their parental strain (PS), BY4741, on Zn2+
-containing media ( and data not shown). Second, if zinc treatments induced apoptosis, we expected the frequency of annexin-binding cells to increase, as occurs when cells are treated with 80 mM acetic acid (Fig. S2
). Instead, the percentage of PI-permeable necrotic cells increased approximately eight-fold after 6 h of Zn2+
treatment, whereas the frequency of annexin-binding, apoptotic ones remained unchanged (Fig. S2
). Although apoptotic genes contributed little to this kind of death, a knockout of a core autophagic gene, ATG8
, actually increased colony formation significantly (). Introducing a yca1
Δ mutation into an atg8
Δ mutant did not change this level of resistance (data not shown).
Figure 2 Zn2+-induced cell death is dependent on ATG8. The parental strain (PS), and two derivatives with deletions of either ATG8 or YCA1, all transformed with the plasmid, YEp, were grown, diluted and stamped as described in onto SD agar with or without (more ...)
Zn2+ partially inhibited the flow of GFP-Atg8 to vacuoles.
The above results implied that zinc triggered a previously unexamined form of cell death mediated, at least in part, by Atg8. Atg8 is essential for the expansion of autophagosomes26
and over the years, has proven to be a reliable monitor for tracking autophagic activity.27
In order to determine whether death was accompanied by a noticeable increase in the number of autophagosomes, a plasmid encoding a GFP-Atg8 fusion protein28
was introduced into the atg8
Δ mutant. This strain was as sensitive as BY4741 when grown on SD medium with 13 mM Zn2+
(data not shown), indicating that the chimeric gene retained its attendant functions. Under normal growing conditions, the fluorescent reporter was distributed throughout the cytoplasm and nearly absent from the vacuoles (). This distribution was reversed when bulk autophagy was induced with rapamycin. Most of these treated cells had comparatively dark cytoplasms and intensely fluorescent vacuoles, together with a characteristic increase in GFP-Atg8 processing (). In contrast, cells treated with zinc were indistinguishable from untreated cells (), apart from the polyphosphate-like inclusions and multibudded appearance. Cells treated with both agents displayed a mixed phenotype in which diffuse, fluorescent vacuoles were surrounded by dark cytoplasm, one to three intensely glowing vesicles docked close to the vacuole (), and levels of GFP-Atg8 processing similar to those seen with rapamycin alone (). The proportion of the population that fell into each of these phenotypic classes is shown in .
Figure 3 GFP-ATG8 accumulated in perivacuolar vesicles in Zn2+-treated cells. The atg8Δ strain transformed with a plasmid encoding GFP-ATG8 was grown to an OD600 = 0.2 and then treated with 1.0 mM PMSF (which did not significantly inhibit yeast growth (more ...)
These studies clearly showed that zinc did not induce autophagic processes like rapamycin. In fact, the most parsimonious interpretation of the results tabulated in appeared to be that Zn2+ partially inhibited a late stage in the autophagic process, so that the reporter was left in the cytoplasm or sequestered in autophagosome-like vesicles that rarely, if ever, delivered their contents to the vacuole for recycling.
Zn2+-treated cells did not harvest common fluorescent reporters of bulk autophagy activity.
It seemed counterintuitive that Zn2+
killed cells in an Atg8-dependent manner without increasing the number of visible autophagosomes. We therefore continued our studies using several different reporter proteins, each one diagnostic for a different form of autophagy. One particularly useful series of reporters has been built recently by fusing an acid-sensitive GFP to an acid-stable RFP.29
When autophagy was not induced, the vacuoles of cells expressing the cytoplasmic variant of this series, ROSELLACyt
,remained dark, even when observations were continued past 24 h (). On the other hand, when bulk autophagy was induced by rapamycin treatment, some of this protein was harvested so that the vacuoles fluoresced red while the cytoplasm glowed red or green, depending on the excitation filters used. Zinc did not have this effect. Instead, ROSELLACyt
remained in the cytoplasm unless cells were treated simultaneously with rapamycin. Note that although GFP-Atg8 revealed autophagosomes in transit during zinc and rapamycin treatment (), no ROSELLACyt
-filled vesicles were seen (). It is possible that ROSELLACyt
accumulation in autophagosomes was too low to highlight them.
Figure 4 Dual fluorescent protein, ROSELLACyt, was restricted to the cytosol in Zn2+-treated cells. PS cells transformed with a plasmid encoding cytoplasmic ROSELLA29 were grown to mid-exponential phase and treated for 16 h with 1.0 mM PMSF together with 13 mM (more ...)
reporter showed that zinc did not induce bulk autophagy like rapamycin. Further experiments with ROSELLA proteins targeted to the nucleus (ROSELLANuc
) or to mitochondria (ROSELLAMit
) demonstrated that zinc similarly failed to induce either piecemeal nuclear autophagy or mitophagy to any noticeable degree (Fig. S3
). Similarly, studies with Rpl25-GFP,30
a fusion between a protein of the 60S ribosomal subunit and GFP, failed to detect signs of ribophagy (data not shown).
Zn2+ treatment inhibited entry of prApe1 into vacuoles.
It is now recognized that cells use different combinations of autophagy proteins to target different parts of the cell. It has even been suggested that there is no such process as nonselective autophagy; rather, many of the treatments that have been used might in fact be causing the simultaneous execution of several (possibly independent) autophagic pathways.31
In particular, at least half of the gene products responsible for inducible bulk autophagy in yeast also participate in the far more selective Cvt pathway that transports a handful of hydrolytic enzymes to the vacuole where they participate in the routine metabolism of the cell.32
One of the enzymes brought there in this way is aminopeptidase I, the product of the gene APE1
. We transformed yeast with plasmids encoding an Ape1-RFP fusion protein33
and used it to monitor the Cvt pathway. Under normal growth conditions, this reporter accumulated in low amounts within the vacuole. Rapamycin treatment increased the amount of fluorescent material in the vacuole considerably (). However, while the subcellular distributions of the ROSELLA and Rpl25-GFP reporter proteins were unaffected by zinc, Ape1-RFP distributions were affected. Zn2+
-treated cells showed a seven-fold reduction in Ape1-RFP processing () and were nine times more likely than untreated cells to have one to two highly fluorescent vesicles, presumably Cvt vesicles, docked onto the surface of otherwise dark vacuoles (). Superficially, the localization and the intensity of fluorescence of Ape1-RFP in zinc-treated cells resembled those seen in mon1
Δ mutants () that are defective in both autophagosome and Cvt vesicle fusion with vacuoles.34
Significantly, this Zn2+
-imposed block was overcome when bulk autophagy became active: vacuolar filling () and Ape1-RFP processing () proceeded equally in cells treated with zinc and rapamycin and in cells treated with rapamycin alone.
Figure 5 Ape1-RFP accumulated in perivacuolar vesicles in Zn2+-treated cells. PS cells transformed with a plasmid encoding Ape1-RFP were grown to an OD600 = 0.2 and then treated with 1.0 mM PMSF together with 13 mM ZnSO4 or 0.22 µM rapamycin (Rap) or both (more ...)
These results indicated that Zn2+ treatment inhibited one or more of the last steps in the delivery of Cvt vesicles to the vacuole. Since rapamycin suppressed this block, it seems likely that bulk autophagy either did not depend on a zinc-sensitive fusion process, or compensated for the reduced efficiency of vesicle-to-vacuole fusion with increased vesicle production. Either of these models would equally accommodate our previous observation that ROSELLACyt, like Ape1-RFP, continued to enter vacuoles when rapamycin-treated cultures were co-challenged with zinc ().
Which autophagic pathway contributed to ziNCD?
The studies with the previous sets of reporter proteins failed to find evidence that zinc treatment induced excessive autophagosome formation and indiscriminate protein harvesting, one possible mechanism leading to cell death.35,36
At the same time, it was counterintuitive to infer that inhibiting a late step in the Cvt pathway would lead to cell death when genetically inactivating the pathway did not.37
This prompted us to investigate whether the atg8
Δ phenotype was representative of all autophagy mutants growing on zinc-rich media. For these experiments, we included 20 mgL−1
uracil in the agar so that we could carry out these tests on plasmid-free strains, and so that we could detect increased sensitivity as well as increased resistance to zinc.
The autophagy-defective strains that we tested fell into four phenotypic classes (), regardless of whether they had been made in BY4741 or BY4742 (Fig. S4
). The majority, including the atg1
Δ and atg2
Δ mutants, behaved like the parental line when each was grown on ZnSO4
-supplemented agar. A smaller number typified by atg12
Δ, were more tolerant, others like atg8
Δ even more so, but a handful, including atg11
Δ, were more sensitive to zinc than their parental strain. The makeup of the four phenotypic classes is summarized in .
Figure 6 Classification of phenotypic responses of mutants to excess zinc. (A) The indicated strains were grown, diluted, and stamped as described in onto SD agar containing 0.002% uracil with or without 13 mM ZnSO4. The mutants shown were representative (more ...)
Classification of knockout mutant phenotypes
These differences in growth on solid media were also seen in liquid medium (). By 12 h, each example that we tested showed signs of necrosis, but strains showing the greatest resistance to zinc (atg8Δ) showed less, while those with the least resistance (atg11Δ) showed more (). Closer examination of these mutants revealed that the number of cells containing polyphosphate-like inclusions correlated with the number of PI-excluding, seemingly viable, cells in each culture (): atg11Δ cultures contained the fewest of these inclusions if one surveyed all cells, but similar numbers if one confined the counts to intact (PI-) cells. This correlation could imply that cells produced polyphosphates defensively, perhaps to immobilize vacuolar zinc. Significantly, although there was also variation in the number of cells accumulating H2O2 after 12 h of zinc treatment (), it seemed that each mutant strain had similar numbers of H2O2-accumulating cells (measured by fluorescence in the presence of H2DCFDA) regardless of their level of zinc resistance. It was as if zinc sensitivity was either unrelated to this source of damage, or instead determined by the subsequent response to that damage.
While the correlation was not perfect, most of the strains in class I were mutated in genes closely associated with the control of nonspecific autophagic responses to starvation and rapamycin treatment. Many of the strains in classes II and III were mutated in genes that encoded proteins making up the core machinery of autophagy, whereas most of the mutants in class IV encoded proteins that were only required for the Cvt pathway and a few specialized forms of autophagy. In an attempt to refine our classification scheme and pinpoint the specific pathways being detected in this assay, we tested mutants that affected a number of protease and ubiquitination processes (APE1, BSD2, DSK2, LAP3, PEP4, TUL1
), as well as mutations specifically associated with pexophagy (PEX3, PEX14
), microphagy (GTR1, GTR2
), mitophagy (AUT1, UTH1
), and early steps in the regulation of autophagy (RAS1, RAS2, SCH9, TOR1
). All of these displayed the same level of sensitivity as their parental strain. On the other hand, strains missing ZRC1
, the principal zinc transporter in the vacuolar membrane, CCZ1, MON1, TLG2
, which help autophagosomes and Cvt vesicles dock with the vacuole,38,39 BRE5
that are needed for ribophagy,30 PEX6
, a peroxisomal protein transporter needed to suppress the buildup of reactive oxygen species that leads to necrotic cell death,40 SLM4
), which is part of the microautophagy pathway and critical for recovery from rapamycin treatments,41 SNF1
, which regulates the cellular response to nutrient availability,42 VPS15
(a regulator of protein sorting43
) and VTC4
(a subunit of the polyphosphate polymerase44
), were all more sensitive to Zn2+
than seen with the wild type (). Surprisingly few of these genes have been associated with zinc tolerance before, despite the fact that several intensive screens for the determinants of zinc homeostasis have previously been carried out. However, most of those screens for zinc sensitivity45
and for zinc-induced genes46
were conducted on YPD medium where the PS strain (), as well as four apoptosis-associated genes and six autophagy genes drawn from all four classes (data not shown), showed no growth inhibition in the presence of 13 mM zinc.
Although most of the autophagy genes in class IV encoded proteins involved specifically in the Cvt pathway, not all Cvt genes were in class IV. Mutants in ATG19
, the key receptor for bringing proteins into Cvt vesicles, fell into class I while mutants in ATG24
, a nexin involved in retrieving SNAREs from endosomes,47
fell into class III. One possible explanation for these exceptions could be that cells use class IV proteins to build a basic vesicle that then interacts modularly either with Atg19 to produce a Cvt vesicle, or with as yet unidentified gene products to select different sets of substrates for a minimum of two alternate forms of selective autophagy. In this scheme, one of these two vesicle-receptor conformations protected cells from the effects of Zn2+
while the other configuration acting together with Atg24 contributed to ziNCD.
One question that lingered throughout this study was why these sets of genes had not shown up in any of the more extensive searches for zinc tolerance pathways.45,46
Some studies may have overlooked the kinds of phenotypes reported here because they were carried out on rich medium (). However, these phenotypes may also have been masked by genetic differences between strains. SEY6210,48
for example, proved to be more sensitive to zinc than BY4741 (compare Fig. S5A
where strains were grown for 5 d with Fig. S4
where strains were grown for 3 d). We do not yet have an explanation for the strain-dependent differences in zinc sensitivity, but we did observe that this sensitivity was recessive in a BY4741/SEY6210 diploid (Fig. S5B
). Despite this difference in responsiveness to zinc, SEY6210 responded qualitatively like BY4741 when its copies of ATG11
were knocked out (Fig. S5A
), but perhaps not dramatically enough to attract attention during a screen of the entire yeast genome. As a side-note, an atg8
Δ double mutant reproducibly grew better than a knockout of ATG8
alone (Fig. S5A
). This synergistic response could indicate that while Atg11 acted primarily to protect cells, it also participated to a small extent in ziNCD.