Joseph V. Gray

“Sleeping Beauty”: Quiescence in Saccharomyces cerevisiae

SUMMARY The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one “model” quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.

A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator

The protein kinase C of Saccharomyces cerevisiae, Pkc1, regulates a MAP kinase, Mpk1, whose activity is stimulated at the G1–S transition of the cell cycle and by perturbations to the cell surface, e.g. induced by heat shock. The activity of the Pkc1 pathway is partially dependent on Cdc28 activity. Swi4 activates transcription of many genes at the G1–S transition, including CLN1 and CLN2. We find that swi4 mutants are defective specifically in bud emergence. The growth and budding defects of swi4 mutants are suppressed by overexpression of PKC1. This suppression requires CLN1 and CLN2. Inhibition of the Pkc1 pathway exacerbates the growth and bud emergence defects of swi4 mutants. We find that another dose‐dependent suppressor of swi4 mutants, the novel gene HCS77, encodes a putative integral membrane protein. Hcs77 may regulate the Pkc1 pathway; hcs77 mutants exhibit phenotypes like those of mpk1 mutants, are partially suppressed by overexpression of PKC1 and are defective in heat shock induction of Mpk1 activity. We propose that the Pkc1 pathway promotes bud emergence and organized surface growth and is activated by Cdc28–Cln1/Cln2 at the G1–S transition and by Hcs77 upon heat shock. Hcs77 may monitor the state of the cell surface.

The Protein Kinase C Pathway Is Required for Viability in Quiescence in Saccharomyces cerevisiae

Protein kinase C, encoded by PKC1, regulates construction of the cell surface in vegetatively growing yeast cells. Pkc1 in part acts by regulating Mpk1, a MAP kinase. Mutants lacking Bck1, a component of the MAP kinase branch of the pathway, fail to respond normally to nitrogen starvation, which causes entry into quiescence. Given that the Tor1 and Tor2 proteins are key inhibitors of entry into quiescence, the Pkc1 pathway may regulate these proteins. We find that pkc1Delta and mpk1Delta mutants rapidly die by cell lysis upon carbon or nitrogen starvation. The Pkc1 pathway does not regulate the TOR proteins: transcriptional changes dependent on inhibition of the TORs occur normally in pkc1Delta and mpk1Delta mutants when starved for nitrogen; pkc1Delta and mpk1Delta mutants die rapidly upon treatment with rapamycin, an inhibitor of the TORs. We find that Mpk1 is transiently activated by rapamycin treatment via a novel mechanism. Finally, we find that rapamycin treatment or nitrogen starvation induces resistance to the cell wall-digesting enzyme zymolyase by a Pkc1-dependent mechanism. Thus, the Pkc1 pathway is not a nutrient sensor but acts downstream of TOR inhibition to maintain cell integrity in quiescence.

Identification of two partners from the bacterial Kef exchanger family for the apical plasma membrane V-ATPase of Metazoa

The vital task of vectorial solute transport is often energised by a plasma membrane, proton-motive V-ATPase. However, its proposed partner, an apical alkali-metal/proton exchanger, has remained elusive. Here, both FlyAtlas microarray data and in situ analyses demonstrate that the bacterial kefB and kefC (members of the CPA2 family) homologues in Drosophila, CG10806 and CG31052, respectively, are both co-expressed with V-ATPase genes in transporting epithelia. Immunocytochemistry localises endogenous CG10806 and CG31052 to the apical plasma membrane of the Malpighian (renal) tubule. YFP-tagged CG10806 and CG31052 both localise to the plasma membrane of Drosophila S2 cells, and when driven in principal cells of the Malpighian tubule, they localise specifically to the apical plasma membrane. V-ATPase-energised fluid secretion is affected by overexpression of CG10806, but not CG31052; in the former case, overexpression causes higher basal rates, but lower stimulated rates, of fluid secretion compared with parental controls. Overexpression also impacts levels of secreted Na+ and K+. Both genes rescue exchanger-deficient (nha1 nhx1) yeast, but act differently; CG10806 is driven predominantly to the plasma membrane and confers protection against excess K+, whereas CG31052 is expressed predominantly on the vacuolar membrane and protects against excess Na+. Thus, both CG10806 and CG31052 are functionally members of the CPA2 gene family, colocalise to the same apical membrane as the plasma membrane V-ATPase and show distinct ion specificities, as expected for the Wieczorek exchanger.

Mpt5p, a Stress Tolerance- and Lifespan-Promoting PUF Protein in Saccharomyces cerevisiae, Acts Upstream of the Cell Wall Integrity Pathway

ABSTRACT Pumilio family (PUF) proteins affect specific genes by binding to, and inhibiting the translation or stability of, their transcripts. The PUF domain is required and sufficient for this function. One Saccharomyces cerevisiae PUF protein, Mpt5p (also called Puf5p or Uth4p), promotes stress tolerance and replicative life span (the maximum number of doublings a mother cell can undergo before entering into senescence) by an unknown mechanism thought to partly overlap with, but to be independent of, the cell wall integrity (CWI) pathway. Here, we found that mpt5Δ mutants also display a short chronological life span (the time cells stay alive in saturated cultures in synthetic medium), a defect that is suppressed by activation of CWI signaling. We found that Mpt5p is an upstream activator of the CWI pathway: mpt5Δ mutants display the appropriate phenotypes and genetic interactions, display low basal activity of the pathway, and are defective in activation of the pathway upon thermal stress. A set of mRNAs that specifically bind to Mpt5p was recently reported. One such putative target, LRG1, encodes a GTPase-activating protein for Rho1p that directly links Mpt5p to CWI signaling: Lrg1p inhibits CWI signaling, LRG1 mRNA contains a consensus Mpt5p-binding site in its putative 3′ untranslated region, loss of Lrg1p suppresses the temperature sensitivity and CWI signaling defects of mpt5Δ mutants, and LRG1 mRNA abundance is inhibited by Mpt5p. We conclude that Mpt5p is required for normal replicative and chronological life spans and that the CWI pathway is a key and direct downstream target of this PUF protein.

Direct and novel regulation of cAMP-dependent protein kinase by Mck1p, a yeast glycogen synthase kinase-3.

The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase.

Functional specialisation of yeast Rho1 GTP exchange factors.

Rho GTPases are regulated in complex spatiotemporal patterns that might be dependent, in part at least, on the multiplicity of their GTP exchange factors (GEFs). Here, we examine the extent of and basis for functional specialisation of the Rom2 and Tus1 GEFs that activate the yeast Rho1 GTPase, the orthologue of mammalian RhoA. First, we find that these GEFs selectively activate different Rho1-effector branches. Second, the synthetic genetic networks around ROM2 and TUS1 confirm very different global in vivo roles for these GEFs. Third, the GEFs are not functionally interchangeable: Tus1 cannot replace the essential role of Rom2, even when overexpressed. Fourth, we find that Rom2 and Tus1 localise differently: Rom2 to the growing bud surface and to the bud neck at cytokinesis; Tus1 only to the bud neck, but in a distinct pattern. Finally, we find that these GEFs are dependent on different protein co-factors: Rom2 function and localisation is largely dependent on Ack1, a SEL1-domain-containing protein; Tus1 function and localisation is largely dependent on the Tus1-interacting protein Ypl066w (which we name Rgl1). We have revealed a surprising level of diversity among the Rho1 GEFs that contributes another level of complexity to the spatiotemporal control of Rho1.

The functional relationships underlying a synthetic genetic network

Here, we focus on synthetic lethal genetic interactions, examples of genetic enhancements, where mutations in two different genes result in lethality but only when present together. We recently identified the synthetic lethal network around the PKC1 gene encoding the essential protein kinase C of yeast. We found that this network is heavily enriched for interactions with genes whose products are closely linked to Pkc1 signallng in vivo. Here, we show that: the PKC1 gene elicits a distinct spectrum of genetic interactions to SLT2, encoding a non-essential component of the very same signaling pathway. We also show that the terminal phenotype underlying the synthetic lethal network around PKC1 is not uniform. Synthetic lethal genetic networks thus appear to be very heterogeneous in nature with important implications for what functional relationships can be discovered from them.

Identifying in vivo pathways using genome-wide genetic networks.

Synthetic genetic interactions occur between two genes when the double mutant displays a phenotype much more severe than does either single mutant alone. Global networks of such interactions are now being systematically determined, spearheaded by the budding yeast genome. Genetic interactions reflect in vivo relationships between gene products. Extracting that functional information from such genetic networks is now possible by exploiting and modifying the key concept of congruence. Here, we focus on synthetic genetic interactions between pairs of null mutations in non-essential yeast genes. We summarize how to identify biological pathways from these emerging networks, using illustrative examples.

Recovery from Rapamycin DRUG-INSENSITIVE ACTIVITY OF YEAST TARGET OF RAPAMYCIN COMPLEX 1 (TORC1) SUPPORTS RESIDUAL PROLIFERATION THAT DILUTES RAPAMYCIN AMONG PROGENY CELLS

Background: The EGO complex activates yeast TORC1 and is somehow required for recovery from rapamycin, a potent inhibitor of TORC1. Results: Rapamycin-insensitive activity of TORC1 partly depends on the EGO complex and supports residual cell proliferation that dilutes the metabolically stable drug among progeny cells. Conclusion: TORC1 activity is required to dilute rapamycin. Significance: Rapamycin only partly inhibits yeast TORC1. The target of rapamycin complex 1 (TORC1) is a key conserved regulator of eukaryotic cell growth. The xenobiotic rapamycin is a potent inhibitor of the yeast complex. Surprisingly, the EGO complex, a nonessential in vivo activator of TORC1, is somehow required for yeast cells to recover efficiently from a period of treatment with rapamycin. Why? Here, we found that rapamycin is only a partial inhibitor of TORC1. We confirmed that saturating amounts of rapamycin do not fully inhibit proliferation of wild-type cells, and we found that the residual proliferation in the presence of the drug is dependent on the EGO complex and on the activity of TORC1. We found that this residual TORC1-dependent proliferation is key to recovery from rapamycin treatment. First, the residual proliferation rate correlates with the ability of cells to recover from treatment. Second, the residual proliferation rate persists long after washout of the drug and until cells recover. Third, the total observable pool of cell-associated rapamycin is extremely stable and decreases only with increasing cell number after washout of the drug. Finally, consideration of the residual proliferation rate alone accurately and quantitatively accounts for the kinetics of recovery of wild-type cells and for the nature and severity of the ego− mutant defect. Overall, our results revealed that rapamycin is a partial inhibitor of yeast TORC1, that persistence of the drug limits recovery, and that rapamycin is not detoxified by yeast but is passively diluted among progeny cells because of residual proliferation.