Genetic analysis of the interface between Cdc42p and the CRIB domain of Ste20p in Saccharomyces cerevisiae

Mutagenesis was used to probe the interface between the small GTPase Cdc42p and the CRIB domain motif of Ste20p. Members of a cluster of hydrophobic residues of Cdc42p were changed to alanine and/or arginine. The interaction of the wild-type and mutant proteins was measured using the two-hybrid assay; many, but not all, changes reduced interaction between Cdc42p and the target CRIB domain. Mutations in conserved residues in the CRIB domain were also tested for their importance in the association with Cdc42p. Two conserved CRIB domain histidines were changed to aspartic acid. These mutants reduced mating, as well as responsiveness to pheromone-induced gene expression and cell cycle arrest, but did not reduce in vitro the kinase activity of Ste20p. GFP-tagged mutant proteins were unable to localize to sites of polarized growth. In addition, these point mutants were synthetically lethal with disruption of CLA4 and blocked the Ste20p-Cdc42p two-hybrid interaction. Compensatory mutations in Cdc42p that reestablished the two-hybrid association with the mutant Ste20p CRIB domain baits were identified. These mutations improved the pheromone responsiveness of cells containing the CRIB mutations, but did not rescue the lethality associated with the CRIB mutant CLA4 deletion interaction. These results suggest that the Ste20p-Cdc42p interaction plays a direct role in Ste20p kinase function and that this interaction is required for efficient activity of the pheromone response pathway.     

Functional molecular masses of vacuolar membrane H+-ATPase from Saccharomyces cerevisiae as studied by radiation inactivation analysis

The functional molecular masses of the vacuolar membrane H+-ATPase in Saccharomyces cerevisiae under two kinetic conditions for ATP hydrolysis were measured by radiation inactivation. When vacuolar membrane vesicles were exposed to gamma-rays from 60Co, the activities catalyzing a single-cycle and multi-cycles of ATP hydrolysis both decreased as single-exponential functions of the radiation dosage. By applying the target theory, the functional molecular masses for single- and multi-cycle hydrolyses of ATP were determined to be approx. 0.9-1.1 X 10(5) and 4.1-5.3 X 10(5) Da, respectively. N,N'-Dicyclohexylcarbodiimide (DCCD) did not inhibit the former reaction but strongly inhibited the latter. It is suggested that the ATPase with a minimal composite of subunits a and b, in which subunit c is not necessarily involved operationally, can catalyze single-cycle hydrolysis of ATP, whereas for multi-cycle hydrolysis of ATP, the ATPase requires a properly organized oligomeric structure with subunits a-c, which may direct a positive cooperative mechanism of ATP hydrolysis and coupled H+ translocation in a DCCD-sensitive manner.     

Vacuolar acidification in Saccharomyces cerevisiae induced by elevated hydrostatic pressure is transient and is mediated by vacuolar H+-ATPase

We analyzed the vacuolar acidification in response to elevated hydrostatic pressure in Saccharomyces cerevisiae. The vacuolar pH, defined using 6-carboxyfluorescein, was directly measured in a hyperbaric chamber with a transparent window under high hydrostatic pressure. The vacuole of strain X2180 became acidified at the onset of pressurization to an extent dependent on the magnitude of pressure applied. A pressure of 40–60 MPa transiently reduced the vacuolar pH by about 0.33 within 4 min. The transient acidification was observed in the presence of D-glucose, D-fructose, or D-mannose as a carbon source, but not 3-o-methyl-D-glucose, ethanol, or glycerol, suggesting that the generation of CO₂ was involved in the process. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH when hydrostatic pressure was applied. This result indicates that the transient vacuolar acidification induced by elevated hydrostatic pressure is mediated through the function of the vacuolar H+-ATPase. Received: August 21, 1996 / Accepted: November 11, 1996     

Cyclic AMP controls the plasma membrane H+-ATPase activity from Saccharomyces cerevisiae

The thermosensitive G1-arrested cdc35-10 mutant from Saccharomyces cerevisiae, defective in adenylate cyclase activity, was shifted to restrictive temperature. After 1 h incubation at this temperature, the plasma membrane H+-ATPase activity of cdc35-10 was reduced to 50%, whereas that in mitochondria doubled. Similar data were obtained with cdc25, another thermosensitive G1-arrested mutant modified in the cAMP pathway. In contrast, the ATPase activities of the G1-arrested mutant cdc19, defective in pyruvate kinase, were not affected after 2 h incubation at restrictive temperature. In the double mutants cdc35-10 cas1 and cdc25 cas1, addition of extracellular cAMP prevented the modifications of ATPase activities observed in the single mutants cdc35-10 and cdc25. These data indicate that cAMP acts as a positive effector on the H+-ATPase activity of plasma membranes and as a negative effector on that of mitochondria.     

Cdc42 regulation of kinase activity and signaling by the yeast p21-activated kinase Ste20

The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated G beta gamma complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway.     

cAMP- and RAS-independent nutritional regulation of plasma-membrane H+-ATPase activity in Saccharomyces cerevisiae

The plasma-membrane ATPase of Saccharomyces cerevisiae is a proton pump whose activity, essential fro proliferation, is subject to regulation by nutritional signals. The previous finding that the CDC25 gene product is required for the glucose-induced H+-ATPase activation suggested that H+-ATPase activity is regulated by cAMP. Analysis of starvation-induced inactivation and glucose-induced activation of the H+-ATPase in mutants affected in activity of the RAS proteins, adenylyl cyclase or cAMP-dependent protein kinase showed that nutritional regulation of H+-ATPase activity does not depend directly on any of these factors. We conclude that adenlyl cyclase does not mediate all nutritional responses. This also indicates that the specific CDC25 requirement for the glucose-induced activation of the H+-ATPase identifies a new function for the CDC25 gene product, a function that appears to be independent of CDC25-mediated modulation of the RAS/adenylyl cyclase/cAMP pathway.     

Saccharomyces cerevisiae Vacuolar H+-ATPase Regulation by Disassembly and Reassembly: One Structure and Multiple Signals

Vacuolar H⁺-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved.     

Activation and significance of vacuolar H+-ATPase in Saccharomyces cerevisiae adaptation and resistance to the herbicide 2,4-dichlorophenoxyacetic acid

The stimulation of the activity of the H(+)-ATPase present in the vacuolar membrane (V-ATPase) of Saccharomyces cerevisiae is here described in response to a moderate stress induced by 2,4-dichlorophenoxyacetic acid (2,4-D). This in vivo activation (up to 5-fold) took place essentially during the adaptation period, preceding cell division under herbicide stress, in coordination with a marked activation of plasma membrane H(+)-ATPase (PM-ATPase) (up to 30-fold) and the decrease of intracellular and vacuolar pH values, suggesting that activation may be triggered by acidification. Single deletion of VMA1 and genes encoding other V-ATPase subunits led to a more extended period of adaptation and to slower growth under 2,4-D stress. Results suggest that a functional V-ATPase is required to counteract, more rapidly and efficiently, the dissipation of the physiological H(+)-gradient across vacuolar membrane registered during 2,4-D adaptation.     

The Salmonella Typhimurium effector SteC inhibits Cdc42-mediated signaling through binding to the exchange factor Cdc24 in Saccharomyces cerevisiae

Intracellular survival of Salmonella relies on the activity of proteins translocated into the host cell by type III secretion systems (T3SS). The protein kinase activity of the T3SS effector SteC is required for F-actin remodeling in host cells, although no SteC target has been identified so far. Here we show that expression of the N-terminal non-kinase domain of SteC down-regulates the mating and HOG pathways in Saccharomyces cerevisiae. Epistasis analyses using constitutively active components of these pathways indicate that SteC inhibits signaling at the level of the GTPase Cdc42. We demonstrate that SteC interacts through its N-terminal domain with the catalytic domain of Cdc24, the sole S. cerevisiae Cdc42 guanine nucleotide exchange factor (GEF). SteC also binds to the human Cdc24-like GEF protein Vav1. Moreover, expression of human Cdc42 suppresses growth inhibition caused by SteC. Of interest, the N-terminal SteC domain alters Cdc24 cellular localization, preventing its nuclear accumulation. These data reveal a novel functional domain within SteC, raising the possibility that this effector could also target GTPase function in mammalian cells. Our results also highlight the key role of the Cdc42 switch in yeast mating and HOG pathways and provide a new tool to study the functional consequences of Cdc24 localization.     

Regulation of neurogenesis by Fgf8a requires Cdc42 signaling and a novel Cdc42 effector protein

Fibroblast growth factor (FGF) signaling is required for numerous aspects of neural development, including neural induction, CNS patterning and neurogenesis. The ability of FGFs to activate Ras/MAPK signaling is thought to be critical for these functions. However, it is unlikely that MAPK signaling can fully explain the diversity of responses to FGFs. We have characterized a Cdc42-dependent signaling pathway operating downstream of the Fgf8a splice isoform. We show that a Cdc42 effector 4-like protein (Cdc42ep4-l or Cep4l) has robust neuronal-inducing activity in Xenopus embryos. Furthermore, we find that Cep4l and Cdc42 itself are necessary and sufficient for sensory neurogenesis in vivo. Furthermore, both proteins are involved in Fgf8a-induced neuronal induction, and Cdc42/Cep4l association is promoted specifically by the Fgf8a isoform of Fgf8, but not by Fgf8b, which lacks neuronal inducing activity. Overall, these data suggest a novel role for Cdc42 in an Fgf8a-specific signaling pathway essential for vertebrate neuronal development.     

Oligomerization regulates the localization of Cdc24, the Cdc42 activator in Saccharomyces cerevisiae

Guanine nucleotide exchange factor activation of Rho G-proteins is critical for cytoskeletal reorganization. In the yeast Saccharomyces cerevisiae, the sole guanine nucleotide exchange factor for the Rho G-protein Cdc42p, Cdc24p, is essential for its site-specific activation. Several mammalian exchange factors have been shown to oligomerize; however, the function of this homotypic interaction is unclear. Here we show that Cdc24p forms oligomers in yeast via its catalytic Dbl homology domain. Mutation of residues critical for Cdc24p oligomerization also perturbs the localization of this exchange factor yet does not alter its catalytic activity in vitro. Chemically induced oligomerization of one of these oligomerization-defective mutants partially restored its localization to the bud tip and nucleus. Furthermore, chemically induced oligomerization of wild-type Cdc24p does not affect in vitro exchange factor activity, yet it results in a decrease of activated Cdc42p in vivo and the presence of Cdc24p in the nucleus at all cell cycle stages. Together, our results suggest that Cdc24p oligomerization regulates Cdc42p activation via its localization.     

Decrease of the plasma membrane H+-ATPase activity during late exponential growth of Saccharomyces cerevisiae

During the last cell division of exponential growth, the H+-ATPase activity from the yeast plasma membrane decreases by a factor of two to three. This "arrest growth control" of ATPase activity is not accompanied by modification of the sensitivity to vanadate.     

Regulation of yeast ectoapyrase ynd1p activity by activator subunit Vma13p of vacuolar H+-ATPase

CD39-like ectoapyrases are involved in protein and lipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. By using a two-hybrid screen, we found that an activator subunit (Vma13p) of yeast vacuolar H(+)-ATPase (V-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase. Interaction of Ynd1p with Vma13p was demonstrated by direct binding and co-immunoprecipitation. Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13Delta mutant was drastically increased compared with that of Ynd1p in VMA13 cells. A similar increase in the apyrase activity of Ynd1p was found in a vma1Delta mutant, in which the catalytic subunit A of V-ATPase is missing, and the membrane peripheral subunits including Vma13p are dissociated from the membranes. However, the E286Q mutant of VMA1, which assembles inactive V-ATPase complex including Vma13p in the membrane, retained wild type levels of Ynd1p activity, demonstrating that the presence of Vma13p rather than the function of V-ATPase in the membrane represses Ynd1p activity. These results suggest that association of Vma13p with the cytoplasmic domain of Ynd1p regulates its apyrase activity in the Golgi lumen.     

Regulation and reversibility of vacuolar H(+)-ATPase

Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.     

The vacuolar H+-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification,mitochondrial function,and iron metabolism

Mutations in the GEF2 gene of the yeast Saccharomyces cerevisiae have pleiotropic effects. The gef2 mutants display a petite phenotype. These cells grow slowly on several different carbon sources utilized exclusively or primarily by respiration. This phenotype is suppressed by adding large amounts of iron to the growth medium. A defect in mitochondrial function may be the cause of the petite phenotype: the rate of oxygen consumption by intact gef2 cells and by mitochondrial fractions isolated from gef2 mutants was reduced 60%–75% relative to wild type. Cytochrome levels were unaffected in gef2 mutants, indicating that heme accumulation is not significantly altered in these strains. The gef2 mutants were also more sensitive than wild type to growth inhibition by several divalent cations including Cu. We found that the cup5 mutation, causing Cu sensitivity, is allelic to gef2 mutations. The GEF2 gene was isolated, sequenced, and found to be identical to VMA3, the gene encoding the vacuolar H ⁺-ATPase proteolipid subunit. These genetic and biochemical analyses demonstrate that the vacuolar H ⁺-ATPase plays a previously unknown role in Cu detoxification, mitochondrial function, and iron metabolism.     

A new destruxin as inhibitor of vacuolar-type H+-ATPase of Saccharomyces cerevisiae

In the course of our screening efforts to discover small molecules as selective inhibitors of vacuolar-type H+-ATPase of Saccharomyces cerevisiae, we have identified eight active destruxins, 1-8, from the fungus Metarhizium anisopliae. The structures were elucidated by extensive 1D- and 2D-NMR spectroscopy, and MS spectrometry. One of these compounds, 8, a regioisomer of chlorohydrin destruxin E (7), is a new destruxin.