A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins.

Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein-protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level "ruler" because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤10nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins.

Greasing the wheels of secretory transport.

Ever since George Palade's pioneering studies of zymogen secretion from pancreatic acinar cells, the underlying molecular mechanisms of vesicle-mediated protein transport have captivated cell biologists and biochemists. A watershed meeting on "Phosphoinositides and the Golgi", held at the Fogarty International Center of the National Institutes of Health in Bethesda, Maryland (March 13-14, 2001), provided reinterpretation and striking new insights about the functions of this phospholipid class in intracellular protein trafficking.

Regulation of G protein-initiated signal transduction in yeast: paradigms and principles.

All cells have the capacity to evoke appropriate and measured responses to signal molecules (such as peptide hormones), environmental changes, and other external stimuli. Tremendous progress has been made in identifying the proteins that mediate cellular response to such signals and in elucidating how events at the cell surface are linked to subsequent biochemical changes in the cytoplasm and nucleus. An emerging area of investigation concerns how signaling components are assembled and regulated (both spatially and temporally), so as to control properly the specificity and intensity of a given signaling pathway. A related question under intensive study is how the action of an individual signaling pathway is integrated with (or insulated from) other pathways to constitute larger networks that control overall cell behavior appropriately. This review describes the signal transduction pathway used by budding yeast (Saccharomyces cerevisiae) to respond to its peptide mating pheromones. This pathway is comprised by receptors, a heterotrimeric G protein, and a protein kinase cascade all remarkably similar to counterparts in multicellular organisms. The primary focus of this review, however, is recent advances that have been made, using primarily genetic methods, in identifying molecules responsible for regulation of the action of the components of this signaling pathway. Just as many of the constituent proteins of this pathway and their interrelationships were first identified in yeast, the functions of some of these regulators have clearly been conserved in metazoans, and others will likely serve as additional models for molecules that carry out analogous roles in higher organisms.

Dynamic localization of the Swe1 regulator Hsl7 during the Saccharomyces cerevisiae cell cycle.

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.

High affinity interaction of yeast transcriptional regulator, Mot1, with TATA box-binding protein (TBP).

Yeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro. Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25-801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802-1867) did not. Complex formation was prevented above 200 mm salt, consistent with electrostatic interaction. Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not growth-inhibitory. C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction. Residues 1-800 of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1. Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled.

Mutations in the YRB1 gene encoding yeast ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects.

We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.

A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo.

Mutational analysis suggests that activation of the yeast pheromone response mitogen-activated protein kinase pathway involves conformational changes in the Ste5 scaffold protein.

Ste5 is essential for pheromone response and binds components of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). Pheromone stimulation releases Gbetagamma (Ste4-Ste18), which recruits Ste5 and Ste20 (p21-activated kinase) to the plasma membrane, activating the MAPK cascade. A RING-H2 domain in Ste5 (residues 177-229) negatively regulates Ste5 function and mediates its interaction with Gbetagamma. Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot complement a ste5Delta mutation, yet supports mating even in ste4Delta ste5Delta cells when artificially dimerized by fusion to glutathione S-transferase (GST). In contrast, wild-type Ste5 fused to GST permits mating of ste5Delta cells, but does not allow mating of ste4Delta ste5Delta cells. This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations. MATa ste4Delta ste5Delta cells expressing Ste5-GST were mutagenized chemically and plasmids conferring the capacity to mate were selected. Three independent single-substitution mutations were isolated. These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when Gbetagamma is absent. The first, Ste5(C226Y), alters the seventh conserved position in the RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function. The second, Ste5(P44L), lies upstream of a basic segment, whereas the third, Ste5(S770K), is situated within an acidic segment in a region that contacts Ste7. None of the mutations increased the affinity of Ste5 for Ste11, Ste7, or Fus3. However, the positions of these novel-activating mutations suggested that, in normal Ste5, the N terminus may interact with the C terminus. Indeed, in vitro, GST-Ste5(1-518) was able to associate specifically with radiolabeled Ste5(520-917). Furthermore, both the P44L and S770K mutations enhanced binding of full-length Ste5 to GST-Ste5(1-518), whereas they did not affect Ste5 dimerization. Thus, binding of Gbetagamma to the RING-H2 domain may induce a conformational change that promotes association of the N- and C-terminal ends of Ste5, stimulating activation of the MAPK cascade by optimizing orientation of the bound kinases and/or by increasing their accessibility to Ste20-dependent phosphorylation (or both). In accord with this model, the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated state and their activation required Ste20.

Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae.

The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca(2+) binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca(2+)-binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca(2+) ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K(d) = 10 microM) was much weaker than that of the third and fourth sites (K(d) = 0.4 microM). Myr-Frq1 bound Ca(2+) with a K(d)app of 3 microM and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca(2+) binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca(2+)-dependent differences, indicating major conformational changes induced upon Ca(2+) binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca(2+)-bound Frq1 were very similar to those of Ca(2+)-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca(2+)-bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca(2+)-free and Ca(2+)-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca(2+)-free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca(2+)-binding contribute to the ability of Frq1 to associate with membranes.

Purification and enzymic properties of Mot1 ATPase, a regulator of basal transcription in the yeast Saccharomyces cerevisiae.

The 1867-residue Mot1 protein is a member of a superfamily of ATPases, some of which are helicases, that interact with protein-nucleic acid assemblies. Mot1 is an essential regulator of RNA polymerase II-dependent transcription in vivo and dissociates TATA box-binding protein (TBP)-DNA complexes in vitro. Mot1-(His)(6) was purified to apparent homogeneity from yeast extracts. The preparation efficiently dissociated TBP.TATA complexes, suggesting that no other protein or cofactor is required. Mot1 behaved as a non-globular monomer in hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs. ATPase activity was stimulated about 10-fold by high ionic strength or alkaline pH, or by deletion of the N-terminal TBP-binding segment, suggesting that the N-terminal domain negatively regulates the C-terminal ATPase domain (Mot1C). Correspondingly, at moderate salt concentration, Mot1 ATPase (but not Mot1C) was stimulated >/=10-fold by yeast TBP, suggesting that interaction with TBP relieves a conformational constraint in Mot1. Double- or single-stranded TATA-containing DNA did not affect ATPase activity of Mot1 or Mot1C, with or without TBP. Mot1 did not exhibit detectable helicase activity in strand displacement assays using substrates with flush ends or 5'- or 3'-overhangs. Mot1-catalyzed dissociation of TBP from DNA was not prevented by a psoralen cross-link positioned immediately preceding the TATA sequence. Thus, Mot1 most likely promotes release of TBP from TATA-containing DNA by causing a structural change in TBP itself, rather than by strand unwinding.

Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1.

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran x GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51(ts)) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran x GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51(ts) yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1(ts) mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1(ts) mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1(ts) cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-beta and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes.

Direct involvement of phosphatidylinositol 4-phosphate in secretion in the yeast Saccharomyces cerevisiae.

The SEC14 gene encodes an essential phosphatidylinositol (PtdIns) transfer protein required for formation of Golgi-derived secretory vesicles in yeast. Suppressor mutations that rescue temperature-sensitive sec14 mutants provide an approach for determining the role of Sec14p in secretion. One suppressor, sac1-22, causes accumulation of PtdIns(4)P. SAC1 encodes a phosphatase that can hydrolyze PtdIns(4)P and certain other phosphoinositides. These findings suggest that PtdIns(4)P is limiting in sec14 cells and that elevation of PtdIns(4)P production can suppress the secretory defect. Correspondingly, we found that PtdIns(4)P levels were decreased significantly in sec14-3 mutants shifted to 37 degrees C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34 degrees C) when carrying a plasmid overexpressing PIK1, encoding one of two essential PtdIns 4-kinases. This effect is specific because overexpression of the other PtdIns 4-kinase gene (STT4) or a PtdIns 3-kinase gene (VPS34) did not rescue sec14-3 cells. To further address Pik1p function in secretion, two different pik1(ts) mutants were examined. Upon shift to restrictive temperature (37 degrees C), the PtdIns(4)P levels dropped by about 60% in both pik1(ts) strains within 1 h. During the same period, cells displayed a reduction (40-50%) in release of a secreted enzyme (invertase). However, similar treatment did not effect maturation of a vacuolar enzyme (carboxypeptidase Y). These findings indicate that, first, PtdIns(4)P limitation is a major contributing factor to the secretory defect in sec14 cells; second, Sec14p function is coupled to the action of Pik1p, and; third, PtdIns(4)P has an important role in the Golgi-to-plasma membrane stage of secretion.

Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase.

In metazoans, certain calmodulin-related calcium-binding proteins (recoverins, neurocalcins and frequenins) are found at highest levels in excitable cells, but their physiological roles are largely uncharacterized. Here we show that Saccharomyces cerevisiae contains a frequenin homologue, Frq1, and that its target is Pik1, a phosphatidylinositol-4-OH kinase. Frq1 binds to a conserved sequence motif in Pik1 outside Pik1's catalytic domain and stimulates its activity in vitro. N-myristoylated Frq1 may also assist in Pik1 localization.

Hsl7 localizes to a septin ring and serves as an adapter in a regulatory pathway that relieves tyrosine phosphorylation of Cdc28 protein kinase in Saccharomyces cerevisiae.

Successful mitosis requires faithful DNA replication, spindle assembly, chromosome segregation, and cell division. In the budding yeast Saccharomyces cerevisiae, the G(2)-to-M transition requires activation of Clb-bound forms of the protein kinase, Cdc28. These complexes are held in an inactive state via phosphorylation of Tyr19 in the ATP-binding loop of Cdc28 by the Swe1 protein kinase. The HSL1 and HSL7 gene products act as negative regulators of Swe1. Hsl1 is a large (1,518-residue) protein kinase with an N-terminal catalytic domain and a very long C-terminal extension. Hsl1 localizes to the incipient site of cytokinesis in the bud neck in a septin-dependent manner; however, the function of Hsl7 was not previously known. Using both indirect immunofluorescence with anti-Hsl7 antibodies and a fusion of Hsl7 to green fluorescent protein, we found that Hsl7 also localizes to the bud neck, congruent with the septin ring that faces the daughter cell. Both Swe1 and a segment of the C terminus of Hsl1 (which has no sequence counterpart in two Hsl1-related protein kinases, Gin4 and Kcc4) were identified as gene products that interact with Hsl7 in a two-hybrid screen of a random S. cerevisiae cDNA library. Hsl7 plus Swe1 and Hsl7 plus Hsl1 can be coimmunoprecipitated from extracts of cells overexpressing these proteins, confirming that Hsl7 physically associates with both partners. Also consistent with the two-hybrid results, Hsl7 coimmunoprecipitates with full-length Hsl1 less efficiently than with a C-terminal fragment of Hsl1. Moreover, Hsl7 does not localize to the bud neck in an hsl1Delta mutant, whereas Hsl1 is localized normally in an hsl7Delta mutant. Phosphorylation and ubiquitinylation of Swe1, preludes to its destruction, are severely reduced in cells lacking either Hsl1 or Hsl7 (or both), as judged by an electrophoretic mobility shift assay. Collectively, these data suggest that formation of the septin rings provides sites for docking Hsl1, exposing its C terminus and thereby permitting recruitment of Hsl7. Hsl7, in turn, presents its cargo of bound Swe1, allowing phosphorylation by Hsl1. Thus, Hsl1 and Hsl7 promote proper timing of cell cycle progression by coupling septin ring assembly to alleviation of Swe1-dependent inhibition of Cdc28. Furthermore, like septins and Hsl1, homologs of Hsl7 are found in fission yeast, flies, worms, and humans, suggesting that its function in this control mechanism may be conserved in all eukaryotes.

Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast.

BACKGROUND: In animal cells, recruitment of phosphatidylinositol 3-kinase by growth factor receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1). Activated PDK1 then phosphorylates and activates downstream protein kinases, including protein kinase B (PKB)/c-Akt, p70 S6 kinase, PKC isoforms, and serum- and glucocorticoid-inducible kinase (SGK), thereby eliciting physiological responses. RESULTS: We found that two previously uncharacterised genes of Saccharomyces cerevisiae, which we term PKH1 and PKH2, encode protein kinases with catalytic domains closely resembling those of human and Drosophila PDK1. Both Pkh1 and Pkh2 were essential for cell viability. Expression of human PDK1 in otherwise inviable pkh1Delta pkh2Delta cells permitted growth. In addition, the yeast YPK1 and YKR2 genes were found to encode protein kinases each with a catalytic domain closely resembling that of SGK; both Ypk1 and Ykr2 were also essential for viability. Otherwise inviable ypk1Delta ykr2Delta cells were fully rescued by expression of rat SGK, but not mouse PKB or rat p70 S6 kinase. Purified Pkh1 activated mammalian SGK and PKBalpha in vitro by phosphorylating the same residue as PDK1. Pkh1 activated purified Ypk1 by phosphorylating the equivalent residue (Thr504) and was required for maximal Ypk1 phosphorylation in vivo. Unlike PKB, activation of Ypk1 and SGK by Pkh1 did not require phosphatidylinositol 3,4,5-trisphosphate, consistent with the absence of pleckstrin homology domains in these proteins. The phosphorylation consensus sequence for Ypk1 was similar to that for PKBalpha and SGK. CONCLUSIONS: Pkh1 and Pkh2 function similarly to PDK1, and Ypk1 and Ykr2 to SGK. As in animal cells, these two groups of yeast kinases constitute two tiers of a signalling cascade required for yeast cell growth.

Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins.

Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs). Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression. Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain. Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs. However, maintenance of repression at PREs is more dependent on Dig1 and/or Dig2 and less dependent on Kss1 than repression at FREs. In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs. Differential utilization of two types of MAPK-mediated regulation (binding-imposed repression and phosphorylation-dependent activation), in combination with distinct Ste12-containing complexes, contributes to the mechanisms by which separate extracellular stimuli that use the same MAPK cascade can elicit two different transcriptional responses.

Repression of yeast Ste12 transcription factor by direct binding of unphosphorylated Kss1 MAPK and its regulation by the Ste7 MEK.

The mitogen-activated protein kinase (MAPK) Kss1 has a dual role in regulating filamentous (invasive) growth of the yeast Saccharomyces cerevisiae. The stimulatory function of Kss1 requires both its catalytic activity and its activation by the MAPK/ERK kinase (MEK) Ste7; in contrast, the inhibitory function of Kss1 requires neither. This study examines the mechanism by which Kss1 inhibits invasive growth, and how Ste7 action overcomes this inhibition. We found that unphosphorylated Kss1 binds directly to the transcription factor Ste12, that this binding is necessary for Kss1-mediated repression of Ste12, and that Ste7-mediated phosphorylation of Kss1 weakens Kss1-Ste12 interaction and relieves Kss1-mediated repression. Relative to Kss1, the MAPK Fus3 binds less strongly to Ste12 and is correspondingly a weaker inhibitor of invasive growth. Analysis of Kss1 mutants indicated that the activation loop of Kss1 controls binding to Ste12. Potent repression of a transcription factor by its physical interaction with the unactivated isoform of a protein kinase, and relief of this repression by activation of the kinase, is a novel mechanism for signal-dependent regulation of gene expression.

Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae.

We recently demonstrated that the S. cerevisiae INP51 locus (YIL002c) encodes an inositol polyphosphate 5-phosphatase. Here we describe two related yeast loci, INP52 (YNL106c) and INP53 (YOR109w). Like Inp51p, the primary structures of Inp52p and Inp53p resemble the mammalian synaptic vesicle-associated protein, synaptojanin, and contain a carboxy-terminal catalytic domain and an amino-terminal SAC1-like segment. Inp51p (108 kD), Inp52p (136 kD) and Inp53p (124 kD) are membrane-associated. Single null mutants (inp51, inp52, or inp53) are viable. Both inp51 inp52 and inp52 inp53 double mutants display compromised cell growth, whereas an inp51 inp53 double mutant does not. An inp51 inp52 inp53 triple mutant is inviable on standard medium, but can grow weakly on media supplemented with an osmotic stabilizer (1 M sorbitol). An inp51 mutation, and to a lesser degree an inp52 mutation, confers cold-resistant growth in a strain background that cannot grow at temperatures below 15 degrees. Analysis of inositol metabolites in vivo showed measurable accumulation of phosphatidylinositol 4,5-bisphosphate in the inp51 mutant. Electron microscopy revealed plasma membrane invaginations and cell wall thickening in double mutants and the triple mutant grown in sorbitol-containing medium. A fluorescent dye that detects endocytic and vacuolar membranes suggests that the vacuole is highly fragmented in inp51 inp52 double mutants. Our observations indicate that Inp51p, Inp52p, and Inp53p have distinct functions and that substrates and/or products of inositol polyphosphate 5-phosphatases may have roles in vesicle trafficking, membrane structure, and/or cell wall formation.

An essential function of a phosphoinositide-specific phospholipase C is relieved by inhibition of a cyclin-dependent protein kinase in the yeast Saccharomyces cerevisiae.

The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1delta cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80delta) mutation and growth on low-phosphate medium, also permitted growth of plc1delta cells at the restrictive temperature. Suppression of the temperature sensitivity of plc1delta cells by pho80delta does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81delta and spl2delta show a synthetic phenotype in combination with plc1delta. Unlike single mutants, plc1delta pho81delta and plc1delta spl2delta double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium.

Inhibitory and activating functions for MAPK Kss1 in the S. cerevisiae filamentous-growth signalling pathway.

Mitogen-activated protein kinase (MAPK) cascades are conserved signalling modules that regulate responses to diverse extracellular stimuli, developmental cues and environmental stresses. A MAPK is phosphorylated and activated by a MAPK kinase (MAPKK), which is activated by an upstream protein kinase, such as Raf, Mos or a MAPKK kinase. Ste7, a MAPKK in the yeast Saccharomyces cerevisiae, is required for two developmental pathways: mating and invasive (filamentous) growth. Kss1 and Fus3, the MAPK targets of Ste7, are required for mating, but their role in invasive growth has been unclear. Because no other S. cerevisiae MAPK has been shown to function in invasive growth, it was proposed that Ste7 may have non-MAPK targets. We show instead that Kss1 is the principal target of Ste7 in the invasive-growth response in both haploids and diploids. We demonstrate further that Kss1 in its inactive form is a potent negative regulator of invasive growth. Ste7 acts to relieve this negative regulation by switching Kss1 from an inhibitor to an activator. These results indicate that this MAPK has a physiologically important function in its unactivated state. Comparison of normal and MAPK-deficient cells indicates that nitrogen starvation and activated Ras stimulate filamentous growth through both MAPK-independent and MAPK-dependent means.