The Liang Bua faunal remains: a 95k.yr. sequence from Flores, East Indonesia.

Excavations at Liang Bua, a limestone cave on the island of Flores, East Indonesia, have yielded a well-dated archaeological and faunal sequence spanning the last 95k.yr., major climatic fluctuations, and two human species -H. floresiensis from 95 to 17k.yr.(1), and modern humans from 11k.yr. to the present. The faunal assemblage comprises well-preserved mammal, bird, reptile and mollusc remains, including examples of island gigantism in small mammals and the dwarfing of large taxa. Together with evidence from Early-Middle Pleistocene sites in the Soa Basin, it confirms the long-term isolation, impoverishment, and phylogenetic continuity of the Flores faunal community. The accumulation of Stegodon and Komodo dragon remains at the site in the Pleistocene is attributed to Homo floresiensis, while predatory birds, including an extinct species of owl, were largely responsible for the accumulation of the small vertebrates. The disappearance from the sequence of the two large-bodied, endemic mammals, Stegodon florensis insularis and Homo floresiensis, was associated with a volcanic eruption at 17 ka and precedes the earliest evidence for modern humans, who initiated use of mollusc and shell working, and began to introduce a range of exotic animals to the island. Faunal introductions during the Holocene included the Sulawesi warty pig (Sus celebensis) at about 7ka, followed by the Eurasian pig (Sus scrofa), Long-tailed macaque, Javanese porcupine, and Masked palm civet at about 4ka, and cattle, deer, and horse - possibly by the Portuguese within historic times. The Holocene sequence at the site also documents local faunal extinctions - a result of accelerating human population growth, habitat loss, and over-exploitation.

Hsp90 chaperone control over transcriptional regulation by the yeast Slt2(Mpk1)p and human ERK5 mitogen-activated protein kinases (MAPKs).

Cell integrity MAPK (mitogen-activated protein kinase) function can be provided in yeast cells by either the native Slt2(Mpk1)p of yeast or by a heterologously expressed human ERK5 (extracellular-signal-regulated kinase 5). Both of these MAPKs need the Hsp90 (heat-shock protein 90) chaperone for their activation, so that when Hsp90 function is compromised their activities are low. This, in turn, affects the capacity of these MAPKs to control the transcription factors that regulate cell integrity genes.

The ZbYME2 gene from the food spoilage yeast Zygosaccharomyces bailii confers not only YME2 functions in Saccharomyces cerevisiae, but also the capacity for catabolism of sorbate and benzoate, two major weak organic acid preservatives.

A factor influencing resistances of food spoilage microbes to sorbate and benzoate is whether these organisms are able to catalyse the degradation of these preservative compounds. Several fungi metabolize benzoic acid by the beta-ketoadipate pathway, involving the hydroxylation of benzoate to 4-hydroxybenzoate. Saccharomyces cerevisiae is unable to use benzoate as a sole carbon source, apparently through the lack of benzoate-4-hydroxylase activity. However a single gene from the food spoilage yeast Zygosaccharomyces bailii, heterologously expressed in S. cerevisiae cells, can enable growth of the latter on benzoate, sorbate and phenylalanine. Although this ZbYME2 gene is essential for benzoate utilization by Z. bailii, its ZbYme2p product has little homology to other fungal benzoate-4-hydroxylases studied to date, all of which appear to be microsomal cytochrome P450s. Instead, ZbYme2p has strong similarity to the matrix domain of the S. cerevisiae mitochondrial protein Yme2p/Rna12p/Prp12p and, when expressed as a functional fusion to green fluorescent protein in S. cerevisiae growing on benzoate, is largely localized to mitochondria. The phenotypes associated with loss of the native Yme2p from S. cerevisiae, mostly apparent in yme1,yme2 cells, may relate to increased detrimental effects of endogenous oxidative stress. Heterologous expression of ZbYME2 complements these phenotypes, yet it also confers a potential for weak acid preservative catabolism that the native S. cerevisiae Yme2p is unable to provide. Benzoate utilization by S. cerevisiae expressing ZbYME2 requires a functional mitochondrial respiratory chain, but not the native Yme1p and Yme2p of the mitochondrion.

Increasing Saccharomyces cerevisiae stress resistance, through the overactivation of the heat shock response resulting from defects in the Hsp90 chaperone, does not extend replicative life span but can be associated with slower chronological ageing of nondividing cells.

Recent studies on Drosophila and Caenorhabditis elegans indicate that increases in stress resistance result in a longer chronological life span, an effect that must operate primarily on the postmitotic tissues of the adult. Stress resistance can be increased through decreases in Hsp90 chaperone activity, since Hsp90 acts to downregulate the activity of heat shock transcription factor. This study investigated whether the increases in stress resistance associated with reduced Hsp90 chaperone activity influence ageing in the budding yeast Saccharomyces cerevisiae, ageing being measured either as the replicative (nonchronological) senescence of budding cells or as the chronological ageing of non-dividing (stationary phase) cultures. Overactivation of the heat shock response caused no slowing of replicative senescence. In some situations though it was associated with a longer chronological life span of stationary cells, the yeast equivalent of the postmitotic state. This is consistent with the idea that stress resistance exerts its life span-extending effects primarily in postmitotic cells and tissues.

Chronological lifespan of stationary phase yeast cells; a model for investigating the factors that might influence the ageing of postmitotic tissues in higher organisms.

Budding yeast can be considered to have two distinct lifespans: (a) a replicative (budding, non-chronological) lifespan, measured as the number of daughters produced by each actively dividing mother cell; and (ii) a chronological lifespan, measured as the ability of stationary cultures to maintain viability over time. In non-dividing cells, essential components that become damaged cannot be diluted out through cell division but must, of necessity, be turned over and renewed. By elevating stress resistances, many of the activities needed for such renewal should be elevated with commensurate reduction in the steady-state levels of damaged cell components. Therefore, chronological lifespan in particular might be expected to relate to stress resistance. For yeast to attain a full chronological lifespan requires the expression of the general stress response. It is more important, though, that the cells should be efficiently adapted to respiratory maintenance, since it is cultures grown to stationary phase on respiratory media that usually display the longest chronological lifespans. For this reason, respiration-adapted cells potentially provide a better model of chronological ageing than cultures pre-grown on glucose.

Targeted gene deletion in Zygosaccharomyces bailii.

Yeasts of the genus Zygosaccharomyces are notable agents of large-scale food spoilage. Despite the economic importance of these organisms, little is known about the stress adaptations whereby they adapt to many of the more severe conditions of food preservation. In this study it was shown that genes of Z. bailii, a yeast notable for its high resistances to food preservatives and ethanol, can be isolated by complementation of the corresponding mutant strains of Saccharomyces cerevisiae. It was also discovered that the acquisition by S. cerevisiae of a single small Z. bailii gene (ZbYME2) was sufficient for the former yeast to acquire the ability to degrade two major food preservatives, benzoic acid and sorbic acid. Using DNA cassettes containing dominant selectable markers and methods originally developed for performing gene deletions in S. cerevisiae, the two copies of ZbYME2 in the Z. bailii genome were sequentially deleted. The resulting Zbyme2/Zbyme2 homozygous deletant strain had lost any ability to utilize benzoate as sole carbon source and was more sensitive to weak acid preservatives during growth on glucose. Thus, ZbYME2, probably the nuclear gene for a mitochondrial mono-oxygenase function, is essential for Z. bailii to degrade food preservatives. This ability to catabolize weak acid preservatives is a significant factor contributing to the preservative resistance of Z. bailii under aerobic conditions. This study is the first to demonstrate that it is possible to delete in Z. bailii genes that are suspected as being important for growth of this organism in preserved foods and beverages. With the construction of further mutant of Z. bailii strains, a clearer picture should emerge of how this yeast adapts to the conditions of food preservation. This information will, in turn, allow the design of new preservation strategies. GenBank Accession Nos: ZbURA3 (AF279259), ZbTIM9 (AF279260), ZbYME2 (AF279261), ZbTRP1 (AF279262), ZbHHT1(AF296170).

The Hsp90 chaperone as a promising drug target.

Geldanamycin and radicicol, antibiotics produced by Streptomycetes and fungi, respectively, were originally discovered many years ago. Only recently was it discovered that they bind with high specificity within the ADP/ATP binding pocket of the Hsp90 molecular chaperone, thereby inhibiting the function of Hsp90. In eukaryotic cells Hsp90 catalyzes the final activation step of many of the most important regulatory proteins. Cells that lose this function are severely compromised and cannot progress through the cell cycle. In cell-culture systems, the administration of geldanamycin induces degradation of several signal transduction proteins of oncological importance. Hsp90 inhibitors are, therefore, now attracting considerable attention as potential antitumor agents; one geldanamycin derivative is already in phase I trials as an anticancer drug. These drugs may also have virucidal, antimalarial and ischemia-protective effects.

Loss of Cmk1 Ca(2+)-calmodulin-dependent protein kinase in yeast results in constitutive weak organic acid resistance, associated with a post-transcriptional activation of the Pdr12 ATP-binding cassette transporter.

Yeast cells display an adaptive stress response when exposed to weak organic acids at low pH. This adaptation is important in the spoilage of preserved foods, as it allows growth in the presence of weak acid food preservatives. In Saccharomyces cerevisiae, this stress response leads to strong induction of the Pdr12 ATP-binding cassette (ABC) transporter, which catalyses the active efflux of weak acid anions from the cytosol of adapted cells. S. cerevisiae cells lacking the Cmk1 isoform of Ca2+-calmodulin-dependent protein kinase are intrinsically resistant to weak acid stress, in that they do not need to spend a long adaptive period in lag phase before resuming growth after exposure to this stress. This resistance of the cmk1 mutant is Pdr12 dependent and, unlike with wild-type S. cerevisiae, cmk1 cells are capable of performing Pdr12-specific functions such as energy-dependent cellular extrusion of fluorescein and benzoate. However, they have neither higher PDR12 gene promoter activity nor higher Pdr12 protein levels. The increased Pdr12 activity in cmk1 cells is therefore caused by Cmk1 exerting a negative post-transcriptional influence over the activity of the Pdr12 ABC transporter, a transporter protein that is constitutively expressed in low-pH yeast cultures. This is the first preliminary evidence that shows a protein kinase, either directly or indirectly, regulating the activity of a yeast ABC transporter.

The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains.

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.

The Saccharomyces cerevisiae weak-acid-inducible ABC transporter Pdr12 transports fluorescein and preservative anions from the cytosol by an energy-dependent mechanism.

Growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid results in the induction of the ATP-binding cassette (ABC) transporter Pdr12 in the plasma membrane (P. Piper, Y. Mahe, S. Thompson, R. Pandjaitan, C. Holyoak, R. Egner, M. Muhlbauer, P. Coote, and K. Kuchler, EMBO J. 17:4257-4265, 1998). Pdr12 appears to mediate resistance to water-soluble, monocarboxylic acids with chain lengths of from C(1) to C(7). Exposure to acids with aliphatic chain lengths greater than C(7) resulted in no observable sensitivity of Deltapdr12 mutant cells compared to the parent. Parent and Deltapdr12 mutant cells were grown in the presence of sorbic acid and subsequently loaded with fluorescein. Upon addition of an energy source in the form of glucose, parent cells immediately effluxed fluorescein from the cytosol into the surrounding medium. In contrast, under the same conditions, cells of the Deltapdr12 mutant were unable to efflux any of the dye. When both parent and Deltapdr12 mutant cells were grown without sorbic acid and subsequently loaded with fluorescein, upon the addition of glucose no efflux of fluorescein was detected from either strain. Thus, we have shown that Pdr12 catalyzes the energy-dependent extrusion of fluorescein from the cytosol. Lineweaver-Burk analysis revealed that sorbic and benzoic acids competitively inhibited ATP-dependent fluorescein efflux. Thus, these data provide strong evidence that sorbate and benzoate anions compete with fluorescein for a putative monocarboxylate binding site on the Pdr12 transporter.

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones.

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.

Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin.

The cellular activity of several regulatory and signal transduction proteins, which depend on the Hsp90 molecular chaperone for folding, is markedly decreased by geldanamycin and by radicicol (monorden). We now show that these unrelated compounds both bind to the N-terminal ATP/ADP-binding domain of Hsp90, with radicicol displaying nanomolar affinity, and both inhibit the inherent ATPase activity of Hsp90 which is essential for its function in vivo. Crystal structure determinations of Hsp90 N-terminal domain complexes with geldanamycin and radicicol identify key aspects of their nucleotide mimicry and suggest a rational basis for the design of novel antichaperone drugs.

Weak organic acid treatment causes a trehalose accumulation in low-pH cultures of Saccharomyces cerevisiae, not displayed by the more preservative-resistant Zygosaccharomyces bailii.

Weak organic acid food preservatives exert pronounced culture pH-dependent effects on both the heat-shock response and the thermotolerance of Saccharomyces cerevisiae. In low-pH cultures, they inhibit this stress response and cause strong induction of respiratory-deficient petites amongst the survivors of lethal heat treatment. In higher pH cultures, 25 degrees C sorbic acid treatment causes a strong induction of thermotolerance without inducing the heat-shock response. In this study we show that trehalose, a major stress protectant, accumulates rapidly in S. cerevisiae exposed to sorbate at low pH. In pH 3.5 cultures, a 25 degrees C sorbate treatment is as effective as a 39 degrees C heat shock in inducing trehalose. This weak-acid-induced trehalose accumulation is enhanced in the pfk1 S. cerevisiae mutant, indicating that it arises through inhibition of glycolysis at the phosphofructokinase step. The more preservative-resistant food spoilage yeast Zygosaccharomyces bailii differs from S. cerevisiae in that: (1) its basal thermotolerance is not strongly affected by culture pH; (2) it does not display trehalose accumulation in response to 25 degrees C sorbate treatment at low pH; and (3) there is no induction of respiratory-deficient petites during lethal heating with sorbate. This probably reflects Z. bailii being both petite-negative and better equipped for maintenance of homeostasis during weak-acid, pH or high-temperature stress.

Yeast superoxide dismutase mutants reveal a pro-oxidant action of weak organic acid food preservatives.

Saccharomyces cerevisiae could provide a simple experimental system for testing the antioxidant or pro-oxidant actions of chemicals, because it has the capacity for aerobic and anaerobic growth and can readily lose its mitochondrial electron transport chain (the major endogenous source of reactive oxygen species [ROS]). This study showed that yeast superoxide dismutase mutants, in a simple petri dish test, readily distinguish a compound that enhances the detrimental effects of endogenous ROS production by the mitochondrial respiratory chain from another chemical that generates oxidative stress by redox cycling. Using this system, weak organic acid food preservatives are shown to exert a strong pro-oxidant action on aerobic yeast cells. In addition these acids are mutagenic toward the yeast mitochondrial genome, even at levels that are subinhibitory to growth. This raises the concern that the large-scale consumption of these preservatives in the human diet may generate oxidative stress within the epithelia of the gastrointestinal tract.

Characterization, crystallization and preliminary X-ray investigation of glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus.

Recombinant Sulfolobus solfataricus glyceraldehyde-3-phosphate dehydrogenase has been purified and found to be a tetramer of 148 kDa. The enzyme shows dual cofactor specificity and uses NADP+ in preference to NAD+. The sequence has been compared with other GAPDH proteins including those from other archaeal sources. The purified protein has been crystallized from ammonium sulfate to produce crystals that diffract to 2.4 A with a space group of P43212 or P41212. A native data set has been collected to 2.4 A using synchrotron radiation and cryocooling.

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding.

The pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast.

Exposure of Saccharomyces cerevisiae to sorbic acid strongly induces two plasma membrane proteins, one of which is identified in this study as the ATP-binding cassette (ABC) transporter Pdr12. In the absence of weak acid stress, yeast cells grown at pH 7.0 express extremely low Pdr12 levels. However, sorbate treatment causes a dramatic induction of Pdr12 in the plasma membrane. Pdr12 is essential for the adaptation of yeast to growth under weak acid stress, since Deltapdr12 mutants are hypersensitive at low pH to the food preservatives sorbic, benzoic and propionic acids, as well as high acetate levels. Moreover, active benzoate efflux is severely impaired in Deltapdr12 cells. Hence, Pdr12 confers weak acid resistance by mediating energy-dependent extrusion of water-soluble carboxylate anions. The normal physiological function of Pdr12 is perhaps to protect against the potential toxicity of weak organic acids secreted by competitor organisms, acids that will accumulate to inhibitory levels in cells at low pH. This is the first demonstration that regulated expression of a eukaryotic ABC transporter mediates weak organic acid resistance development, the cause of widespread food spoilage by yeasts. The data also have important biotechnological implications, as they suggest that the inhibition of this transporter could be a strategy for preventing food spoilage.

The C-terminus of yeast plasma membrane H+-ATPase is essential for the regulation of this enzyme by heat shock protein Hsp30, but not for stress activation.

Several stresses cause additional activation to the glucose-stimulated plasma membrane H+-ATPase activity of yeast, but it is not clear how this is achieved. We recently reported that cells lacking the integral plasma membrane heat shock protein Hsp30 display enhanced increases in plasma membrane H+-ATPase activity with either heat shock or weak organic acid stress (Piper, P.W., Ortiz-Calderon, C., Holyoak, C., Coote, P. and Cole, M. (1997) Cell Stress and Chaperones 2, 12-24), indicating that the stress induction of Hsp30 acts to reduce stress stimulation of the H+-ATPase. In this study it is shown that Hsp30 acts to reduce the Vmax of H+-ATPase in heat shocked cells. Its action is lost with deletion of the C-terminal 11 amino acids of the H+-ATPase, a deletion that does not abolish the stress stimulation of this enzyme. Mutation of the Thr-912 residue within the C-terminal domain of H+-ATPase, a potential site of phosphorylation by the Ca2+-calmodulin-dependent protein kinase, also abolishes any effect of Hsp30. Hsp30 may therefore be acting on a Thr-912 phosphorylated form of the H+-ATPase.

Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone.

Hsp90 molecular chaperones in eukaryotic cells play essential roles in the folding and activation of a range of client proteins involved in cell cycle regulation, steroid hormone responsiveness, and signal transduction. The biochemical mechanism of Hsp90 is poorly understood, and the involvement of ATP in particular is controversial. Crystal structures of complexes between the N-terminal domain of the yeast Hsp90 chaperone and ADP/ATP unambiguously identify a specific adenine nucleotide binding site homologous to the ATP-binding site of DNA gyrase B. This site is the same as that identified for the antitumor agent geldanamycin, suggesting that geldanamycin acts by blocking the binding of nucleotides to Hsp90 and not the binding of incompletely folded client polypeptides as previously suggested. These results finally resolve the question of the direct involvement of ATP in Hsp90 function.