The biochemical properties of the ATPase activity of a 70-kDa heat shock protein (Hsp70) are governed by the C-terminal domains

The cytosolic 70-kDa heat shock proteins (Hsp70s), Ssa and Ssb, of Saccharomyces cerevisiae are functionally distinct. Here we report that the ATPase activities of these two classes of Hsp70s exhibit different kinetic properties. The Ssa ATPase has properties similar to those of other Hsp70s studied, such as DnaK and Hsc70. Ssb, however, has an unusually low steady-state affinity for ATP but a higher maximal velocity. In addition, the ATPase activity of Hsp70s, like that of Ssa1, depends on the addition of K+ whereas Ssb activity does not. Suprisingly, the isolated 44-kDa ATPase domain of Ssb has a Km and Vmax for ATP hydrolysis similar to those of Ssa, rather than those of full length Ssb. Analysis of Ssa/Ssb fusion proteins demonstrates that the Ssb peptide-binding domain fused to the Ssa ATPase domain generates an ATPase of relatively high activity and low steady-state affinity for ATP similar to that of native Ssb. Therefore, at least some of the biochemical differences between the ATPases of these two classes of Hsp70s are not intrinsic to the ATPase domain itself. The differential influence of the peptide-binding domain on the ATPase domain may, in part, explain the functional uniqueness of these two classes of Hsp70s.

The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene

The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (Citrullus vulgaris) cotyledons two hsps with molecular masses of 70 and 72 kDa. Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of glyoxysomes and plastids. Affinity purification and partial amino acid determination revealed the 70-kDa protein to share high sequence identity with cytosolic hsp70s from a number of plant species, while the 72 kDa protein was very similar to plastid hsp70s from pea and cucumber. A full-length cDNA clone encoding the 72-kDa hsp70 was isolated and identified two start methionines in frame within the N-terminal presequence leading either to an N-terminal extension of 67 amino acids or to a shorter one of 47 amino acids. The longer presequence was necessary and sufficient to target a reporter protein into watermelon proplastids in vitro. The shorter extension starting from the second methionine within the long version harbored a consensus peroxisomal targeting signal (RT-X5-KL) that directed in vivo a reporter protein into peroxisomes of the yeast Hansenula polymorpha. Peroxisomal targeting was however prevented, when the 67-residue presequence was fused to the reporter protein, indicating that the peroxisomal targeting signal 2 information is hidden in this context. We propose that the 72-kDa hsp70 is encoded by a single gene, but targeted alternatively into two organelles by the modulated use of its presequence.

Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes

Coordination between the activities of organelles and the nucleus requires the exchange of signals. Using Chlamydomonas, we provide evidence that plastid-derived chlorophyll precursors may replace light in the induction of two nuclear heat-shock genes (HSP70A and HSP70B) and thus qualify as plastidic signal. Mutants defective in the synthesis of Mg-protoporphyrin IX were no longer inducible by light. Feeding of Mg-protoporphyrin IX or its dimethyl ester to wild-type or mutant cells in the dark resulted in induction. The analysis of HSP70A promoter mutants that do or do not respond to light revealed that these chlorophyll precursors specifically activate the light signaling pathway. Activation of gene expression was not observed when protoporphyrin IX, protochlorophyllide, or chlorophyllide were added. A specific interaction of defined chlorophyll precursors with factor(s) that regulate nuclear gene expression is suggested.

Mutations in Drosophila heat shock cognate 4 are enhancers of Polycomb

The homeotic genes controlling segment identity in Drosophila are repressed by the Polycomb group of genes (PcG) and are activated by genes of the trithorax group (trxG). An F1 screen for dominant enhancers of Polycomb yielded a point mutation in the heat shock cognate gene, hsc4, along with mutations corresponding to several known PcG loci. The new mutation is a more potent enhancer of Polycomb phenotypes than an apparent null allele of hsc4 is, although even the null allele occasionally displays homeotic phenotypes associated with the PcG. Previous biochemical results had suggested that HSC4 might interact with BRAHMA, a trxG member. Further analyses now show that there is no physical or genetic interaction between HSC4 and the Brahma complex. HSC4 might be needed for the proper folding of a component of the Polycomb repression complex, or it may be a functional member of that complex.

Vipp1 deletion mutant of Synechocystis: A connection between bacterial phage shock and thylakoid biogenesis?

Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.

The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity

The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix–turn–helix proteins, HSF’s wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein–protein interactions, possibly within a trimer or between adjacent trimers. To understand the function of the wing in the HSF DNA-binding domain, a Saccharomyces cerevisiae strain was created that expresses a wingless HSF protein. This strain grows normally at 30°C, but shows a decrease in reporter gene expression during constitutive and heat-shocked conditions. Removal of the wing does not affect the stability or trimeric nature of a protein fragment containing the DNA-binding and trimerization domains. Removal of the wing does result in a decrease in DNA-binding affinity. This defect was mainly observed in the ability to form the first trimer-bound complex, as the formation of larger complexes is unaffected by the deletion. Our results suggest that the wing is not involved in the highly co-operative nature of HSF binding, but may be important in stabilizing the first trimer bound to DNA.

Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomyces cerevisiae

Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2/4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G1 and represses expression of CLN1 and CLN2. Mutants with reduced G1 cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3–2 protein prevents G1 arrest upon AZC treatment and temperature up-shift. Finally, we find that the EXA3–1 mutation, encoding a defective HSF, prevents efficient G1 arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G1 arrest.

High-Molecular-Weight FK506-Binding Proteins Are Components of Heat-Shock Protein 90 Heterocomplexes in Wheat Germ Lysate1

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249–15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

The Heat-Shock Element Is a Functional Component of the Arabidopsis APX1 Gene Promoter1

Ascorbate peroxidases are important enzymes that detoxify hydrogen peroxide within the cytosol and chloroplasts of plant cells. To better understand their role in oxidative stress tolerance, the transcriptional regulation of the apx1 gene from Arabidopsis was studied. The apx1 gene was expressed in all tested organs of Arabidopsis; mRNA levels were low in roots, leaves, and stems and high in flowers. Steady-state mRNA levels in leaves or cell suspensions increased after treatment with methyl viologen, ethephon, high temperature, and illumination of etiolated seedlings. A putative heat-shock cis element found in the apx1 promoter was shown to be recognized by the tomato (Lycopersicon esculentum) heat-shock factor in vitro and to be responsible for the in vivo heat-shock induction of the gene. The heat-shock cis element also contributed partially to the induction of the gene by oxidative stress. By using in vivo dimethyl sulfate footprinting, we showed that proteins interacted with a G/C-rich element found in the apx1 promoter.

Expression of Small Heat-Shock Proteins at Low Temperatures1 : A Possible Role in Protecting against Chilling Injuries

We previously reported that short exposure of tomato (Lycopersicon esculentum L.) fruits to high temperature protects them from chilling injury. To study the involvement of heat-shock proteins (HSPs) in the acquisition of low-temperature tolerance, we cloned two heat-shock-induced genes that are also expressed at low temperatures. The cloned cDNAs belong to the small HSP group. Sequence analyses of the clones showed perfect homology to the tomato-ripening gene tom66 and to the tomato chloroplastic HSP21 gene tom111. The expression of both genes was induced by high temperature in fruits, flowers, leaves, and stems, but not by low or ambient temperatures or by other stresses such as drought and anaerobic conditions. When the heated fruits were transferred to low temperature, tom66 and tom111 mRNA levels first decreased but were then reinduced. Induction was not observed in nonheated fruits at low temperature. Immunodetection of tom111-encoded protein indicated that this protein is present at low temperatures in the heated fruits. The results of this study show that the expression of tom66 and tom111 is correlated with protection against some, but not all, symptoms of chilling injury.

Polypeptides of the Maize Amyloplast Stroma1 : Stromal Localization of Starch-Biosynthetic Enzymes and Identification of an 81-Kilodalton Amyloplast Stromal Heat-Shock Cognate

In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast.

The Chloroplast Small Heat-Shock Protein Oligomer Is Not Phosphorylated and Does Not Dissociate during Heat Stress in Vivo1

Plants synthesize several classes of small (15- to 30-kD monomer) heat-shock proteins (sHSPs) in response to heat stress, including a nuclear-encoded, chloroplast-localized sHSP (HSP21). Cytosolic sHSPs exist as large oligomers (approximately 200–800 kD) composed solely or primarily of sHSPs. Phosphorylation of mammalian sHSPs causes oligomer dissociation, which appears to be important for regulation of sHSP function. We examined the native structure and phosphorylation of chloroplast HSP21 to understand this protein's basic properties and to compare it with cytosolic sHSPs. The apparent size of native HSP21 complexes was > 200 kD and they did not dissociate during heat stress. We found no evidence that HSP21 or the plant cytosolic sHSPs are phosphorylated in vivo. A partial HSP21 complex purified from heat-stressed pea (Pisum sativum L.) leaves contained no proteins other than HSP21. Mature recombinant pea and Arabidopsis thaliana HSP21 were expressed in Escherichia coli, and purified recombinant Arabidopsis HSP21 assembled into homo-oligomeric complexes with the same apparent molecular mass as HSP21 complexes observed in heat-stressed leaf tissue. We propose that the native, functional form of chloroplast HSP21 is a large, oligomeric complex containing nine or more HSP21 subunits, and that plant sHSPs are not regulated by phosphorylation-induced dissociation.