Expression of members of the Saccharomyces cerevisiae hsp70 multigene family

The hsp70 multigene family of Saccharomyces cerevisiae is a complex multigene family, composed of members exhibiting complex patterns of regulation. Expression of some members is induced after a heat shock, whereas expression of others is repressed. Some members of the family are expressed during exponential growth. One gene, SSA3, shows an unusual pattern of expression during approach to stationary phase. While most RNAs decrease in abundance, SSA3 RNA levels dramatically increase. The constitutive expression of SSA3 in cells lacking adenylate cyclase activity suggests that cAMP modulates SSA3 expression.     

Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: correlation with cAMP levels and growth arrest.

Levels of cyclic 3',5'-cyclic monophosphate (cAMP) play an important role in the decision to enter the mitotic cycle in the yeast, Saccharomyces cerevisiae. In addition to growth arrest at stationary phase, S. cerevisiae transiently arrest growth as they shift from fermentative to oxidative metabolism (the diauxic shift). Experiments examining the role of cAMP in growth arrest at the diauxic shift show the following: 1) yeast lower cAMP levels as they exhaust their glucose supply and shift to oxidative metabolism of ethanol, 2) a reduction in cAMP is essential for traversing the diauxic shift, 3) the decrease in adenylate cyclase activity is associated with a decrease in the expression of CYR1 and CDC25, two positive regulators of cAMP levels and an increase in the expression of IRA1 and IRA2, two negative regulators of intracellular cAMP, 4) mutants carrying disruptions in IRA1 and IRA2 were unable to arrest cell division at the diauxic shift and were unable to progress into the oxidative phase of growth. These results indicate that changes cAMP levels are important in regulation of growth arrest at the diauxic shift and that changes in gene expression plays a role in the regulation of the Ras/adenylate cyclase system.     

Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis

6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.     

Ssa1p chaperone interacts with the guanine nucleotide exchange factor of ras Cdc25p and controls the cAMP pathway in Saccharomyces cerevisiae

We have found that the guanine nucleotide exchange factor for ras, Cdc25p, interacts with Ssa1p in Saccharomyces cerevisiae. This interaction was observed with GST-fused Cdc25p polypeptides and confirmed by coimmunoprecipitation with the endogenous Cdc25p. Hsp82 appeared also to be co-immunoprecipitated with Cdc25p, albeit to a lower level than Hsp70. In a strain deleted for SSA1 and SSA2, we observed a reduced cellular content of Cdc25p. Consistent with a reduced activity of the cAMP-dependent PKA pathway, the rate of accumulation of both trehalose and glycogen was stimulated in the ssa-deleted strain. Expression of SSA1 reversed these effects, whereas co-expression of SSA1 and PDE2 restored high accumulation. The expression of genes repressed by cAMP, GAC1 and TPS1, fused to beta-galactosidase, was also stimulated by deletion of SSA genes. The effect of ssa deletion on glycogen accumulation was lost in a strain deleted for CDC25 rescued by the RAS2ile152 allele. Altogether, these results lead to the conclusion that Ssa1p positively controls the cAMP pathway through Cdc25p. We propose that this connection plays a critical role in the adaptation of cells to stress conditions.     

Studies on the nature and role of polyadenylated RNA in spore development of Bacillus subtilis

Summary cDNA probes synthesized on poly(A)RNAs isolated from sporulating cells of Bacillus subtilis were used for hybridization studies with RNAs derived from cells at different stages of growth and sporulation. It was shown that these cDNAs hybridized only to RNA from sporulating cells. No hybridization was observed if total RNA isolated from vegetative cells or from stationary phase cells of a zero stage asporogenic mutant was used. The hybridization studies also indicate that at each sporulation stage different poly(A)RNA species are synthesized. Furthermore, the hybridization kinetics have clearly demonstrated the existence of three distinct abundance classes of poly(A)RNA similar to those observed in eukaryotic cells. BamHI endonuclease restriction fragments of B. subtilis DNA that were found to hybridize to labeled poly(A)RNA were ligated to the pHV33 vector and hybrid clones that hybridized efficiently to poly(A)RNA were selected. Among these, three have been found to carry the spoOB gene.These results strongly suggest that the appearance of poly(A)RNA can be correlated to the expression of spore genes.     

Camptothecin-somatostatin conjugates inhibit the growth of small cell lung cancer cells

The effects of camptothecin-somatostatin (CPT-SS) conjugates were investigated on small cell lung cancer (SCLC) cells. CPT was coupled to a SS agonist (SSA), c(Cys-Phe-DTrp-Lys-Thr-Cys)Thr-NH2 using the built in nucleophile assisted-releasing group (L1) N-methyl-aminoethyl-Gly-Dser-Nle-Dtyr-Dser or (L2) aminoethyl-Gly-Dser-Nle-Dtyr-Dser. The resulting CPT-L1-SSA and CPT-L2-SSA inhibited the specific binding of [125I-Tyr11]SS to NCI-H69 cell membranes with IC50 values of 0.2 and 2.1 nM, respectively. [125I]CPT-L1-SSA was internalized by SCLC cells at 37 degrees C but not at 4 degrees C. CPT-L1-SSA and CPT-L2-SSA inhibited in a dose-dependent manner the increase in adenylylcyclase activity caused by 25 microM forskolin. In vitro, 0.3 microM CPT-L1-SSA half-maximally inhibited the clonal growth of SCLC cells and 1 microM CPT-L1-SSA strongly inhibited 3H-thymidine incorporation into DNA and trypan-blue exclusion. These results suggest that CPT conjugated peptides such as CPT-L1-SSA may prove useful for exploring the efficacy of receptor-directed cytotoxicity to inhibit the proliferation of SCLC cells.     

Impact of the growth phase on the activity of multidrug resistance pumps and membrane potential of S. cerevisiae: effect of pump overproduction and carbon source

The potentiometric fluorescence probe diS-C3(3) is expelled from S. cerevisiae by ABC pumps Pdr5 and Snq2 and can conveniently be used for studying their performance. The activity of these pumps in a strain with wild-type PDR1 allele was shown to drop sharply on glucose depletion from the medium and then again at the end of the diauxic shift when the cells are adapted to growth on respiratory substrates. The presence of the PDR1-3 allele causing pump overproduction prevented this second drop and the pump activity typical for diauxic cells was largely retained. Growth phase-dependent changes of membrane potential measured by the same probe in pump-free mutants included a Deltapsi drop in the late exponential and diauxic growth phase, indicating lowered activity of H+ -ATPase. Suppression of activity of both ABC pumps and H+ -ATPase obviously signifies cell transition to an energy-saving mode. Challenging respiration-adapted cells with glucose showed a novel feature of yeast ABC pumps--a strong dependence of pump activity on the type of the carbon source.     

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.     

Hsp110 cooperates with different cytosolic HSP70 systems in a pathway for de novo folding

Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bind mostly post-translationally to a more restricted subset of newly translated polypeptides, suggesting a downstream function in the folding pathway. Notably, loss of SSB dramatically enhances the cotranslational association of SSA with nascent chains, suggesting SSA can partially fulfill an SSB-like function. On the other hand, the absence of SSE1 enhances polypeptide binding to both SSB and SSA and impairs cell growth. It, thus, appears that Hsp110 is an important regulator of Hsp70-substrate interactions. Based on our data, we propose that Hsp110 cooperates with the SSB and SSA Hsp70 subfamilies, which act sequentially during de novo folding.     

Functionally redundant isoforms of a yeast Hsp70 chaperone subfamily have different antiprion effects

Why eukaryotes encode multiple Hsp70 isoforms is unclear. Saccharomyces cerevisiae Ssa1p and Ssa2p are constitutive 98% identical Hsp70's. Stress-inducible Ssa3p and Ssa4p are 80% identical to Ssa1/2p. We show Ssa1p-4p have distinct functions affecting [PSI(+)] and [URE3] prions. When expressed as the only Ssa, Ssa1p antagonized [URE3] and Ssa2p antagonized [PSI(+)]. Ssa3p and Ssa4p influenced [URE3] and [PSI(+)] somewhat differently but overall their effects paralleled those of Ssa1p and Ssa2p, respectively. Additionally, Ssa3p suppressed a prion-inhibitory effect of elevated temperature. Our previously described Ssa1-21p mutant weakens [PSI(+)] in SSA1-21 SSA2 cells and abolishes it in SSA1-21 ssa2Delta cells. To test if the same mutation affected other prions or altered Ssa2p similarly, we compared effects of a constructed Ssa2-21p mutant and Ssa1-21p on both prions. Surprisingly, [URE3] was unaffected in SSA1-21 SSA2 cells and could propagate in SSA1-21 ssa2Delta cells. Ssa2-21p impaired [URE3] considerably and weakened [PSI(+)] strongly but in a manner distinct from Ssa1-21p, highlighting functional differences between these nearly identical Hsp70's. Our data uncover exquisite functional differences among isoforms of a highly homologous cytosolic Hsp70 subfamily and point to a possibility that variations in Hsp70 function that might improve fitness under optimal conditions are also important during stress.     

RNA synthesis during the stationary growth phase in the Bacillus subtilis Cgr4 mutant

RNA synthesis was studied in Bacillus subtilis Cgr4 grown in the mineral sporulation medium enriched with glucose up to 2% and amino acids up to 1%. To study mRNA synthesis, a method of transfer of the 3H-uridine pulse-labeled culture to the supernatant of physiologically identical, not labeled culture, followed by further incubation was used, the amount of 3H-uridine in the supernatant as well as in cells being measured. RNA was also analysed electrophoretically and distribution of the label among the fractions was determined. It is shown that mRNA synthesized in the logarithmic phase degrades up to 12% on the 2nd hour of growth during 10 min; the mRNA in the stationary phase is stable on the 7th hour of growth; no degradation is observed in the course of 2-3 hours. The beginning of degradation coincides in time with secondary induction of the synthesis of serine proteases and with the onset of sharp decrease in incorporation of 3H-uridine in RNA as well as with induction of spore morphogenesis. On the basis of electrophoretical analysis of pulse-labeled RNA, it was demonstrated that, prior to the transfer, labeled uridine was included and preserved in RNA fraction for 2-3 hours after the transfer, this fraction corresponding in mobility with mRNA in polyacrylamide gel. The following conclusion may be drawn: stable mRNAs are synthesized in the stationary phase and may be used for the translation of extracellular serine protease.