Differential association of products of alternative transcripts of the candidate tumor suppressor ING1 with the mSin3/HDAC1 transcriptional corepressor complex.

The candidate tumor suppressor ING1 was identified in a genetic screen aimed at isolation of human genes whose expression is suppressed in cancer cells. It may function as a negative growth regulator in the p53 signal transduction pathway. However, its molecular mechanism is not clear. The ING1 locus encodes alternative transcripts of p47(ING1a), p33(ING1b), and p24(ING1c). Here we report differential association of protein products of ING1 with the mSin3 transcriptional corepressor complex. p33(ING1b) associates with Sin3, SAP30, HDAC1, RbAp48, and other proteins, to form large protein complexes, whereas p24(ING1c) does not. The ING1 immune complexes are active in deacetylating core histones in vitro, and p33(ING1b) is functionally associated with HDAC1-mediated transcriptional repression in transfected cells. Our data provide basis for a p33(ING1b)-specific molecular mechanism for the function of the ING1 locus.

SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex.

We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function. Sgt1p physically associates with Skp1p in vivo and in vitro. SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content. Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function. Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p. Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase. sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro. Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution.

Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase.

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers. The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination. Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins. The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition. These findings provide a further link between VHL and the cellular ubiquitination machinery.

Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1.

Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.

F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex.

We have reconstituted the ubiquitination pathway for the Cdk inhibitor Sic1 using recombinant proteins. Skp1, Cdc53, and the F-box protein Cdc4 form a complex, SCFCdc4, which functions as a Sic1 ubiquitin-ligase (E3) in combination with the ubiquitin conjugating enzyme (E2) Cdc34 and E1. Cdc4 assembled with Skp1 functions as the receptor that selectively binds phosphorylated Sic1. Grr1, an F-box protein involved in Cln destruction, forms complexes with Skp1 and Cdc53 and binds phosphorylated Cln1 and Cln2, but not Sic1. Because the constituents of the SCF complex are members of protein families, SCFCdc4 is likely to serve as the prototype for a large class of E3s formed by combinatorial interactions of related family members. SCF complexes couple protein kinase signaling pathways to the control of protein abundance.

GrpE alters the affinity of DnaK for ATP and Mg2+. Implications for the mechanism of nucleotide exchange.

DnaK, DnaJ, and GrpE heat shock proteins of Escherichia coli activate site-specific DNA binding by the RepA replication initiator protein of plasmid P1 in a reaction dependent on ATP and Mg2+. We previously showed that GrpE is essential for in vitro RepA activation specifically at about 1 microM free Mg2+. In this paper, we demonstrate that GrpE lowers the requirement of DnaK ATPase for Mg2+, resulting in a large stimulation of ATP hydrolysis at about 1 microM Mg2+ with and without DnaJ and RepA. In contrast to its effect on the Mg2+ requirement, GrpE increases the ATP requirement for DnaK ATPase and dramatically lowers the affinity of DnaK for ATP in the absence of Mg2+. We propose that GrpE not only lowers the affinity of DnaK for nucleotide but, by increasing affinity of DnaK for Mg2+, also weakens the interactions of Mg2+ with nucleotide prior to its release.

A molecular chaperone, ClpA, functions like DnaK and DnaJ.

The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp60s (GroEL) and Hsp70s (DnaK). Clp proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. We discovered that ClpA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone. ClpA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein. RepA is activated by the conversion of dimers to monomers. We show that ClpA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis. In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase.

Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity.

In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can. In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required. GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP. The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP. GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES. The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated. We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay. However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein. Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex.

The interplay of the GrpE heat shock protein and Mg2+ in RepA monomerization by DnaJ and DnaK.

Genetic and biochemical studies have established that the sole function of the Escherichia coli DnaJ, DnaK, and GrpE heat shock proteins in plasmid P1 DNA replication is to convert RepA dimers to monomers. Monomers bind avidly to oriP1 DNA and initiate DNA replication. However, with purified heat shock proteins, only DnaJ, DnaK, and ATP were required for the monomerization of RepA; GrpE was not required. We have found reaction conditions that mimic the physiological situation. GrpE function is absolutely necessary for RepA activation in vitro with DnaJ and DnaK when the free Mg2+ concentration is maintained at a level of approximately 1 microM by a metal ion buffer system. EDTA or physiological metabolites, including citrate, phosphate, pyrophosphate, and ATP, all elicit the GrpE requirement. With these metal ion-buffering systems, GrpE specifically lowers the concentration of Mg2+ required for the RepA activation reaction. The absence of Mg2+ blocks activation and high levels of Mg2+ in solution bypass the requirement for GrpE but not for the other two heat shock proteins. Our results imply that GrpE facilitates the utilization of Mg2+ for an essential step in RepA activation.

DnaJ, DnaK, and GrpE heat shock proteins are required in oriP1 DNA replication solely at the RepA monomerization step.

We have found that three Escherichia coli heat shock proteins, DnaK (the hsp70 homolog), DnaJ, and GrpE, function in oriP1 DNA replication in vitro solely to activate DNA binding by the replication initiator protein RepA. Activation results from the conversion of P1 or P7 RepA dimers to monomers that bind with high affinity to the origin of replication of plasmid P1. Thus, the essential role of these three heat shock proteins in this replication system is to change the quaternary structure of a single protein, RepA.

The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein.

The DnaK protein of Escherichia coli and its eukaryotic hsp70 analogues are known to bind some polypeptides and to release or dissociate from them following ATP hydrolysis. Here we demonstrate that hydrolysis (and not simply binding) of nucleotide triphosphates leads to a change in the DnaK protein, from the "closed" to the "open" conformation. A conformational change is not observed with the mutant DnaK756 protein, which is always found in the open conformation. Although ATP is the preferred substrate, the hydrolysis of CTP, GTP, UTP, and dATP also results in DnaK's conversion from a closed to an open conformation. The ability of DnaK to hydrolyze various triphosphates correlates perfectly with its ability to release the bound denatured bovine pancreatic trypsin inhibitor polypeptide.

The E. coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner.

Pelham previously proposed that the hsp70 family of heat shock proteins could prevent the formation and/or allow the dissolution of protein aggregates created during stress conditions. We confirmed this hypothesis by showing that the E. coli hsp70 homolog, the dnaK gene product, protects the host RNA polymerase enzyme from heat inactivation in an ATP-independent reaction. In addition, we show that heat-inactivated and aggregated RNA polymerase is both disaggregated and reactivated following simultaneous incubation with DnaK protein and hydrolyzable ATP. The DnaK756 mutant protein has lost the ability to disaggregate the inactivated RNA polymerase enzyme. Our results demonstrate that the DnaK protein contributes to E. coli's growth not only by protecting some enzymes from denaturation but also by reactivating some once they are misfolded or aggregated.

Enzymology of the pre-priming steps in lambda dv DNA replication in vitro.

We have examined some of the early pre-priming steps of bacteriophage lambda dv DNA replication in vitro. Previous experiments have shown that bacteriophage lambda replication requires host RNA polymerase-dependent RNA synthesis near or at the origin of replication (ori lambda) to initiate DNA synthesis. Using a crude Fraction II enzymatic system we have shown that during RNA polymerase action, at least the bacteriophage lambda O and lambda P replication proteins as well as the host dnaB protein must be present to initiate ori lambda-specific DNA replication. The presence of three other host initiation proteins, dnaG primase, dnaJ, and dnaK, is not required during RNA polymerase action. Because of the apparent absence of a requirement for the dnaJ and dnaK pre-priming proteins during the transcriptional activation step, we propose that the early events of lambda dv DNA replication, prior to action by the dnaG primase, can be divided into two recognizable steps: an early step which requires at least RNA polymerase, lambda O, lambda P, and dnaB, and a subsequent step which requires the action of at least the dnaJ and dnaK proteins.