Cytomegalovirus assemblin: the amino and carboxyl domains of the proteinase form active enzyme when separately cloned and coexpressed in eukaryotic cells.

The cytomegalovirus (CMV) serine proteinase assemblin is synthesized as a precursor that undergoes three principal autoproteolytic cleavages. Two of these are common to the assemblin homologs of all herpes group viruses: one at the maturational site near the carboxyl end of the precursor and another at the release site near the midpoint of the precursor. Release-site cleavage frees the proteolytic amino domain, assemblin, from the nonproteolytic carboxyl domain of the precursor. In CMV, a third autoproteolytic cleavage at an internal site divides assemblin into an amino subunit (An) and a carboxyl subunit (Ac) of approximately the same size that remain associated as an active "two-chain" enzyme. We have cloned the sequences encoding An and Ac as separate genes and expressed them by transfecting human cells with recombinant plasmids and by infecting insect cells with recombinant baculoviruses. When An and Ac from either simian CMV or human CMV were coexpressed in human or insect cells, active two-chain assemblin was formed. This finding demonstrates that An and Ac do not require synthesis as single-chain assemblin to fold and associate correctly in these eukaryotic systems, and it suggests that they may be structurally, if not functionally, distinct domains. An interaction between the independently expressed An and Ac subunits was demonstrated by coimmunoprecipitation experiments, and efforts to disrupt the complex indicate that the subunit interaction is hydrophobic. Cell-based cleavage assays of the two-chain assemblin formed from independently expressed An and Ac also indicate that (i) its specificity for both CMV and herpes simplex virus native substrates is similar to that of single-chain assemblin, (ii) R-site cleavage is not essential for the activity of two-chain recombinant assemblin, and (iii) the human CMV and simian CMV An and Ac recombinant subunits are functionally interchangeable.     

Cytomegalovirus assemblin (pUL80a): cleavage at internal site not essential for virus growth; proteinase absent from virions

The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called assemblin. I-site cleavage cuts assemblin in half without detected effect on its enzymatic behavior in vitro. To investigate the requirement for this cleavage during virus infection, we used the CMV-bacterial artificial chromosome system (E. M. Borst, G. Hahn, U. H. Koszinowski, and M. Messerle, J. Virol. 73:8320-8329, 1999) to construct a virus encoding a mutant I site (Ala143 to Val) intended to be blocked for cleavage. Characterizations of the resulting mutant (i) confirmed the presence of the mutation in the viral genome and the inability of the mutant virus to effect I-site cleavage in infected cells; (ii) determined that the mutation has no gross effect on the rate of virus production or on the amounts of extracellular virions, noninfectious enveloped particles (NIEPs), and dense bodies; (iii) established that assemblin and its cleavage products are present in NIEPs but are absent from CMV virions, an apparent difference from what is found for virions of herpes simplex virus; and (iv) showed that the 23-kDa protein product of C-site cleavage is more abundant in mutant virus-than in wild-type virus-infected cells and NIEPs. We conclude that the production of infectious CMV requires neither I-site cleavage of assemblin nor the presence of assemblin in the mature virion.     

Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a

Chemical rescue is an established approach that offers a directed strategy for designing mutant enzymes in which activity can be restored by supplying an appropriate exogenous compound. This method has been used successfully to study a broad range of enzymes in vitro, but its application to living systems has received less attention. We have investigated the feasibility of using chemical rescue to make a conditional-lethal mutant of the cytomegalovirus (CMV) maturational protease. The 28-kDa CMV serine protease, assemblin, has a Ser-His-His catalytic triad and an internal (I) cleavage site near its midpoint. We found that imidazole can restore I-site cleavage to mutants inactivated by replacing the critical active site His with Ala or with Gly, which rescued better. Comparable rescue was observed for counterpart mutants of the human and simian CMV assemblin homologs and occurred in both living cells and in vitro. Cleavage was established to be at the correct site by amino acid sequencing and proceeded at approximately 11%/h in bacteria and approximately 30%/h in vitro. The same mutations were unresponsive to chemical rescue in the context of the assemblin precursor, pUL80a. This catalytic difference distinguishes the two forms of the CMV protease.     

Herpesvirus proteinase: site-directed mutagenesis used to study maturational, release, and inactivation cleavage sites of precursor and to identify a possible catalytic site serine and histidine.

The cytomegalovirus maturational proteinase is synthesized as a precursor that undergoes at least three processing cleavages. Two of these were predicted to be at highly conserved consensus sequences--one near the carboxyl end of the precursor, called the maturational (M) site, and the other near the middle of the precursor, called the release (R) site. A third less-well-conserved cleavage site, called the inactivation (I) site, was also identified near the middle of the human cytomegalovirus 28-kDa assemblin homolog. We have used site-directed mutagenesis to verify all three predicted sequences in the simian cytomegalovirus proteinase, and have shown that the proteinase precursor is active without cleavage at these sites. We have also shown that the P4 tyrosine and the P2 lysine of the R site were more sensitive to substitution than the other R- and M-site residues tested: substitution of alanine for P4 tyrosine at the R site severely reduced cleavage at that site but not at the M site, and substitution of asparagine for lysine at P2 of the R site reduced M-site cleavage and nearly eliminated I-site cleavage but had little effect on R-site cleavage. With the exception of P1' serine, all R-site mutations hindered I-site cleavage, suggesting a role for the carboxyl end of assemblin in I-site cleavage. Pulse-chase radiolabeling and site-directed mutagenesis indicated that assemblin is metabolically unstable and is degraded by cleavage at its I site. Fourteen amino acid substitutions were also made in assemblin, the enzymatic amino half of the proteinase precursor. Among those tested, only 2 amino acids were identified as essential for activity: the single absolutely conserved serine and one of the two absolutely conserved histidines. When the highly conserved glutamic acid (Glu22) was substituted, the proteinase was able to cleave at the M and I sites but not at the R site, suggesting either a direct (e.g., substrate recognition) or indirect (e.g., protein conformation) role for this residue in determining substrate specificity.     

Cleavage of human cytomegalovirus protease pUL80a at internal and cryptic sites is not essential but enhances infectivity

The cytomegalovirus (CMV) maturational protease, assemblin, contains an "internal" (I) cleavage site absent from its homologs in other herpesviruses. Blocking this site for cleavage did not prevent replication of the resulting I(-) mutant virus. However, cells infected with the I(-) virus showed increased amounts of a fragment produced by cleavage at the nearby "cryptic" (C) site, suggesting that its replication may bypass the I-site block by using the C site as an alternate cleavage pathway. To test this and further examine the biological importance of these cleavages, we constructed two additional virus mutants-one blocked for C-site cleavage and another blocked for both I- and C-site cleavage. Infectivity comparisons with the parental wild-type virus showed that the I(-) mutant was the least affected for virus production, whereas infectivity of the C(-) mutant was reduced by approximately 40% and when both sites were blocked virus infectivity was reduced by nearly 90%, providing the first evidence that these cleavages have biological significance. We also present and discuss evidence suggesting that I-site cleavage destabilizes assemblin and its fragments, whereas C-site cleavage does not.     

Enzymatic activities of human cytomegalovirus maturational protease assemblin and its precursor (pPR, pUL80a) are comparable: [corrected] maximal activity of pPR requires self-interaction through its scaffolding domain

Herpesviruses encode an essential, maturational serine protease whose catalytic domain, assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered "tail" (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.     

Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad.

The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-amino-acid precursor that undergoes at least three autoproteolytic cleavages. The mature proteinase, called assemblin, is one of the products of autoproteolysis and is composed of the first 256 amino acids of the precursor. HCMV assemblin and its homologs in other herpes group viruses contain five highly conserved domains (CD1 through CD5). An absolutely conserved serine in CD3 has been shown by site-directed mutagenesis of the simian cytomegalovirus (SCMV) and herpes simplex virus type 1 (HSV-1) enzymes and by inhibitor affinity labeling of the HSV-1 and HCMV enzymes to be the active-site nucleophile of assemblin. An absolutely conserved histidine in CD2 has also been demonstrated by site-directed mutagenesis of the SCMV and HSV-1 enzymes to be essential for proteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of site-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 histidine residues implicated as active-site components, and for other amino acids whose influence on enzyme activity was of interest. The mutant proteinases were tested in a transient transfection assay for their ability to cleave their natural substrate, the assembly protein precursor. Results of these experiments verified that HCMV CD3 serine (Ser-132) and CD2 histidine (His-63) are essential for proteolytic activity and identified a glutamic acid (Glu-122) within CD3 that is also essential for proteolytic activity and may be conserved among all herpesvirus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase.     

Cytomegalovirus protein substrates are not cleaved by the herpes simplex virus type 1 proteinase.

The herpesvirus maturational proteinase, assemblin, is made as a precursor that undergoes at least two autoproteolytic cleavages--one in a sequence toward its carboxyl end, called the maturational (M) site, and one in a sequence toward its midpoint, called the release (R) site. The M- and R-site sequences are both well conserved among the herpesvirus proteinase homologs, suggesting that the proteinase of one herpesvirus might be able to cleave the substrates of another. To test this possibility, we cloned and expressed in human cells the long (i.e., full-length open reading frame of proteinase gene) and short (i.e., proteolytic domain, assemblin) forms of the proteinase from human and simian cytomegalovirus (HCMV and SCMV, respectively) and from herpes simplex virus type 1 (HSV-1), as well as the genes for their respective assembly protein precursor substrates. Data from cotransfections of these proteinase genes with appropriate homologous and heterologous substrates showed that although the SCMV and HCMV enzymes cleaved the M-sites of the assembly protein substrates of all three viruses and an SCMV R-site substrate, the HSV-1 proteinase cleaved only its own substrate. This finding demonstrates that the substrate specificity properties of the HSV-1 enzyme differ from those of the two CMV enzymes.     

Two-Step Autocatalytic Processing of the Glutaryl 7-Aminocephalosporanic Acid Acylase from Pseudomonas sp. Strain GK16

The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 is an (αβ)₂ heterotetramer of two nonidentical subunits. These subunits are derived from nascent polypeptides that are cleaved proteolytically between Gly198 and Ser199 after the nascent polypeptides have been translocated into the periplasm. The activation mechanism of the GL-7-ACA acylase has been analyzed by both in vivo and in vitro expression studies, site-directed mutagenesis, in vitro renaturation of inactive enzyme precursors, and enzyme reconstitution. An active enzyme complex was found in the cytoplasm when its translocation into the periplasm was suppressed. In addition, the in vitro-expressed GL-7-ACA acylase was processed into α and β subunits, and the inactive enzyme aggregate of the precursor was also processed and became active during the renaturation step. Mutation of Ser199 to Cys199 and enzyme reconstitution allowed us to identify the secondary processing site that resides in the α subunit and to show that Ser199 of the β subunit is essential for these two sequential processing steps. Mass spectrometry clearly indicated that the secondary processing occurs at Gly189-Asp190. All of the data suggest that the enzyme is activated through a two-step autocatalytic process upon folding: the first step is an intramolecular cleavage of the precursor between Gly198 and Ser199 for generation of the α subunit, containing the spacer peptide, and the β subunit; the second is an intermolecular event, which is catalyzed by the N-terminal Ser (Ser199) of the β subunit and results in a further cleavage and the removal of the spacer peptide (Asp190 to Gly198).     

Subunit complementation of thymidylate synthase.

Each of the two active sites of thymidylate synthase contains amino acid residues contributed by the other subunit. For example, Arg-178 of one monomer binds the phosphate group of the substrate dUMP in the active site of the other monomer [Hardy et al. (1987) Science 235, 448-455]. Inactive mutants of such residues should combine with subunits of other inactive mutants to form heterodimeric hybrids with one functional active site. In vivo and in vitro approaches were used to test this hypothesis. In vivo complementation was accomplished by cotransforming plasmid mixtures encoding pools of inactive Arg-178 mutants and pools of inactive Cys-198 mutants into a host strain deficient in thymidylate synthase. Individual inactive mutants of Arg-178 were also cotransformed with the C198A mutant. Subunit complementation was detected by selection or screening for transformants which grew in the absence of thymidine, and hence produced active enzyme. Many mutants at each position representing a wide variety of size and charge supported subunit complementation. In vitro complementation was accomplished by reversible dissociation and unfolding of mixtures of purified individual inactive Arg-178 and Cys-198 mutant proteins. With the R178F + C198A heterodimer, the Km values for dUMP and CH2H4folate were similar to those of the wild-type enzyme. By titrating C198A with R178F under unfolding-refolding conditions, we were able to calculate the kcat value for the active heterodimer. The catalytic efficiency of the single wild-type active site of the C198A + R178F heterodimer approaches that of the wild-type enzyme.     

Cloning, expression and characterization of the proteinase from human herpesvirus 6.

After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus. Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site.     

Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro

An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.     

Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival.

Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic-multifunctional proteinase found in higher eukaryotic cells. We have isolated three mutants affecting the proteolytic activity of proteinase yscE. The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2. The PRE1 gene was cloned and shown to be essential. The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms. Two-dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits. Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome. Diploids homozygous for pre1-1 are defective in sporulation. Strains carrying the pre1-1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature. Under these stress conditions pre1-1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin-protein conjugates.     

Expression and characterization of recombinant murine cytomegalovirus protease.

The protease domain of the murine cytomegalovirus (MCMV) M80 open reading frame was expressed in and purified from Escherichia coli. The recombinant enzyme was recovered as a mixture of active one- and two-chain forms. The two-chain enzyme was formed by internal cleavage of the one-chain enzyme at the I site. Activity measurements showed that MCMV protease cleaves R- and M-site peptide mimics with kinetics similar to those of recombinant human cytomegalovirus (HCMV) protease. Both the MCMV and HCMV proteases cleave I-site peptide substrates very poorly, but the crystal structure of the HCMV protease indicates that the cytomegalovirus I site likely resides on a solvent-exposed loop close to the active site.     

Overlapping binding sites in protein phosphatase 2A for association with regulatory A and alpha-4 (mTap42) subunits

Diverse functions of protein Ser/Thr phosphatases depend on the distribution of the catalytic subunits among multiple regulatory subunits. In cells protein phosphatase 2A catalytic subunit (PP2Ac) mostly binds to a scaffold subunit (A subunit or PR65); however, PP2Ac alternatively binds to alpha-4, a subunit related to yeast Tap42 protein, which also associates with phosphatases PP4 or PP6. We mapped alpha-4 binding to PP2Ac to the helical domain, residues 19-165. We mutated selected residues and transiently expressed epitope-tagged PP2Ac to assay for association with A and alpha-4 subunits by co-precipitation. The disabling H118N mutation at the active site or the presence of the active site inhibitor microcystin-LR did not interfere with binding of PP2Ac to either the A subunit or alpha-4, showing that these are allosteric regulators. Positively charged side chains Lys(41), Arg(49), and Lys(74) on the back surface of PP2Ac are unique to PP2Ac, compared with phosphatases PP4, PP6, and PP1. Substitution of one, two, or three of these residues with Ala produced a progressive loss of binding to the A subunit, with a corresponding increase in binding to alpha-4. Conversely, mutation of Glu(42) in PP2Ac essentially eliminated PP2Ac binding to alpha-4, with an increase in binding to the A subunit. Reciprocal changes in binding because of mutations indicate competitive distribution of PP2Ac between these regulatory subunits and demonstrate that the mutated catalytic subunits retained a native conformation. Furthermore, neither the Lys(41)-Arg(49)-Lys(74) nor Glu(42) mutations affected the phosphatase-specific activity or binding to microcystin-agarose. Binding of PP2Ac to microcystin and to alpha-4 increased with temperature, consistent with an activation energy barrier for these interactions. Our results reveal that the A subunit and alpha-4 (mTap42) require charged residues in separate but overlapping surface regions to associate with the back side of PP2Ac and modulate phosphatase activity.     

Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta2 crystallin

Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.     

Complementation of subunits from glycogen phosphorylases of frog and rabbit skeletal muscle and rabbit liver.

Activity can be induced in potentially active rabbit skeletal muscle phosphorylase monomers covalently bound to Sepharose by noncovalent interaction with soluble subunits carrying inactive pyridoxal 5'-phosphate analogs or even salicyladlehyde. These analogs are themselves incapable of reconstituting active holophorphorylase from apophosphorylase. Phosphorylases with one intrinsically inactive and one potentially active subunit have about one half of the activity of the native phosphorylase dimer. The usefulness of this technique for subunit complementation was demonstrated by forming hybrid phosphorylases with inactive Sepharose-bound rabbit skeletal muscle subunits containing pyridoxal 5'-phosphate monomethylester and soluble activatable frog muscle and rabbit liver phosphorylase monomers. The inactive Sepharose-bound subunit induced in each case activity in the soluble subunit. But whereas the inactive rabbit muscle phosphorylase subunit even transmitted its characteristic temperature dependence of the rate of the reaction to the frog muscle subunit, it could not propagate its control properties to the liver enzyme. Differences of hybrid phosphorylases are related to immunological and amino acid divergencies among the component enzymes.     

Complementation in vitro of mutant and wild-type ATPase of Escherichia coli using isolated subunits.

1. The inactive ATPases of four different mutant strains of Escherichia coli have been purified to homogeneity. 2. Molecular weights, subunit patterns in sodium dodecylsulfate electrophoresis and immunological properties of mutant and wild-type proteins are identical. The mutant enzymes compete with the wild-type enzyme for the binding sites on the membrane. 3. On freezing and thawing in salt solutions, the ATPase is split into subunits IA (alpha, gamma, epsilon), IB (delta; alpha, gamma, epsilon), and II (beta). By complementation in vitro of the isolated subunits, it is shown that subcomplex IA (alpha, gamma, epsilon) is altered in the mutant strains described here.     

Interleukin-1 beta converting enzyme requires oligomerization for activity of processed forms in vivo.

Interleukin-1 beta converting enzyme (ICE) is composed of 10' (p10) and 20 kDa (p20) subunits, which are derived from a common 45 kDa precursor. Recent crystallographic studies have shown that ICE exists as a tetramer (p20/p10)2 in the crystal lattice. We provide evidence that the p10 and p20 subunits of ICE associate as oligomers in transfected COS cells. Using intragenic complementation, we show that the activity of a p10/p10 interface mutant defective in autoprocessing can be restored by co-expression with active site ICE mutants. Different active site mutants can also complement each other by oligomerization to form active ICE. These studies indicate that ICE precursor polypeptides may associate in different quaternary structures and that oligomerization is required for autoprocessing. Furthermore, integenic complementation of active site mutants of ICE and an ICE homolog restores autoprocessing activity, suggesting that hetero-oligomerization occurs between ICE homologs.     

Intersubunit location of the active site of ribulose-bisphosphate carboxylase/oxygenase as determined by in vivo hybridization of site-directed mutants

Ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is a homodimer of 50.5-kDa subunits with two substrate binding sites per molecule of dimer. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interactions, we have used a novel in vivo approach for producing heterodimers from catalytically inactive, site-directed mutants of the carboxylase. When the alleles encoding these mutant proteins are placed separately into compatible plasmids and coexpressed in the same Escherichia coli host, activity is observed at about 20% of the wild-type level. Analysis of the carboxylase purified from these cells reveals the presence of heterodimers of the two mutant proteins. This interallelic complementation demonstrates that domains from each of the subunits interact to form a shared active site.