The Sup35 Omnipotent Suppressor Gene Is Involved in the Maintenance of the Non-Mendelian Determinant [Psi(+)] in the Yeast Saccharomyces Cerevisiae

The SUP35 gene of yeast Saccharomyces cerevisiae encodes a 76.5-kD ribosome-associated protein (Sup35p), the C-terminal part of which exhibits a high degree of similarity to EF-1α elongation factor, while its N-terminal region is unique. Mutations in or overexpression of the SUP35 gene can generate an omnipotent suppressor effect. In the present study the SUP35 wild-type gene was replaced with deletion alleles generated in vitro that encode Sup35p lacking all or a part of the unique N-terminal region. These 5'-deletion alleles lead, in a haploid strain, simultaneously to an antisuppressor effect and to loss of the non-Mendelian determinant [psi(+)]. The antisuppressor effect is dominant while the elimination of the [psi(+)] determinant is a recessive trait. A set of the plasmid-borne deletion alleles of the SUP35 gene was tested for the ability to maintain [psi(+)]. It was shown that the first 114 amino acids of Sup35p are sufficient to maintain the [psi(+)] determinant. We propose that the Sup35p serves as a trans-acting factor required for the maintenance of [psi(+)].     

String of Pearls Encodes Drosophila Ribosomal Protein S2, Has Minute-like Characteristics, and Is Required during Oogenesis

The first allele of string of pearls (sop) was isolated as a recessive female sterile mutant in a P element enhancer trap screen. Oogenesis in homozygous sop females arrests at approximately stage 5. In addition, homozygous flies of both sexes have Minute-like characteristics that include reduced bristles, delayed development and larval lethality. sop maps to 30D/E on chromosome 2L and encodes the Drosophila homolog of eukaryotic ribosomal protein S2. The gene is present in a single copy in the Drosophila genome and the level of mRNA present in mutant animals is reduced. The identification of a mutant allele that blocks development at a mid-stage of oogenesis may indicate that sop has a specific developmental role during oogenesis in addition to its general role in protein synthesis as a component of the small ribosomal subunit.     

Functional Expression of hMYH, a Human Homolog of the Escherichia coli MutY Protein

We have previously described the hMYH cDNA and genomic clones (M. M. Slupska et al., J. Bacteriol. 178:3885-3892, 1996). Here, we report that the enzyme expressed from an hMYH cDNA clone in Escherichia coli complements the mutator phenotype in a mutY mutant and can remove A from an A. 8-hydroxydeoxyguanine mismatch and to a lesser extent can remove A from an A. G mismatch in vitro.     

Interaction of the Yeast Omnipotent Suppressors Sup1(sup45) and Sup2(sup35) with Non-Mendelian Factors

The SUP1 and SUP2 genes code for protein factors intimately involved in the control of translational accuracy. The disrupted alleles of these genes confer a recessive lethal phenotype in both [psi+] and [psi-] genetic backgrounds, indicating an essential function for the corresponding proteins. In [psi+] diploids, heterozygous for the SUP1 null allele, several dominant phenotypes were evident with slow growth and inability to sporulate. These dominant phenotypes disappear after transformation with the multicopy plasmid carrying the wild-type allele of the SUP1 gene. Such dominant phenotypes were not observed for the SUP2 null allele. The incompatibility of multicopy plasmids carrying the SUP2 gene with guanidine hydrochloride-curable cytoplasmic factor(s) was also demonstrated. The possible mechanisms of interaction of the SUP1 and SUP2 genes with the [psi] determinant are discussed.     

Histidine Methylation of Yeast Ribosomal Protein Rpl3p Is Required for Proper 60S Subunit Assembly

Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine 243, a residue that contacts the 25S rRNA near the P site. Rpl3p methylation is dependent upon the presence of Hpm1p, a candidate seven-beta-strand methyltransferase. In this study, we elucidated the biological activities of Hpm1p in vitro and in vivo. Amino acid analyses reveal that Hpm1p is responsible for all of the detectable protein histidine methylation in yeast. The modification is found on a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nucleus-containing organelle fraction but was not detected in proteins of the ribosome-free cytosol fraction. In vitro assays demonstrate that Hpm1p has methyltransferase activity on ribosome-associated but not free Rpl3p, suggesting that its activity depends on interactions with ribosomal components. hpm1 null cells are defective in early rRNA processing, resulting in a deficiency of 60S subunits and translation initiation defects that are exacerbated in minimal medium. Cells lacking Hpm1p are resistant to cycloheximide and verrucarin A and have decreased translational fidelity. We propose that Hpm1p plays a role in the orchestration of the early assembly of the large ribosomal subunit and in faithful protein production.     

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Functional domains of Escherichia coli Ribosomal Protein S1. Formation and characterization of a fragment with ribosome-binding properties.

Treatment of Escherichia coli ribosomal protein S1 with TPCK-treated trypsin under mild conditions (0 °C, 1 to 2 μig trypsin/mg S1 protein) results in the production of a high molecular weight fragment in yields of up to 80% within a few minutes. The fragment is relatively resistant to further degradation. We have isolated the fragment in pure form for structural and functional characterization. The fragment (denoted S1-F1) has a molecular weight of 48,500 as shown by sodium dodecyl sulphate gel electrophoresis, and therefore it contains approximately 60% of the amino acid residues of S1. The N-terminal sequence of the fragment is different from that of intact S1.The fragment binds to the 30 S ribosomal subunit and to polyuridylic acid in approximately the same manner as intact S1, indicating that the active centres of S1 concerned with these two characteristic binding properties are localized within the fragment. In spite of the above properties, the fragment was completely unable to support protein synthesis. The significance of these results in relation to the structure and function of S1 is discussed.     

Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation

Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process.     

The mdoC Gene of Escherichia coli Encodes a Membrane Protein That Is Required for Succinylation of Osmoregulated Periplasmic Glucans

Osmoregulated periplasmic glucans (OPGs) of Escherichia coli are anionic oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by phosphoglycerol originating from the membrane phospholipids and by succinyl residues from unknown origin. A phosphoglycerol-transferase-deficient mdoB mutant was subjected to Tn5 transposon mutagenesis, and putative mutant clones were screened for changes in the anionic character of OPGs by thin-layer chromatography. One mutant deficient in succinylation of OPGs was obtained, and the gene inactivated in this mutant was characterized and named mdoC. mdoC, which encodes a membrane-bound protein, is closely linked to the mdoGH operon necessary for the synthesis of the OPG backbone.     

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain

Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn²⁺ cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn²⁺ is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.     

The Heat Shock Protein YbeY Is Required for Optimal Activity of the 30S Ribosomal Subunit

The highly conserved bacterial ybeY gene is a heat shock gene whose function is not fully understood. Previously, we showed that the YbeY protein is involved in protein synthesis, as Escherichia coli mutants with ybeY deleted exhibit severe translational defects in vivo. Here we show that the in vitro activity of the translation machinery of ybeY deletion mutants is significantly lower than that of the wild type. We also show that the lower efficiency of the translation machinery is due to impaired 30S small ribosomal subunits.Many heat shock proteins are chaperones and proteases that constitute the protein quality control system (4, 5, 13, 18). Recent studies demonstrated that beyond protein quality control, the heat shock response includes proteins implemented in the translation machinery (16, 17), such as FtsJ (2, 3) and Hsp15 (11).FtsJ catalyzes methylation of U2552 in 23S rRNA (3). This modification occurs during the final steps of 50S biogenesis and is important for the structural stability of the 50S subunit (2). ftsJ deletion mutants accumulate ribosomal subunits at the expense of polysomes (2). Consequently, crude ribosome extracts prepared from ftsJ deletion mutants are far less active than wild-type preparations (3). Hsp15 recognizes and binds with high affinity to the aberrant state of the 50S subunit in complex with peptidyl tRNA positioned at the A site (10), which is more frequent at high temperatures (10). It has been proposed that Hsp15 participates in releasing the bound peptide and thereby helps recycle the 50S subunit (8, 10). Thus, heat shock proteins play a significant role both in the biogenesis of ribosomes and in the translation process.YbeY is a 17-kDa heat shock protein, highly conserved among bacteria, that belongs to the UPF0054 family of metal-dependent hydrolases, suggesting that it may have a potential hydrolytic function (14, 21). In Aquifex aeolicus, analysis of YbeY structure homology showed similarity to eukaryotic extracellular proteinases such as collagenase and gelatinase. However, in vitro experiments could not detect collagenase, gelatinase, or other hydrolase activity in YbeY (14).Recently, we showed that ybeY deletion mutants exhibit severe translational defects manifested by a very low level of polysomes and accumulation of free ribosomes and ribosomal subunits, indicating that most ribosomes in the cell are not engaged in translation. This translational defect intensifies at elevated temperatures (42°C) and results in growth arrest (17).Here we present in vitro studies indicating that the activity of the translation machinery prepared from ybeY deletion mutants is lower than in the wild type. In addition, we show that this lower activity stems specifically from a defective 30S ribosomal subunit.     

Mapping of Functional Domains in Herpesvirus Saimiri Complement Control Protein Homolog: Complement Control Protein Domain 2 Is the Smallest Structural Unit Displaying Cofactor and Decay-Accelerating Activities

Herpesvirus saimiri encodes a functional homolog of human regulator-of-complement-activation proteins named CCPH that inactivates complement by accelerating the decay of C3 convertases and by serving as a cofactor in factor I-mediated inactivation of their subunits C3b and C4b. Here, we map the functional domains of CCPH. We demonstrate that short consensus repeat 2 (SCR2) is the minimum domain essential for classical/lectin pathway C3 convertase decay-accelerating activity as well as for factor I cofactor activity for C3b and C4b. Thus, CCPH is the first example wherein a single SCR domain has been shown to display complement regulatory functions.The complement system is an ancient and yet highly evolved effector mechanism of immune defense that forms an imperative branch of innate immunity (23, 46). In addition, recent findings have clearly revealed its role as a vital viaduct between the innate and acquired immune systems (6, 18). Thus, it is not surprising that the system helps in purging a wide array of invaders, including viruses. Consequently, for their successful survival, many viruses have developed mechanisms to subvert the host complement system (7, 24, 26, 29, 39, 45). Herpesviruses and poxviruses, in particular, subvert host complement by encoding structural and/or functional homologs of human complement regulators belonging to the regulator-of-complement-activation (RCA) family, by capturing host membrane complement regulators and by using cellular receptors for entering cells (1, 8, 15, 23).The RCA proteins are formed by multiple tandem repeats of bead-like complement control protein (CCP) domains or short consensus repeats (SCRs) separated by short linkers. It has been suggested that the sequence variations enforced upon these SCR domain folds and the interdomain dynamics dictate the functionality of the complement regulators (17, 19, 44, 49). Because sequence similarity in herpesviral complement regulators varies between 43% and 89% and in poxviral complement regulators exceeds 91%, it is likely that the structural diversity in herpesviral complement regulators may have resulted in functional differences in these proteins and/or have resulted in variation in structural requirements for complement regulation. In the herpesviridae family, detailed functional characterization has been performed for complement regulators of Kaposi''s sarcoma-associated herpesvirus (Kaposica/KCP) (28, 42), herpesvirus saimiri (HVS) (CCPH) (10, 38), and rhesus rhadinovirus (RCP) (31). All these proteins showed conservation of complement regulatory activities, indicating thereby that structural diversity has not resulted in loss of complement regulatory functions in these proteins. However, it is not clear whether sequence variations within the herpesviral complement regulators have resulted in differences in the domain requirements for complement regulatory activities, since mapping of functional domains has been performed only for Kaposica (30, 43). In the present study, we therefore have mapped the complement regulatory domains of HVS CCPH to get further insight into diversity in domain requirements for functional activities.HVS is a classical prototype of the gamma 2-herpesviruses or rhadinoviruses. It causes rapidly progressing fulminant lymphoma, lymphosarcoma, and leukemia of T-cell origin in marmosets, owl monkeys, and other species of New World primates but not in its natural host, the squirrel monkey (9, 16). Unlike other herpesviruses, it encodes two complement regulators: an RCA homolog (ORF 4; CCPH) that regulates the early steps of complement activation (2, 10) and a CD59 homolog (ORF 15) that inhibits the late steps of complement activation (4, 36). The RCA homolog is formed of four SCR modules (Fig. ​(Fig.1).1). As a result of alternative splicing, the protein is expressed as a full-length membrane-bound form (mCCPH) containing the transmembrane region as well as a spliced secretory form (sCCPH) lacking the transmembrane region (2). Earlier, we showed that sCCPH inhibits complement by targeting C3 convertases: (i) it supports serine protease factor I-mediated inactivation of C3b and C4b, the subunits of C3 convertases (cofactor activity), and (ii) it accelerates the irreversible decay of the classical pathway (CP)/lectin pathway and to a limited extent the alternative pathway (AP) C3 convertases (decay-accelerating activity [DAA]) (38).Open in a separate windowFIG. 1.Schematic illustration of sCCPH and SDS-PAGE analysis of purified recombinant sCCPH and its deletion mutants. (Top) Schematic representation of the structure of the soluble form of CCPH (sCCPH), which is composed of four SCRs. The domains are numbered, and the minimum domains shown to be important for C3b and C4b cofactor activities (CFA) and CP DAA are identified. (Bottom) Expressed and purified sCCPH and its deletion mutants were analyzed by 12% (left) and 13% (right) SDS-PAGE under reducing conditions and stained with Coomassie blue. Molecular weights as determined by SDS-PAGE: for sCCPH, 32,000; for SCR1-3, 26,000; for SCR2-4, 27,500; for SCR1-2, 17,000; for SCR2-3, 17,500; for SCR3-4, 16,500; for SCR1, 9,500; for SCR2, 7,000; for SCR3, 8,000; and for SCR4, 8,000. Molecular mass is expressed as kilodaltons in the figure.(This work was done in partial fulfillment of the Ph.D. thesis requirements of A.K.S., University of Pune, Pune, India.)In order to map the functional domains of sCCPH, we have generated a series of soluble triple, double, and single SCR deletion mutants. In brief, the deletion mutants of sCCPH comprising SCR1-3, -2-4, -1-2, -2-3, and -3-4 as well as SCR1, -2, -3, and -4 were constructed from the full-length HVS sCCPH clone (38) by PCR amplification and cloning into the bacterial expression vector pET29. The authenticity of each of the clones was confirmed by DNA sequencing, and then they were transformed into the Escherichia coli BL21 strain for expression. The mutants carried the histidine tag at the C terminus and hence were purified to homogeneity by using histidine affinity chromatography. Refolding of the purified proteins was performed by using the rapid dilution method as previously described (38, 47, 48), and the refolded proteins were loaded onto a Superose 12 gel filtration column (Pharmacia) to obtain monodisperse populations of the expressed mutants (38, 48). The preservation of various functions in mutants (see below) suggests that the mutants have maintained their proper conformation. The expressed proteins were >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. ​(Fig.11).To identify the domains required for cofactor activities of sCCPH against C3b and C4b, we utilized a fluid phase assay wherein C3b or C4b was incubated with each of the deletion mutants and factor I, and inactivation of C3b/C4b (cleavage of the α′-chain) was determined by running the samples on SDS-PAGE gels. It is clear from the data presented in Fig. ​Fig.22 that sCCPH and the mutants SCR1-3, -2-4, and -1-2 supported the cleavage of the α′-chain of C3b. A very weak cleavage was also supported by SCR2-3 and -3-4. The cleavage of the α′-chain of C4b, however, was supported by sCCPH and the mutants SCR1-3, -2-4, -1-2, and -2-3 but not by SCR3-4 (Fig. ​(Fig.2).2). Together, these data point out that SCR1 and -2 considerably contribute to the C3b and C4b cofactor activities of sCCPH but that SCR3 and SCR4 in the case of C3b cofactor activity and SCR3 in the case of C4b cofactor activity contribute to its optimal activity. These results, however, did not elucidate whether a single domain(s) could impart the cofactor activities. We therefore expressed the single-domain mutants (SCR1, SCR2, SCR3, and SCR4) and analyzed their cofactor activities. The results presented in Fig. ​Fig.33 indicate that SCR2, by itself, possesses the ability to support factor I-mediated inactivation of C3b and C4b; SCR3 also displayed very weak cofactor activity against C3b when used at higher concentrations (88 μM; data not shown). These results suggest that structural elements involved in the interaction of sCCPH with factor I are primarily located within SCR2 and -3. Admittedly, the single-domain mutants possess very weak cofactor activities and other domains too contribute to the optimal activity; the cofactor activities of SCR2 for C3b and C4b were 781- and 212-fold lower than that for sCCPH (Fig. ​(Fig.3).3). It should be mentioned here that earlier observations on mapping of the human RCA proteins (factor H, C4b-binding protein, membrane cofactor protein, and complement receptor 1) (3, 11-13, 21), Kaposica (30), and vaccinia virus CCP (VCP) (27) indicated that a minimum of two (in Kaposica) or three (in all other RCA proteins) successive SCR domains are necessary for factor I cofactor activities. Thus, sCCPH is the first complement regulator in which a single SCR domain has been shown to display the factor I cofactor function.Open in a separate windowFIG. 2.Analysis of factor I cofactor activity of sCCPH and its deletion mutants for human complement proteins C3b and C4b. Cofactor activity was assessed by incubating 3.0 μg of human C3b (upper panels) or C4b (lower panels) with sCCPH/SCR1-3/SCR2-4 (4.0 μM) or SCR1-2/2-3/3-4 (24 μM) in the presence or absence of factor I (100 ng) for the indicated time periods at 37°C in 10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl. The reactions were stopped by addition of sample buffer containing dithiothreitol, and the amount of C3b or C4b cleaved was visualized by subjecting the samples to SDS-PAGE analysis on 10% or 11.5% gel, respectively, and staining with Coomassie blue. During C3b cleavage, the α′-chain is cleaved into N-terminal 68-kDa and C-terminal 46-kDa fragments. The 46-kDa fragment is then cleaved into a 43-kDa fragment. These cleavages indicate inactivation of C3b. In the case of C4b, the α′-chain is cleaved into N-terminal 27-kDa, C-terminal 16-kDa (not visible in the gel), and central C4d fragments. These cleavages indicate the inactivation of C4b.Open in a separate windowFIG. 3.Analysis of factor I cofactor activity (CFA) of single SCR mutants of sCCPH for human complement proteins C3b and C4b. (Upper panels) Cofactor activity was assessed by incubating 3.0 μg of human C3b or C4b with the single SCR mutants (44 μM) in the presence or absence of factor I (100 ng) for 4 h at 37°C in PBS (10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl). The reactions were stopped by addition of sample buffer containing dithiothreitol, and the amount of C3b or C4b cleaved was visualized by subjecting the samples to 13% SDS-PAGE and stained with Coomassie blue. Cleavage of the α′-chain of C3b and C4b and generation of cleavage products indicate the inactivation of these proteins. (Middle panels) Human C3b (3.0 μg) or C4b (3.0 μg) and factor I (100 ng) were incubated in PBS with increasing concentrations of sCCPH or the SCR2 mutant at 37°C for 1 h, and the cleavage products were analyzed as described above. (Lower panels) The intensity of the α′-chains of C3b and C4b in the middle panels was determined densitometrically and is represented graphically. The closed and open circles represent sCCPH and the SCR2 mutant, respectively.As discussed above, in addition to the inactivation of subunits of C3 convertases (C3b and C4b), sCCPH also regulates C3 convertases by accelerating their decay. It possesses considerable DAA for the CP/lectin pathway C3 convertase (C4b,2a) and a poor decay activity for the AP C3 convertase (C3b,Bb). Thus, we next examined the DAAs of the various sCCPH mutants to map the domains required for this function. To measure the CP C3 convertase decay activity, the C4b,2a enzyme was formed on sheep erythrocytes and allowed to decay in the presence of various mutants. The remaining enzyme activity was then measured by incubating the reaction mixture with EDTA sera (a source of C3 to C9) and measuring hemolysis. Apart from sCCPH, mutants SCR1-3, -1-2, and -2-3 showed substantial DAA for the CP C3 convertase (Fig. ​(Fig.4).4). These data suggested that SCR1-3 is primarily responsible for this activity. On a molar basis, SCR1-3 was 1.6-fold less efficient than sCCPH. Because both SCR1-2 and SCR2-3 possessed the decay activity, it was likely that similar to the cofactor activities, a single SCR domain of sCCPH might also possess the DAA for the CP C3 convertase. Hence, we also assessed the DAAs of the single-domain mutants. Interestingly again, SCR2 was the only single domain that distinctly displayed CP DAA (Fig. ​(Fig.4);4); however, on a molar basis, it was 26-fold less active than sCCPH. Previous data on the involvement of SCR domains in decay acceleration of CP C3 convertase in human RCA proteins (decay-accelerating factor, complement receptor 1, and C4b-binding protein) (3, 5, 20) and viral RCA homologs (Kaposica and VCP) (27, 30) have shown that a minimum of two or three consecutive domains are necessary for the activity. Thus, sCCPH is the only prototype to date in which a single SCR is adequate to impart the CP DAA.Open in a separate windowFIG. 4.Analysis of CP and AP C3 convertase DAAs of sCCPH and its mutants. (Upper panel) The CP C3 convertase C4b,2a was formed on antibody-coated sheep erythrocytes (EA) by sequentially incubating them with human C1, C4, and C2 (Calbiochem). The C3 convertase on the cells was then allowed to decay by incubating EA-C4b,2a with various concentrations of sCCPH or its mutants for 5 min at 22°C, and the activity of the remaining enzyme was assessed by measuring the cell lysis following incubation for 30 min at 37°C with Guinea pig sera containing 40 mM EDTA (27, 32). (Lower panel) The AP C3 convertase C3b,Bb was formed on sheep erythrocytes (ES) by incubating them with human C3 (Calbiochem) and factors B and D in the presence of NiCl₂. The C3 convertase on the cells was then allowed to decay by incubating ES-C3b,Bb with various concentrations of sCCPH or its mutants for 10 min at 37°C, and the activity of the remaining enzyme was assessed by measuring the cell lysis following incubation with EDTA-sera for 30 min at 37°C (35, 37). The data obtained were normalized by considering the lysis that occurred in the absence of an inhibitor as 100% lysis.Although sCCPH is known to possess limited AP C3 convertase DAA, we sought to determine whether this limited activity is localized in a specific region or the full-length protein. To measure the AP DAA, the C3 convertase C3b,Bb was formed on the sheep erythrocytes and incubated with sCCPH or with each of its deletion mutants. The decay of the AP C3 convertase was assessed by adding EDTA sera and measuring hemolysis. Although the full-length protein displayed a limited AP C3 convertase, none of the deletion mutants exhibited any activity (Fig. ​(Fig.44).Inactivation of C3 convertases by the RCA proteins, owing to their cofactor and decay activities, requires interaction of these proteins with C3b and C4b. The ligand binding activity of the RCA proteins, however, does not always correlate with their cofactor and decay activities (12, 34), as apart from ligand binding, cofactor activity involves interaction of the RCA protein with factor I (40), and decay activity involves interaction of the RCA protein with C2a or Bb (22, 25). In order to determine whether cofactor and decay activity data of sCCPH and the various mutants correlate with the ligand binding data, we measured binding of these proteins to C3b and C4b by using a surface plasmon resonance-based assay (38). As observed earlier (38), sCCPH displayed higher affinity for C4b than for C3b (Fig. ​(Fig.55 and Table ​Table1).1). When we measured binding of various deletion mutants to C3b and C4b, only SCR2-4 showed binding to C3b, and SCR1-3 showed binding to C4b (Fig. ​(Fig.5).5). However, there were reductions of about 16- and 14-fold in the affinities of these deletion mutants for C3b and C4b, respectively, compared to that for sCCPH (Table ​(Table1),1), suggesting that all the four domains contribute to binding to C3b and C4b. Because most of the deletion mutants that displayed complement regulatory activities possessed negligible binding to C3b and C4b, it is clear that binding of the mutants does not correlate with their cofactor and decay activities. It is likely that during cofactor activity, interaction of the mutants with C3b and C4b is stabilized by the interaction of factor I with C3b/C4b and the mutants. Similarly, during DAAs, the mutants may possess better affinity for the convertases than their subunits C3b and C4b. Consistent with this argument, decay-accelerating factor has previously been shown to bind to CP C3 convertase with 1,000-fold higher affinity than to C4b (33).Open in a separate windowFIG. 5.Binding of sCCPH and its mutants to C3b and C4b. Binding was determined by a surface plasmon resonance-based assay (38). Sensograms were generated by immobilizing biotinylated C3b (1,200 response units [RUs]) and C4b (940 RUs) on streptavidin chips (Sensor Chip SA; Biacore AB; additional RUs of C3b [∼6,000 RUs] were deposited by forming AP C3 convertase on the chip and flowing native C3 [14]) and injecting sCCPH or its mutants in PBS-T (10 mM sodium phosphate and 145 mM NaCl, pH 7.4, containing 0.05% Tween 20) over the chip. Flow cells immobilized with bovine serum albumin-biotin (Sigma) served as control flow cells. (Left panels) Binding of sCCPH and its various mutants to C3b (top) and C4b (bottom). The sensograms were generated by injecting 500 nM and 2 μM of sCCPH and its various mutants over C3b and C4b chips, respectively. (Middle panels) Sensogram overlay for the interaction between sCCPH and C3b (top) or sCCPH and C4b (bottom). (Right panels) Sensogram overlay for the interaction between SCR2-4 and C3b (top) and SCR1-3 and C4b (bottom). The concentrations of proteins injected are indicated at the right of the sensograms. The solid lines in the top middle and top right panels represent the global fitting of the data to a 1:1 Langmuir binding model with a drifting baseline (A + B ↔ AB; Biaevaluation 4.1). The small arrows in the bottom middle and right panels indicate the time points used for evaluating the steady-state affinity data.

TABLE 1.

Kinetic and affinity data for the interactions of sCCPH and the deletion mutants with human complement proteins C3b and C4b^a

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity Is a Multi-domain Intrinsically Disordered Protein

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.     

The Conserved Bud20 Zinc Finger Protein Is a New Component of the Ribosomal 60S Subunit Export Machinery

The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C₂H₂-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.     

Amino Acid Differences in a 30S Ribosomal Protein from Two Strains of Escherichia coli

The 30S ribosomal proteins of the K-12 and B strains of Escherichia coli differ in at least one protein component. This component, which is allelic in the two strains, has been isolated from both organisms. Amino acid analyses show that the protein from strain B contains between 20 and 28 more amino acids than does the analogue protein from strain K-12.