The translation machinery and 70 kd heat shock protein cooperate in protein synthesis.

The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide. Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis. The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes.     

The yeast Rgd1p is a GTPase activating protein of the Rho3 and rho4 proteins.

The RGD1 gene, identified during sequencing of the Saccharomyces cerevisiae genome, encodes a protein with a Rho-GTPase activating protein (GAP) domain at the carboxy-terminal end. The Rgd1 protein showed two-hybrid interactions with the activated forms of Rho2p, Rho3p and Rho4p. Using in vitro assays, we demonstrated that Rgd1p stimulated the GTPase activity of both Rho3p and Rho4p; no stimulation was observed on Rho2p. In addition, the rho3Deltargd1Delta double mutant exhibited a dramatic growth defect compared to the single mutants, suggesting that Rgd1p has a GAP activity in vivo. The present study allowed the identification of the first GAP of Rho3p and Rho4p.     

The structure and function of the replication initiator protein (Rep) of pSC101: an analysis based on a novel positive-selection system for the replication-deficient mutants

Plasmid pSC101 encodes a 37.5 kDa Rep (RepA) protein, which binds to three 21-base repeats (DR-1, DR-2, and DR-3) in the replication origin region (ori) of the plasmid to initiate replication. Rep also binds to two palindromic sequences (IR-1 and IR-2) which overlap the rep promoter. The binding of Rep to IR-2 represses the production of Rep itself. It is highly likely that the balance of these functions of Rep plays a major role in controlling the copy number of pSC101. In this study, we developed a positive-selection system for replication-deficient mutants of the initiator protein. This system can be applied to the study of other replication systems by changing ori and rep of pSC101 to the corresponding genes. Thirty-four replication-deficient (Ini(-)) mutants were isolated with this system, and analyzed as to the relation between the structure and function of the Rep protein. Seventeen of these 34 Ini(-) mutants were found to lack auto-repressor activity as well as initiator activity. DNA sequence analysis showed that one-third (from the C-terminus) of Rep is dispensable for the auto-repressor activity, while the initiator activity seems to require the whole protein.     

Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing

Condensin is required for chromosome dynamics and diverse DNA metabolism. How condensin works, however, is not well understood. Condensin contains two structural maintenance of chromosomes (SMC) subunits with the terminal globular domains connected to coiled-coil that is interrupted by the central hinge. Heterotrimeric non-SMC subunits regulate SMC. We identified a novel fission yeast SMC hinge mutant, cut14-Y1, which displayed defects in DNA damage repair and chromosome segregation. It contains an amino acid substitution at a conserved hinge residue of Cut14/SMC2, resulting in diminished DNA binding and annealing. A replication protein A mutant, ssb1-418, greatly alleviated the repair and mitotic defects of cut14-Y1. Ssb1 protein formed nucleolar foci in cut14-Y1 cells, but the number of foci was diminished in cut14-Y1 ssb1-418 double mutants. Consistent with the above results, Ssb1 protein bound to single-strand DNA was removed by condensin or the SMC dimer through DNA reannealing in vitro. Similarly, RNA hybridized to DNA may be removed by the SMC dimer. Thus, condensin may wind up DNA strands to unload chromosomal components after DNA repair and prior to mitosis. We show that 16 suppressor mutations of cut14-Y1 were all mapped within the hinge domain, which surrounded the original L543 mutation site.     

The mitochondrial nucleoid protein, Mgm101p, of Saccharomyces cerevisiae is involved in the maintenance of rho(+) and ori/rep-devoid petite genomes but is not required for hypersuppressive rho(-) mtDNA

The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional rho(+) genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a rho(+) strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have rho(-) genomes that are stably maintained. Interestingly, all surviving rho(-) mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS rho(-) mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of rho(+) and ori/rep-devoid rho(-) mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS rho(-) genome is stably maintained in a mgm101, rpo41 double mutant.     

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E. coli rho protein

Summary Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1–116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)₁₀ to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1–130 of rho protein (Rho130) is found to be stably folded, both in the absence and presence of nucleic acid. NMR studies of the complex of Rho 130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho 130 are similar to those of intact rho protein; therefore, Rho 130 is a suitable model of the RNA-binding domain of rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggest that Rho130 forms oligomers in the presence of longer oligonucleotides. ¹H, ¹⁵N and ¹³C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.Abbreviations Rho116, Rho130 protein containing the first 116 (130) residues of rho - CSD cold shock domain - RRM RNA recognition motif - RBD RNA-binding domain - IPTG isopropyl -D-thiogalactopyranoside - EDTA ethylenediaminetetraacetic acid - NOE nuclear Overhauser enhancement     

Complementing yeast rho1 mutation groups with distinct functional defects

Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-beta-glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-beta-glucan synthase activity.     

Identification of rho as a substrate for botulinum toxin C3-catalyzed ADP-ribosylation

Recombinant Aplysia rho and a GTP-binding protein purified from human neutrophil membranes (G22K) were ADP-ribosylated by botulinum toxin C3 with stoichiometries of 0.8 and 0.6, respectively. Rho and G22K appeared to be different proteins since (i) rho migrated faster on polyacrylamide gels, (ii) unlike G22K, rho did not require the presence of cytosol to be ADP-ribosylated, (iii) G22K was not recognized by an anti-rho antiserum, and (iv) antibody 142-24E05 recognized G22K effectively but only poorly cross reacted with rho. ADP-ribosylation had no effect on the ability of rho to bind or hydrolyse GTP. Therefore, it appears that there are multiple botulinum toxin C3 substrates and that the toxin exerts its effects on cell function by a mechanism other than modulating the GTPase activity of rho.     

Relative map location of the rep and rho genes of Escherichia coli.

The rep gene of Escherichia coli was mapped between ilvC and rho by three-factor P1 transductional crosses and also by complementation with a set of lambda transducing phages that contain known amounts of bacterial DNA linked to ilvC. The physical distance between ilvC and rep and between rep and rho were calculated with an accuracy of +/- 0.4 kilobase to be 0 less than or equal to ilvC-rep less than or equal to 3.4 kilobases and 2.0 less than or equal to rep-rho less than or equal to 6.0 kilobases. It was shown that rho-15 is Gro+ for phage ST-1. An ilv::Tn10 mutation was located in ilvY.     

Schizosaccharomyces pombe rho2p GTPase regulates cell wall alpha-glucan biosynthesis through the protein kinase pck2p

Schizosaccharomyces pombe rho1(+) and rho2(+) genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton. Although both GTPases interact with each of the two S. pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other. It is known that Rho1p regulates (1,3)beta-D-glucan synthesis both directly and through Pck2p. In this paper, we have investigated Rho2p signaling and show that pck2 delta and rho2 delta strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway. We also show that Rho2 GTPase regulates the synthesis of alpha-D-glucan, the other main structural polymer of the S. pombe cell wall, primarily through Pck2p. Although overexpression of rho2(+) in wild-type or pck1 delta cells is lethal and causes morphological alterations, actin depolarization, and an increase in alpha-D-glucan biosynthesis, all of these effects are suppressed in a pck2 delta strain. In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1-3)alpha-D-glucan synthase. Thus, a rho2 delta mutation, like pck2 delta, is synthetically lethal with mok1-664, and the mutant partially fails to localize Mok1p to the growing areas. Moreover, overexpression of mok1(+) in rho2 delta cells causes a lethal phenotype that is completely different from that of mok1(+) overexpression in wild-type cells, and the increase in alpha-glucan is considerably lower. Taken together, all of these results indicate the presence of a signaling pathway regulating alpha-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.     

Characterization of copy number mutants of plasmid pSC101

We have characterized three copy number mutants of the plasmid pSC101. These mutations caused single amino acid substitutions at the 46th, 83rd and 115th codons in the rep gene and an increase in the copy number by 4- to 8-fold. Although the in vivo and in vitro repressor activities of these mutated Rep proteins were quite different from each other, the intracellular concentrations of the proteins were maintained at higher levels than the wild-type protein. It has been reported that excess amounts of Rep inhibit pSC101 replication (Ingmer and Cohen, 1993). This inhibitory activity of Rep was markedly decreased in all three mutants. When both the wild-type and one of the mutated rep genes were retained in the same plasmids, the copy number of these plasmids was decreased compared with plasmids retaining a single mutated rep gene. These results support the theory that the inhibitory activity of Rep for its own replication plays an important role in copy number regulation.     

Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12

The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb. This plasmid does not fully complement a uvrA mutation in a Rec+ background. Plasmids which contain the uvrA and ssg genes, but not the lexA gene, show a higher but still only partial complementation. Full complementation achieved when the ssb gene us inactivated by insertion of Tn5. Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light. The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed.