Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.     

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport.

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.     

New Mutants of Saccharomyces Cerevisiae Affected in the Transport of Proteins from the Endoplasmic Reticulum to the Golgi Complex

We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.     

Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli.

Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly.     

Temperature-sensitive mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 genes result in defects in mRNA turnover.

In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37 degrees. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p.     

Nucleophosmin serves as a rate-limiting nuclear export chaperone for the Mammalian ribosome

Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.     

Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export.

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.     

Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae

Using molecular genetic techniques, we have generated and characterized six temperature sensitive (ts) alleles of nop2. All failed to support growth at 37°C and one was also formamide sensitive (fs) and failed to grow on media containing 3% formamide. Conditional lethality is not due to rapid turnover of mutant Nop2p proteins at 37°C. Each allele contains between seven and 14 amino acid substitutions and one possesses a nonsense mutation near the C-terminus. Mapping experiments with one allele, nop2-4, revealed that a subset of the amino acid substitutions conferred the ts phenotype and that these mutations have an additive effect. All six mutants exhibited dramatic reductions in levels of 60S ribosome subunits under non-permissive conditions as well as some reduction at permissive temperature. Processing of 27S pre-rRNA to mature 25S rRNA was defective in all six mutants grown under non-permissive conditions. Levels of the 40S ribosomal subunit and 18S rRNA were not significantly affected. Amino acid substitutions in nop2 conditional alleles are discussed in the context of the hypothesis that Nop2p functions both as an RNA methyltransferase and a trans-acting factor in rRNA processing and large ribosomal subunit biogenesis.     

Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene

The translocation stage of protein synthesis is a highly conserved process in all cells. Although the components necessary for translocation have been delineated, the mechanism of this activity has not been well defined. Ribosome movement on template mRNA must allow for displacement of tRNA-mRNA complexes from the ribosomal A to P sites and P to E sites, while ensuring rigid maintenance of the correct reading frame. In Escherichia coli, translocation of the ribosome is promoted by elongation factor G (EF-G). To examine the role of EF-G and rRNA in translocation we have characterized mutations in rRNA genes that can suppress a temperature-sensitive (ts) allele of fusA, the gene in E. coli that encodes EF-G. This analysis was performed using the ts E. coli strain PEM100, which contains a point mutation within fusA. The ts phenotype of PEM100 can be suppressed by either of two mutations in the decoding region of the 16S rRNA when present in combination with a mutation at position 2058 in the peptidyltransferase domain of the 23S rRNA. Communication between these ribosomal domains is essential for coordinating the events of the elongation cycle. We propose a model in which EF-G promotes translocation by modulating this communication, thereby increasing the efficiency of this fundamental process.     

Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein

The nucleolar Saccharomyces cerevisiae protein Nep1 was previously shown to bind to a specific site of the 18S rRNA and to be involved in assembly of Rps19p into pre-40S ribosome subunits. Here we report on the identification of tma23 and nop6 mutations as recessive suppressors of a nep1(ts) mutant allele and the nep1 deletion as well. Green fluorescent protein fusions localized Tma23p and Nop6p within the nucleolus, indicating their function in ribosome biogenesis. The high lysine content of both proteins and an RNA binding motif in the Nop6p amino acid sequence suggest RNA-binding functions for both factors. Surprisingly, in contrast to Nep1p, Tma23p and Nop6p seem to be specific for fungi as no homologues could be found in higher eukaryotes. In contrast to most other ribosome biogenesis factors, Tma23p and Nop6p are nonessential in S. cerevisiae. Interestingly, the tma23 mutants showed a considerably increased resistance against the aminoglycoside G418, probably due to a structural change in the 40S ribosomal subunit, which could be the result of incorrectly folded 18S rRNA gene, missing rRNA modifications or the lack of a ribosomal protein.     

Structure and function of MRP20 and MRP49, the nuclear genes for two proteins of the 54 S subunit of the yeast mitochondrial ribosome.

MRP20 and MRP49 are proteins of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae. Their genes were identified through immunological screening of a genomic library in the expression vector lambda gt11. Nucleotide sequencing revealed that MRP49 is tightly linked to TPK3 and encodes a 16-kDa, basic protein with no significant relatedness to any other known protein. MRP20 specifies a 263-amino-acid polypeptide with sequence similarity to members of the L23 family of ribosomal proteins. The levels of the mRNAs and proteins for both MRP20 and MRP49 were regulated in response to carbon source. In [rho0] strains lacking mitochondrial rRNA, the levels of the two proteins were reduced severalfold, presumably because the unassembled proteins are unstable. Null mutants of MRP20 converted to [rho-] or [rho0], a characteristic phenotype of mutations in essential genes for mitochondrial translation. Inactivation of MRP49 caused a cold-sensitive respiration-deficient phenotype, indicating that MRP49 is not an essential ribosomal protein. The mrp49 mutants were defective in the assembly of stable 54 S ribosomal subunits at the nonpermissive temperature. With the results presented here, there are now published sequences for 14 yeast mitochondrial ribosomal proteins, only five of which bear discernable relationships to eubacterial ribosomal proteins.     

A temperature-sensitive mutation affecting the mammalian 60 S ribosome.

A temperature-sensitive Chinese hamster cell mutant, ts14, is unable to synthesize protein in tissue culture at 39 degrees. That mutant's protein biosynthetic machinery has been characterized in cell-free, biologically active extracts. Similar to the mutant's phenotype in tissue culture, ts14 extracts cease protein synthesis in vitro within 15 min at 40 degrees. In contrast, at 25 degrees both ts14 and wild type extracts synthesize protein for more than 2 hours. Fractionation of mutant extracts and complementation with comparable wild type preparations indicate that ts14 possesses a thermolabile component associated with its polyribosomes. In preparation of ts14 ribosomes that are free of mRNA and bound protein factors, the defective factor is complemented functionally only by 60 S ribosomal subunits prepared from the wild type parent. Sedimentation analyses in sucrose gradients demonstrate that ts14's mutation specifically affects stability of the mutant's 60 S ribosome. Treatment with high ionic strength buffers preferentially disrupts the mutant's 60 S ribosomal subunit and results in preparations of mutant ribosomes that contain biologically active 40 S subunits only. These studies demonstrate the applicability of a genetic approach to analyzing structure-function relationships in the eukaryotic ribosome.     

Functional link between ribosome formation and biogenesis of iron-sulfur proteins

In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in extramitochondrial iron-sulfur (Fe-S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe-S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe-S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre-rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre-40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe-S proteins.