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

Abstract

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 heatsensitive 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 rpll6b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rp116b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rp1l6b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.Most eukaryotic ribosomes are assembled from approximately 75 different ribosomal proteins (r-proteins) and 4 rRNA molecules in a process involving multiple cellular compartments. Newly synthesized r-proteins make their way from the cytoplasm to the nucleolus by a largely unknown mechanism and there associate with nascent rRNA. By analogy to Escherichia coli, the different r-proteins probably associate with assembling ribosomes in a strict order (reviewed in reference 60). Nearly all of the r-proteins assemble into subunits in the nucleolus. The ribosomes then are exported to the cytoplasm, where a few late-assembling proteins join them (72). The ribosomes then can assume their role as the protein-synthesizing machinery of the cell (reviewed in reference 70).Very little is known about how eukaryotic ribosomes assemble, although maintaining the stoichiometry of ribosomal components appears to be important. In yeasts, r-proteins that are synthesized in excess of their counterparts are rapidly degraded, presumably in the absence of their ability to assemble into ribosomes (14,39,65,71). Similarly, if the synthesis of a single r-protein or rRNA is terminated, most other proteins and RNAs of the same subunit of which that molecule is a part are synthesized and then degraded (1,15,41,42,68,73). In these cases, turnover of ribosomal constituents may result upon disassembly of incorrectly or incompletely assembled subunits.Temperature-sensitive mutants defective for ribosome assembly have also been isolated. In particular, cold-sensitive mutants have been a rich source of assembly mutants in bacteria, probably because ribosome assembly has a high activation energy (6,21,44,46,62). Heat-sensitive assembly mutants have been isolated as well (10,40,43,49,55

DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.

“…The TEF3 gene is identical to the CAMI gene, recently cloned by Kambouris et al (23). Tef3p (Camlp) was found to be tightly associated with a polypeptide of 34 kDa, which is identical to the published molecular mass for S. cerevisiae elongation factor EF-13 (11). There anism to direct components of the translational apparatus toward membranous components of the cell has been postulated since the discovery that many secretory proteins are cotranslationally translocated across the endoplasmic reticulum (39).…”

“…S. cerevisiae mutants blocked in rRNA processing or unable to synthesize rRNAs or r-proteins have been obtained. Inactivation or depletion of individual r-proteins or rRNAs blocks proper assembly of the subunit of which that rRNA or protein is a part and leads to rapid degradation of the other rRNAs and proteins of that subunit (1,33,34,37,64). Heat-and cold-sensitive mutants that have ribosomal subunit deficits have been identified by monitoring changes in the steady-state levels of ribosomal subunits, although in most cases the corresponding genes have not been cloned (3,7,17,54,56).…”

Ribosome Concentration Contributes to Discrimination against Poly(A)− mRNA during Translation Initiation in Saccharomyces cerevisiae

“…Therefore, we constructed a second pap1-1 derivative by deleting RPL16B, one of the duplicated genes encoding Rpl16p (38). Deletion of only one of two copies of RPL16 allowed us to lower the 60 S subunit levels while producing ribosomes with a normal complement of ribosomal proteins (33,36,39).…”

Global Screening of Genes Essential for Growth in High-Pressure and Cold Environments: Searching for Basic Adaptive Strategies Using a Yeast Deletion Library

“…It is well-known that bacterial and yeast Saccharomyces cerevisiae mutants defective in ribosome assembly are cold sensitive, probably because ribosome assembly has high activation energy in the absence of certain subunits (Friedman et al 1969;Bryant and Sypherd 1974;Singh et al 1978;Ursic and Davies 1979). Some yeast mutants that have altered sensitivities to the antibiotics trichodermin and cycloheximide are also cold sensitive (Moritz et al 1991;Dresios et al 2000Dresios et al , 2001Dresios et al , 2003. A substitution of aspartic acid for leucine in actin at position 266 confers cold sensitivity at temperatures between 9°and 15°due to polymerization defects of actin at low temperatures (Chen et al 1993).…”