Identification of a role for actin in translational fidelity in yeast.

Numerous studies have suggested a role for actin in translation, but the molecular details of this role are unknown. To elucidate the function(s) of actin in translation, we have studied 25 isogenic, conditional yeast actin mutants. Strikingly, analysis of these mutants indicates that none of those tested have conditional growth defects caused by reduced rates of protein synthesis; and analysis of latrunculin A-treated wild-type cells indicates that even complete disruption of the actin cytoskeleton has no significant effect on the rate of translation. However, analysis of the effect of the 25 actin mutations on fidelity and sensitivity to translation inhibitors identified two mutations ( act1-2 and act1-122) that cause a significant reduction in the fidelity of translation, as assayed by nonsense suppression, and several mutants that are sensitive to paromomycin, which affects translational fidelity. Translation elongation factor 1A (eEF1A) also has a role in fidelity, and in the presence of excess eEF1A four of the mutants ( act1-2, act1-20, act1-120, and act1-125) are even more sensitive to paromomycin, while one mutant ( act1-122) becomes less sensitive. Together, these findings suggest that actin may not be important for the rate of translation, but may have a critical role in ensuring translational fidelity.

Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast.

The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1B alpha, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

The dynein genes of Paramecium tetraurelia: the structure and expression of the ciliary beta and cytoplasmic heavy chains.

The genes encoding two Paramecium dynein heavy chains, DHC-6 and DHC-8, have been cloned and sequenced. Sequence-specific antibodies demonstrate that DHC-6 encodes ciliary outer arm beta-chain and DHC-8 encodes a cytoplasmic dynein heavy chain. Therefore, this study is the first opportunity to compare the primary structures and expression of two heavy chains representing the two functional classes of dynein expressed in the same cell. Deciliation of paramecia results in the accumulation of mRNA from DHC-6, but not DHC-8. Nuclear run-on transcription experiments demonstrate that this increase in the steady state concentration of DHC-6 mRNA is a consequence of a rapid induction of transcription in response to deciliation. This is the first demonstration that dynein, like other axonemal components, is transcriptionally regulated during reciliation. Analyses of the sequences of the two Paramecium dyneins and the dynein heavy chains from other organisms indicate that the heavy chain can be divided into three regions: 1) the sequence of the central catalytic domain is conserved among all dyneins; 2) the tail domain sequence, consisting of the N-terminal 1200 residues, differentiates between axonemal and cytoplasmic dyneins; and 3) the N-terminal 200 residues are the most divergent and appear to classify the isoforms. The organization of the heavy chain predicts that the variable tail domain may be sufficient to target the dynein to the appropriate place in the cell.

The dynein genes of Paramecium tetraurelia. Sequences adjacent to the catalytic P-loop identify cytoplasmic and axonemal heavy chain isoforms.

Paramecium tetraurelia is a unicellular organism that utilizes both axonemal and cytoplasmic dyneins. The highly conserved region containing the catalytic P-loop of the dynein heavy chain was amplified by RNA-directed polymerase chain reaction. Eight different P-loop-containing cDNA fragments were cloned. Southern hybridization analysis indicated that each fragment corresponds to a separate dynein gene and that there are at least 12 dynein heavy chain genes expressed in Paramecium. Seven of the eight cloned contain sequence motif A, which is found in axonemal dyneins, and one contains sequence motif B, which is found in the dyneins from cell types that do not have cilia or flagella. Two of the Paramecium dynein genes were further investigated: DHC-6 which contains motif A, and DHC-8 which contains motif B. Additional sequencing of the central portions of these genes showed that DHC-6 most closely matches sea urchin ciliary beta heavy chain and DHC-8 is similar to the cytoplasmic dynein from Dictyostelium. Deciliation of the cells resulted in a substantial increase in the steady state concentration of DHC-6 mRNA but only a small change in DHC-8 mRNA. Antisera were produced against synthetic peptides derived from sequence motifs A and B. Competitive solid-phase binding assays demonstrated that each antiserum was peptide-specific. In western blots, the antiserum to motif A reacted with both ciliary and cytoplasmic dyneins. In contrast, the antiserum to motif B reacted with the cytoplasmic dyneins of Paramecium and bovine brain but did not react with ciliary dynein.(ABSTRACT TRUNCATED AT 250 WORDS)