Eur. J. Biochem. zyxwvutsrqpon 107, 173-183 (1980) zyxwvutsrq A Trichodermin-Resistant Mutant of Saccharomyces cerevisiae with an Abnormal Distribution of Native Ribosomal Subunits Christopher J. CARTER, Michael CANNON, and Anlonio JIMENEZ Department of Biochemistry, University of London King’s College, and Instituto de Bioquimica de MacromolCculas, Centro de Biologia Molecular, Universidad Autonoma de Madrid (Received November 7, 1979) 1. A yeast mutant (CLP-8), resistant at the ribosome level to the trichothecene antibiotic trichodermin, differs from its parent in having an unusual distribution of native ribosomal subunits. Sucrose gradient analysis of cytoplasmic extracts from this mutant revealed a large excess of material sedimenting at 60 S with little or no material sedimenting at 40 S. 2. The excess 60-S material consists predominantly of functionally active 60-S ribosomal subunits, as indicated by both analysis of ribosomal RNA and studies in vitro using a poly(U)- directed protein-synthesizing system. 3. Using the poly(U) system it was found that high-salt-washed particles derived from either the excess 60-S peak or 80-S ribosomes of CLP-8 exhibited very similar levels of resistance to the antibiotic fusarenon-X, a drug closely related chemically to trichodermin. The same level of resistance to fusarenon-X was also shown by high-salt-washed 60-S ribosomal particles obtained from a further trichodermin-resistant yeast strain (TR-l), although this strain has a normal distribution of native ribosomal subunits. In addition, both CLP-8 and TR-1 are equally resistant to inhibition of protein synthesis by trichothecene antibiotics, as assayed in vivo. 4. Genetic analysis of CLP-8 indicates that the trichodermin-resistant trait can be segregated from the lesion responsible for the inbalance of native ribosomal subunits. However, the latter defect is only expressed phenotypically in cells that retain the trichodermin-resistant character. 5. CLP-8 has a further defect in that both in vivo and in vitro it fails to generate native 40-S ribosomal subunits from 80-S particles. There may be a lesion in the protein factor normally required for this process. lsolation and analysis of antibiotic-resistant mu- tants from bacteria has greatly facilitated the study of ribosome structure and function in these organisms [l-31. The basis of resistance has often been de- termined [3 zyxwvutsrqp - 51 and this has provided insight into the molecular nature of certain drug receptor sites on ribosomes. Similar studies in eukaryotes have involved analysis, mainly, of antibiotic-resistant mutants of Scirchuvomyces cerevisiue some of which have ribo- somal lesions that have been genetically mapped [6- 81. However, there is little convincing evidence for the precise identification of a specifically-altered ribosomal component controlling antibiotic resis- tance in any of these mutants. Abbreviution. Poly(U), poly(uridy1ic acid). Drfl’nition. Native 60-S* subunits, material sedimenting at 60 S observed in or isolated from extracts of zyxwvutsrq Succhuromycrs zyxwvuts cerr- visiue (strain CLP-8). Enzyme. Creatine kinase (EC 2.7.3.2). Drug-resistant mutants are frequently cross re- sistant to different antibiotics that block (apparently) the same ribosomal function, and the ribosomal target site(s) for these antibiotics is presumed to be in- volved intimately with the function inhibited. For example, two independent mutants (TR-1 and CLP-1) of zyxwvu S. cerevisiae, isolated as spontaneously resistant to trichodermin, exhibit cross resistance in vitro to many other structurally-related trichothecene antibiotics with the resistance residing in each case in the 60-S ribosomal subunit [9 - 121. Both mutants also show cross resistance, both in vivo and in vitro, to other compounds, including anisomycin, narciclasine, har- ringtonin and bruceantin [12- 151, although these compounds are very different chemically from the trichothecene gi-oup. Ncvertheless all thc inhibitors appear to block selectively the activity of the pcptidyl- transferase centre located on 60-S subunits of eukaryotic ribosomes.