The mechanism by which the aminoglycoside antibiotic streptomycin permeabilizes the cytoplasmic membrane of Escherichiu coli cells was reinvestigated. For this purpose, the extent of streptomycin-induced K+ loss from cells growing at low external K+ concentrations was taken as a measure of membrane permeabilization. Experiments with different K+-uptake mutants showed that the antibiotic specifically increased the passive permeability of the cell membrane to K+ and other ions. These permeability changes were small and the membrane potential of the treated cells remained high. The membrane permeabilization was not due to a direct interaction of the antibiotic with the cell membrane, since cells that carry an rpsL mutation and synthesize proteins in a streptomycininsensitive way did not lose K+ after the addition of the antibiotic. Due to misreading and premature termination of translation the cells synthesized aberrant proteins under the conditions where membrane permeabilization occurred. Two conditions are described under which the cells both degraded these mistranslated proteins rapidly and reaccumulated K+, lending support to the hypothesis that membrane permeabilization is due to the presence of the mistranslated proteins in the cell membrane. Evidence is presented that the irreversibility of (dihydro)streptomycin uptake by cells washed free from the antibiotic might also be due to rapid degradation of the mistranslated proteins, leading to 'caging' of the antibiotic inside the cells.

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All eukaryotes investigated so far contain multiple copies of ubiquitin genes, most of which are arranged in fusions coding for either polyubiquitin or ubiquitin-ribosomal protein constructs; the former are normally under the control of a heat shock promoter. Giardia Zamblia, an intestinal parasite, is the most primitive eukaryote known to date We have investigated the arrangement and expression of ubiquitin genes in this organism by Southern and Northern blotting. Our data strongly suggest that G. Iamblia contains just one ubiquitin gene, which consists of a single copy of the coding sequence and the expression of which is not enhanced by heat shock. By pulsed-field gel electrophoresis we localized this gene on the largest of the five giardial chromosomes. These data imply that the ubiquitin system in Giardia has probably been trapped at an original stage.

The antino acid tmquencc of ubiquilin from ~tmm~elm histMyticu, as dedu~d from a eDNA nucl¢otid¢ Paluen~. deviated at six positions from the consensus of all other known ubiquitins (run,rig from To '/umosm;u, ¢ru:i to Hmm, x#pienx). The corresponding re,..~idaes were r,e.~ttcre..d over the primary ~quence. but came ¢lorm together on the surfa= of tl~e folded prot~'in structure. We conclude that (i) E. hixtd.vtiea bnumhed olT wry early from the main eakaryoti¢ line, and (ii) thi:¢ or~'mism may yield clues as to the evolutionary development of the ubiquitin sy=t¢m. Against this background we thought it interesting to investigate the amebal ubiquitin, Ubiquitin is a 76. amino acid protein found in all eukaryotic cells where people have looked for it; it has not been found in any prokaryote yet, The amino acid sequence of ubiquitin has been highly conserved throughout evolution, which may be a necessary consequence of its astonishing multifunctionality. The best.characterized biological role of ubiquitin is that of a covalently bound recognition signal for non-lysosomal proteolysis; ubiquitin is also found in linear ubiquitin-prot¢in fusions. Putative functions of ubiquitination include DNA repair, ¢¢11 cycle control, stress response and ribosome biogenesis. In a recent review [3] it has been estimated that over forty Below we present the coding sequence for ubiquitin derived from an amebal eDNA done. The amino acid sequence deduced from this nucleotide sequence devi. ated substantially from that of all other ubiquitins ann. lyzed so far-in particular, six of the variant positions were unique for the E, histolytica ubiquitin. We discuss the implications of this finding for the evolutionary history of' both E, hl#tolytica and the ubiquitin system. EXPERIMENTAL I. Cell~E, hixtMj,rir# HM hIMSS trophozoites were 8town ax=nically at 36*C in TY1-$.33 medium [5] with 15% serum, supplemgnted with penicillin (lO0/Jgml "~) and streptomycin .~ulfate (i00 mg.ml'~), The ameba= were harvested in lat¢-IoiPtrithmi¢ growth by chilling on ice and a 10.rain ccnzrifuBation at 400 x g, and washed twice in phos. phate-buffered laiia=. "E~ridmwnl of ubiqultia io amebal protoitJ extractThe ~lis were suspended in phozphatc-bu•wd saline with iodoacctamid¢ (2 raM) to suppress amebal protear¢ activity [6]. and disrupted with a Branson sonifyer (25 puir,¢,~ of 0.5 s at 40.-50 W), The pro~dur¢ for the enrichment of ubiquilin followed that describ¢d in [7] up to and including the ammonium sulfate fractionations. The ubiquitin.comaining material pr~ipitatcd by 80% (w/v) ammonium sulfate was diraoived in 50 mM Tris.HCl, pH 7,4, and subjected to SOS-PAGE and immunoblot analysis, SDS.PAGE and hmmmoblot analj,aisProteins were separated by SDS-PAG E ( 16% acwlamide. 6 M urea) a~ordin= to ~cMngor and yon Ja~ow [8]. and transferred to nitrootllulose or hnmohilon.P membrane by ¢lcciroblotting. Ubiquitia was detected with polyclonal antibodies ag:tin~t SDS-denatured bovine 54Puh/ished hy Etaevter Science Puhlixher$ B, V,

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