Dynamin is a 100-kDa GTPase that is believed to be involved in the constriction of clathrin-coated pits and the fission of clathrin-coated vesicles during receptormediated endocytosis and during membrane retrieval in nerve termini. It has been shown that purified dynamin incubated under low salt conditions forms rings and spirals that, in dimension and appearance, resemble the dense material occasionally observed at the necks of coated pits. In this report we show that purified dynamin forms spirals under physiological salt conditions when incubated with GDP and ␥-phosphate analogues (beryllium and aluminum fluoride) or when dialyzed into guanosine 5 -3-O-(thio)triphosphate. Moreover, spirals still form when dynamin is proteolyzed to either a predominant ϳ90-kDa species, lacking the C terminus, or to two smaller fragments, a ϳ55-kDa species originating from the N-terminal half of the protein and a ϳ30-kDa species lacking both the N and C termini. This work indicates that the addition of GDP and ␥-phosphate analogues arrests dynamin in a GTP or transition state that markedly stabilizes the spiral conformation under physiological ionic strength conditions and thereby suggests that dynamin in the absence of a receptor is capable of assembly into spirals at the necks of coated pits prior to vesicle fission.

Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibioticresistant mutants of the extremely thermophilic bacterium Thermus thermophilus, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in T. thermophilus 16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that T. thermophilus may be an ideal model system for the study of ribosome structure and function.Members of the bacterial genus Thermus are extreme thermophiles first described by Brock and Freeze in 1969 (2) and have since been found in terrestrial and marine thermal environments throughout the world (60). Together with Deinococcus, Meiothermus, Marinithermus, Oceanithermus, and Vulcanithermus, they form a deeply branching phylum now known to be monophyletic (23,59). The close affiliation between Thermus and Deinococcus has been confirmed by complete genome sequences of Thermus thermophilus (30) and Deinococcus radiodurans (59). The finding of slightly thermophilic species related to Deinococcus, together with the thermophilic nature of the other genera of this phylum, suggests that thermophily is a primitive character of this clade (59).Recent advances in structural biology have produced a vast reservoir of high-resolution structural information regarding components of the protein synthetic machinery from members of the Deinococcus-Thermus phylum, including high-resolution crystal structures of the T. thermophilus 30S subunit (47, 61), medium-resolution structures of the entire T. thermophilus 70S ribosome (65), and high-resolution structures of the D. radiodurans 50S subunit (26). The value of these structures is magnified by their interpretation in light of several decades of genetics and biochemistry using ribosomes from mesophiles such as Escherichia coli. However, the ability to make such interpretations is potentially compromised by the absence of genetic and biochemical data obtained for ribosomes from members of the Deinococcus-Thermus phylum. It is assumed that nucleotide sequence c...

We characterized endocytosis of iron-saturated (holo) and iron-depleted (apo) 125I-labeled bovine lactoferrin (Lf) by isolated rat hepatocytes. Hepatocytes ingested both Lf forms--determined by EGTA/dextran sulfate removal of surface-bound Lf--at maximal endocytic rates of 1.85 and 1.52 fmol cell-1 min-1 for 125I-apo-Lf and 125I-holo-Lf, respectively. First-order endocytic rate constants (37 degrees C) for 125I-apo-Lf and 125I-holo-Lf were 0.276 and 0.292 min-1, respectively. Regardless of Lf's iron content, hyperosmotic media (approximately 500 mmol/kg) inhibited Lf uptake by approximately 90%, indicating endocytosis of both Lf forms was primarily clathrin-dependent. Endocytosis of both Lf forms was not altered significantly in the presence of excess iron chelator desferrioxamine or rat holo-transferrin, or by cycloheximide treatment. Fluorescein isothiocyanate- and cyclohexanedione-modified Lf competed fully with native Lf for binding and endocytosis, indicating that, unlike human Lf, modification of lysine or arginine residues does not block the interaction of bovine Lf with cells. After binding Lf at 4 degrees C, cells at 37 degrees C internalized approximately 90% of Lf bound to Ca(2+)-dependent sites but not Lf bound to Ca(2+)-independent sites. Following uptake, hepatocytes released acid-soluble (degraded) products of 125I-Lf biphasically at 37 degrees C, an initial rapid phase within the first 20 min--more pronounced with 125I-holo-Lf--followed by a sustained linear release of 298 and 355 molecule equiv cell-1 min-1 for 125I-apo-Lf and 125I-holo-Lf, respectively. At 4 degrees C, both digitonin-permeabilized and intact cells bound approximately 1.1 x 10(6) 125I-Lf molecules to Ca(2+)-dependent sites per cell, indicating that hepatocytes do not contain a sizeable intracellular pool of these sites. Moreover, cells retained > 70% of Ca(2+)-dependent sites on the surface during sustained Lf endocytosis. Thus, these Lf binding sites recycle during endocytosis at an estimated 4-5 min/circuit.

Codon recognition by aminoacyl-tRNA on the ribosome triggers a process leading to GTP hydrolysis by elongation factor Tu (EF-Tu) and release of aminoacyl-tRNA into the A site of the ribosome. The nature of this signal is largely unknown. Here, we present genetic evidence that a specific set of direct interactions between ribosomal protein S12 and aminoacyl-tRNA, together with contacts between S12 and 16S rRNA, provide a pathway for the signaling of codon recognition to EF-Tu. Three novel amino acid substitutions, H76R, R37C, and K53E in Thermus thermophilus ribosomal protein S12, confer resistance to streptomycin. The streptomycin-resistance phenotypes of H76R, R37C, and K53E are all abolished by the mutation A375T in EF-Tu. A375T confers resistance to kirromycin, an antibiotic freezing EF-Tu in a GTPase activated state. H76 contacts aminoacyl-tRNA in ternary complex with EF-Tu and GTP, while R37 and K53 are involved in the conformational transition of the 30S subunit occurring upon codon recognition. We propose that codon recognition and domain closure of the 30S subunit are signaled through aminoacyl-tRNA to EF-Tu via these S12 residues.

Dysmorphic pulmonary vascular growth and abnormal endothelial cell (EC) proliferation are paradoxically observed in premature infants with bronchopulmonary dysplasia (BPD) despite vascular pruning. The pentose phosphate pathway (PPP), a metabolic pathway parallel to glycolysis, generates NADPH as a reducing equivalent and ribose 5-phosphate for nucleotide synthesis. It is unknown whether hyperoxia, a known mediator of BPD in rodent models, alters glycolysis and the PPP in lung ECs. We hypothesized that hyperoxia increases glycolysis and the PPP, resulting in abnormal EC proliferation and dysmorphic angiogenesis in neonatal mice. To test this hypothesis, lung ECs and newborn mice were exposed to hyperoxia and allowed to recover in air. Hyperoxia increased glycolysis and the PPP. Increased PPP, but not glycolysis, caused hyperoxia-induced abnormal EC proliferation. Blocking the PPP reduced hyperoxia-induced glucose-derived deoxynucleotide synthesis in cultured ECs. In neonatal mice, hyperoxia-induced abnormal EC proliferation, dysmorphic angiogenesis, and alveolar simplification were augmented by nanoparticle-mediated endothelial overexpression of phosphogluconate dehydrogenase, the second enzyme in the PPP. These effects were attenuated by inhibitors of the PPP. Altogether, neonatal hyperoxia augments the PPP, causing abnormal lung EC proliferation, dysmorphic vascular development, and alveolar simplification. These novel observations provide mechanisms and potential metabolic targets to prevent BPD-associated vascular dysgenesis.

The structural basis for the streptomycin dependence phenotype of ribosomal protein S12 mutants is poorly understood. Here we describe the application of site-directed mutagenesis and gene replacement of Thermus thermophilus rpsL to assess the importance of side chain identity and tertiary interactions as phenotypic determinants of drug-dependent mutants.Ribosomal protein S12 is a highly conserved protein located at the functional center of the 30S subunit of the ribosome. A direct role for S12 in the tRNA selection process has been extracted from high-resolution X-ray crystal structures of the 30S ribosomal subunit of the extremely thermophilic bacterium Thermus thermophilus (4,5,13,15). Direct contacts between S12 and several important structural elements of 16S rRNA are alternately made and broken during the tRNA selection process (13), and mutations in S12 known to influence the accuracy of decoding are predicted to do so by affecting the stability of these interactions.

The ribosome decodes mRNA by monitoring the geometry of codon-anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EFTu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.

Ribosomal protein S12 contains a highly conserved aspartic acid residue that is posttranslationally ␤-methylthiolated. Using mass spectrometry, we have determined the modification states of several S12 mutants of Thermus thermophilus and conclude that ␤-methylthiolation is not a determinant of the streptomycin phenotype.Streptomycin and streptomycin-resistant mutants have played a seminal role in the elucidation of the decoding process of protein synthesis (reviewed in reference 19). Genetic and biochemical analyses of ribosomes from streptomycin-resistant mutants implicated ribosomal protein S12 as the determinant of the various streptomycin phenotypes, including resistance, dependence, and pseudodependence (reviewed in references 11 and 17). Such mutations have been localized in the three-dimensional structure of the Thermus thermophilus 30S ribosomal subunit to reside within two highly conserved loops centered around residues P90 and K42 (Escherichia coli numbering used throughout) ( Fig. 1) (7).It has only recently been discovered by use of mass spectrometry that E. coli ribosomal protein S12 is posttranslationally modified via a ␤-methylthiolation at position D88 ( Fig. 2A), near the streptomycin binding site and in the midst of residues altered in streptomycin-resistant mutants (14). This modification has also been found to occur in the phototrophic bacterium Rhodopseudomonas palustris (22), and we have identified it for the extremely thermophilic bacterium T. thermophilus (24). Posttranscriptional modifications of rRNA residues have been shown to affect resistance to various antibiotic classes (reviewed in reference 8). For example, ksgA mutants are resistant to kasugamycin due to the loss of N6-dimethylation of two conserved adenosines in 16S rRNA (13), while methylation of rRNA in the peptidyltransferase center (21) or in the decoding region (3) confers resistance to erythromycin and aminoglycosides, respectively (reviewed in references 20 and 26).Interestingly, loss of modification of a ribosomal protein has not yet been shown to affect antibiotic sensitivity. Nevertheless, the proximity of S12 residue D88 to residues altered in streptomycin-resistant mutants raises the possibility that such mutations might confer resistance indirectly by inhibiting ␤-methylthiolation of D88. To address this question, we performed matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to establish the modification status of ribosomal protein S12 in a series of streptomycinresistant and streptomycin-dependent mutants of T. thermophilus.Loss of ␤-methylthiolation in a subset of S12 mutants. We examined the modification states of several S12 mutants of T. thermophilus IB-21 (ATCC 43815) (16) by MALDI-TOF MS as described previously (23). Wild-type S12 was determined to have a mass of 14,519 Ϯ 6 Da (Table 1), consistent with our previous report (24), indicating loss of the initial methionine and the presence of the ␤-methylthiolation. Considering the proximity to D88, we sought to deter...