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The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15-42 represent an island of "normal" random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and atomic force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. Atomic force microscopy height images at successive time points during a single growth experiment showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphology (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h ‫؍‬ 2.6 ؎ 0.5 nm) and granular particles corresponding to higher order oligomers (h ‫؍‬ 4 -12 nm) could also be detected. The mutants 15Ure2 and ⌬15-42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addition, ⌬15-42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates.

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2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F. ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the I8 subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed 13-subunit Tyr-368 to be present at noncatalytic sites and /8 Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.The ATP synthase FtFl, found in mitochondria, chloroplasts, and bacteria, couples proton translocation to ATP synthesis during oxidative phosphorylation and photophosphorylation. The F1 component can be readily solubilized and has a a3f33y&e subunit structure. F1 contains the catalytic sites for ATP synthesis, but when detached from the membrane it is only capable of net ATP hydrolysis (1, 2).Of the six nucleotide binding sites present on the beef heart mitochondrial enzyme, MF1, only three sites exchange bound nucleotide rapidly during hydrolysis of MgATP at pH 8.0 (3). Evidence has been presented that at least two (4-6) and probably all three (7-12) of the exchangeable sites participate sequentially in catalysis at high substrate concentration. The three nonexchangeable sites, referred to here as noncatalytic sites, have been suggested to play a structural or regulatory role (for example, see refs. 13 and 14).A valuable approach to the characterization of nucleotide binding sites on F1 has been the use of affinity and photoaffinity analogs (15). Perhaps the most promising probes are the 2-azidoadenine nucleotides (11,16,17). Their anti conformation resembles that of F1-bound ADP and ATP; thus, they bind very tightly and serve as good substrates (18). Procedures described by Kironde and Cross (19) Garin et al. (22). We also report here the effects of derivatization on catalytic activity. MATERIALS AND METHODSPreparation of MF1. MF1 was isolated from beef heart mitochondria as described (23) Photoaffinity Labeling. To label a noncatalytic nucleotide site, in a typical experiment 60 ,ul of 14.5 mM 2-azido-[,B,y-32P]ATP was added with rapid mixing to 450 ul of 75 ,uM MF1[2,1] and incubated for 15 min. Unbound ligand was removed on a 3-ml centrifuge column, and MgATP was added to a final concentration of 15 mM to displace 2-azido-ANP from catalytic sites. After 1 min, the enzyme was passed through a second 3-ml centrifuge column and diluted to 2 ml. Four 500-,ul aliquots were placed on a shallow plastic tray 8 mm from a Minerallight lamp and photolyzed at the short wavelength setting for 30 min.To 5715The publication costs of this article were defrayed in part by page charge payment. This article must therefore be here...

To investigate genetic mechanisms of high altitude adaptations of native mammals on the Tibetan Plateau, we compared mitochondrial sequences of the endangered Pantholops hodgsonii with its lowland distant relatives Ovis aries and Capra hircus, as well as other mammals. The complete mitochondrial genome of P. hodgsonii (16,498 bp) revealed a similar gene order as of other mammals. Because of tandem duplications, the control region of P. hodgsonii mitochondrial genome is shorter than those of O. aries and C. hircus, but longer than those of Bos species. Phylogenetic analysis based on alignments of the entire cytochrome b genes suggested that P. hodgsonii is more closely related to O. aries and C. hircus, rather than to species of the Antilopinae subfamily. The estimated divergence time between P. hodgsonii and O. aries is about 2.25 million years ago. Further analysis on natural selection indicated that the COXI (cytochrome c oxidase subunit I) gene was under positive selection in P. hodgsonii and Bos grunniens. Considering the same climates and environments shared by these two mammalian species, we proposed that the mitochondrial COXI gene is probably relevant for these native mammals to adapt the high altitude environment unique to the Tibetan Plateau.

Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single wobble base pair, G3-U70, of tRNA Ala as a major determinant. However, this base pair is divergent in human mitochondrial tRNA Ala , but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNA Ala , in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNA Ala in a G3-U70-independent manner, requiring several elements in the acceptor stem. In addition, we found that hmtAlaRS misactivates noncognate Gly and catalyzes strong transfer RNA (tRNA)-independent pre-transfer editing for Gly. A completely conserved residue outside of the editing active site, Arg 663 , likely functions as a tRNA translocation determinant to facilitate tRNA entry into the editing domain during editing. Finally, we investigated the effects of the severe infantile-onset cardiomyopathy-associated R592W mutation of hmtAlaRS on the canonical enzymatic activities of hmtAlaRS. Overall, our results provide fundamental information about tRNA recognition and deepen our understanding of translational quality control mechanisms by hmtAlaRS.

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