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A A Komar

Publications and source records attributed to A A Komar.

15 recordsLinked to original sources
PubMed2001

Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p.

The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999) J. Biol. Chem. 274, 13666-13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions. We show here that Ure2p dimerizes through its C-terminal region. We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils. A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein. This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils. Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies.

Amyloid↗
PubMed1999

Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation.

To investigate the possible influence of the local rates of translation on protein folding, 16 consecutive rare (in Escherichia coli) codons in the chloramphenicol acetyltransferase (CAT) gene have been replaced by frequent ones. Site-directed silent mutagenesis reduced the pauses in translation of CAT in E. coli S30 extract cell-free system and led to the acceleration of the overall rate of CAT protein synthesis. At the same time, the silently mutated protein (with unaltered protein sequence) synthesized in the E. coli S30 extract system was shown to possess 20% lower specific activity. The data suggest that kinetics of protein translation can affect the in vivo protein-folding pathway, leading to increased levels of protein misfolding.

Amino Acid Sequence↗
PubMed1999

Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2.

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.

Base Sequence↗
PubMed1999

Characterization of the interaction domains of Ure2p, a prion-like protein of yeast.

In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.

Catalytic Domain↗
PubMed1999

The [URE3] yeast prion: from genetics to biochemistry.

[URE3] is a non-Mendelian genetic element of the yeast Saccharomyces cerevisiae, an altered prion form of Ure2 protein. We show that recombinant Ure2p is a soluble protein that can assemble in vitro into dimers, tetramers, and octamers or form insoluble fibrils observed for PrP in its filamentous form or for Sup35p upon self-assembling, suggesting a similar mechanism for all prions. Computational, genetic, biochemical, and structural data allow us to specify a new boundary between the so-called prion-forming and nitrogen regulator (catalytic) domains of the protein and to map this boundary to Met-94. We bring strong evidence that the COOH-terminal (94-354) part of the protein forms a tightly folded domain, while the NH2-terminal (1-94) part is unstructured. These domains (or various parts of these domains) were shown (by means of the two-hybrid system approach and affinity binding experiments) to interact with each other (both in vivo and in vitro). We bring also evidence that the COOH-terminal (94-354) catalytically active part of the protein can be synthesized (both in vitro and in vivo) via an internal ribosome-binding mechanism, independently of the production of the full-length protein. We finally show that Ure2p aggregation in vivo (monitored by fluorescence of Ure2p--GFP fusion) does not necessarily give rise to [URE3] phenotype. The significance of these findings for the appearance and propagation of the yeast prion [URE3] is discussed.

Fungal Proteins↗
PubMed1998

Enhanced expression of the yeast Ure2 protein in Escherichia coli: the effect of synonymous codon substitutions at a selected place in the gene.

The expression of the yeast Ure2 protein and its two N- and C-terminal HA-(YPYPVDYA) epitope and His-tag fusions has been enhanced in E. coli by selected silent mutagenesis of the URE2 gene. The two Arg-AGA codons at positions 253 and 254 of the URE2 gene coding sequence were exchanged by CGT codons accordingly. This has allowed an increased yield (up to 100-fold) of the full-length protein synthesized. Western blotting with HA-epitope-specific antibodies using N- and C-terminal Ure2p-HA(epitope)-His-tag fusion constructs confirmed the integrity of the recombinant proteins. The N-(C-) terminal tagged proteins were shown to possess biological activity of the natural Ure2 protein.

Amino Acid Sequence↗
PubMed1997

Differential resistance to proteinase K digestion of the yeast prion-like (Ure2p) protein synthesized in vitro in wheat germ extract and rabbit reticulocyte lysate cell-free translation systems.

The Ure2p yeast prion-like protein was translated in vitro in the presence of labeled [35S]methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell-free systems. When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was proteolysed much more slowly compared to that synthesized in RRL; this displays fragments of about 31-34 kDa, persisting over 8 min. Thus, the digestion rate and pattern of the protein synthesized in WGE, unlike that synthesized in RRL, revealed characteristic features of the [URE3] prion-like isoform of the Ure2p protein [Masison, D.C. and Wickner, R.B. (1995) Science 270, 93-95]. Chloramphenicol acetyltransferase, synthesized under the same conditions, differed fundamentally in its proteolytic sensitivity toward proteinase K (PK); in the RRL system it was more slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems. Posttranslational addition of the WGE to the RRL-synthesized Ure2p does not protect Ure2p from efficient PK degradation either. The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesized in WGE; obviously, they yield a protein that mimics the behavior of the Ure2p in [URE3] yeast strains. The present data suggest that particular conditions of the Ure2p protein translation and/or certain cellular components (accessory proteins and extrinsic factors), as well as the nature of the translation process itself, could affect the intracellular folding pathway of Ure2p leading to the de novo formation of the prion [URE3] isoform.

Animals↗
PubMed1997

Cotranslational folding of globin.

Globin synthesis in a wheat germ cell-free translation system was performed in the presence of [3H]hemin and [35S]methionine to determine the minimal length of the nascent ribosome-bound globin chain capable of heme binding. Nascent polypeptides of predetermined size were synthesized on ribosomes by translation of truncated mRNA molecules. Analysis with the use of sucrose gradient centrifugation and puromycin reaction revealed that the ribosome-bound N-terminal alpha-globin fragments of 140, 100, and 86 amino acid residues are capable of an efficient heme binding, whereas those of 75, 65, and 34 amino acid residues display a significantly weaker, or just nonspecific, affinity to heme. This indicates that the ribosome-bound nascent chain of 86 amino acid residues has already acquired a spatial structure that allows its interaction with the heme group or that heme attachment promotes the formation of the proper tertiary structure in the ribosome-bound nascent peptide. In any case the cotranslational folding of globin is suggested.

Animals↗
PubMed1995

Kinetics of translation of gamma B crystallin and its circularly permutated variant in an in vitro cell-free system: possible relations to codon distribution and protein folding.

Analysis of nascent gamma B-crystallin peptides accumulating during in vitro translation in a rabbit reticulocyte lysate cell-free system was carried out. As a consequence of the irregular distribution of rare codons along the polypeptide chain of gamma B-crystallin, translation of the two-domain protein is a non-uniform process characterized by specific pauses. One of the major delays occurs during the translation of the connecting peptide between the domains. Comparing the kinetics of translation of natural gamma B-crystallin and its circularly permutated variant (with the order of the N- and C-terminal domains exchanged) reveals that the natural N-terminal domain is translated faster than the C-terminal one. Since the N-terminal domain in natural gamma B-crystallin is known to be more stable and to fold faster than the C-terminal one [E.-M. Mayr et al. (1994) J. Mol. Biol. 235, 84-88], the present data suggest that the translation rates are optimized to tune the synthesis and folding of the nascent polypeptide chain. In this connection, the pause in the linker region between the domains provides a delay allowing the correct folding of the N-terminal domain and its subsequent assistance in the stabilization of the C-terminal one.

Animals↗
PubMed1993

Cotranslational heme binding to nascent globin chains.

Globin synthesis in cell-free extracts of rabbit reticulocytes was carried out in the presence of 3H-labeled hemin. Sucrose gradient centrifugation analysis revealed [3H]hemin in the polyribosome fraction. The addition of puromycin resulted in the release of both [3H]hemin- and [14C]leucine-labeled polypeptide from the polyribosomes. The data suggest cotranslational folding of the globin molecule on the ribosome and cotranslational heme binding to the nascent globin chain.

Animals↗
PubMed1991

Nonuniform size distribution of nascent globin peptides, evidence for pause localization sites, and a contranslational protein-folding model.

Examination of nascent globin peptides accumulating in vitro during globin synthesis in rabbit reticulocyte lysates was carried out. A view was supported that nonrandom distribution of codons with different usage frequencies in mRNA may determine the messenger's translation kinetics. Regions of reduced translation of alpha- and beta-globin polypeptide chains were localized, and the cotranslational protein-folding model suggested previously was substantiated. An active conjunction of synthesis and folding of proteins was proposed as one of the main destinations of a translation nonuniformity.

Animals↗
PubMed1989

[Role of the code redundancy in determining cotranslational protein folding].

It has been demonstrated earlier in our laboratory that rare codon clusters can determine the boundaries of the polypeptide chain fragments of the same secondary structure type during the co-translational protein folding. According to this data, co-translational protein folding can occur under condition of a correlation between the frequency of codon choice in mRNAs and the relative abundance of their isoaccepting tRNAs. The alterations in the spectrum and concentrations of the isoaccepting tRNAs in different cells were demonstrated by many authors. The existence of a mechanism of the coordinate regulation of the levels (activities) of the isoaccepting tRNAs, corresponding aminoacyl-tRNA synthetases and mRNAs predominantly translated at a given moment of time can be suggested. Such a mechanism can ensure the needed accuracy of the protein folding process. Analysis of gene sequences of various pro- and eukaryotic organisms carried out in the present work revealed that the codon usage frequency spectra of simultaneously synthesized proteins are similar. The relative appearance of the most rare and frequent codons in investigated gene sequences displays a high degree of conservatism. It has also been found that structural-homologous proteins from different organisms (cytochromes c, myoglobins) have very similar codon frequency distribution profiles. This property retains despite the significant variations in the codon usage spectra in the investigated gene sequences. The data obtained indicate that the codon distribution in mRNAs whose diversity is mainly conditioned by the genetic code redundance is a program that determines translational rates of different mRNA parts thus controlling the spatial folding of the synthesized peptide chain.

Animals↗