Making the match and breaking it: values, perceptions, and obstacles of trainees applying into physician-scientist training programs.

BACKGROUND: Replenishing the physician-scientist workforce constitutes a central mission of medical education, but the loss of qualified trainees to non-academic positions remains an ongoing threat. Among the barriers facing physician-scientists today is the game-like model of U.S. medical residency matching through the National Research Matching Program (NRPM), which applies several assumptions regarding the comparability of applicant qualifications, cohort size, and the institutional breadth of applicants' training needs. METHODS: The current report therefore summarizes the survey-based views and experiences of physician-scientist trainees obtained following the 2021-2022 application cycle for research-oriented residency programs, or physician-scientist training programs (PSTPs). From among this small cohort of applicants, we obtained survey-based feedback of 27 PSTP applicants across 17 U.S. medical universities, among whom 85% (23/27) matched into a PSTP. RESULTS: Among these PSTP applicants, 25/27 (93%) recognized "scientific community" as the most important feature of a postgraduate training program, with applicants identifying as female placing a higher value on the program's infrastructure of personal and/or family support. Most (18/27) respondents found "waiting for interviews" as the most stressful phase of their application cycle, and roughly half of all respondents encountered at least one NRMP policy violation through post-interview communication. Specifically, 93% (25/27) respondents were contacted by at least one PSTP following interviews, and 1/3 of them admitted to feeling pressured into sharing their ranking preferences. CONCLUSION: We highlight many previously unrecognized priorities among applicants to PSTPs, which include fostering community among its trainees and reinforcing structured mentoring. We uncover an inconsistency among PSTPs regarding the post-interview process, which represents an opportunity to better support applicants seeking to gauge programs according to their clinical, scientific, and academic interests as physician-scientists, while still adhering to NRMP policies.

Purification and properties of cysteinyl-tRNA synthetase from rabbit liver.

Cysteinyl-tRNA synthetase (CRS) from rabbit liver was purified 8300-fold to a constant specific activity. SDS-PAGE revealed the presence of two polypeptides of 86 kDa and 92 kDa, in the proportions of 60% and 40% respectively. The SDS-electrophoretic migration of the major 86 kDa component was indistinguishable from that of the single polypeptide previously found in CRS from S. cerevisiae. The two polypeptides from rabbit CRS were inaccessible to Edman degradation, but internal peptides generated from each by in-gel proteolysis after SDS-electrophoretic separation, yielded sequences found in the deduced protein sequence of human CRS. Moreover, subjecting the two polypeptides separated by SDS-PAGE to a renaturation treatment showed that CRS activity was associated with both. The structure of the native enzyme was probed by limited proteolysis with elastase. The strikingly simple degradation pattern observed supported a model according to which the two polypeptides derive from the same gene, differing only by a approximately 6 kDa extension located at the C-terminal extremity of the 92 kDa component. Moreover, the finding that notwithstanding the presence of the two polypeptides, the behaviour of rabbit CRS upon gel-filtration or chemical cross-linking was indistinguishable from that of homodimeric yeast CRS, indicated that the 6 kDa C-terminal extension on the 92 kDa polypeptide does not impede dimerisation. The origin of the two components of rabbit CRS is discussed in light of the deduced protein sequence of human CRS derived from the published cDNA sequence and the recently released genomic sequence of the human enzyme.

Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae. Purification, characterization and assignment to the genomic sequence YNL247w.

Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to a high specific activity (kcat4.3 s-1 at 25 degrees C for the aminoacylation of yeast tRNACys). SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa. Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa in a yeast extract subjected to SDS-PAGE followed by immunoblotting. In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking. Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w. The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources. Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment. Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.

Expression of rat aspartyl-tRNA synthetase in Saccharomyces cerevisiae. Role of the NH2-terminal polypeptide extension on enzyme activity and stability.

Cytoplasmic aspartyl-tRNA synthetase from mammals is one of the components of a multienzyme complex comprising nine synthetase activities. The presence of an amino-terminal extension composed of about 40 residues is a characteristic of the eukaryotic enzyme. We report here the expression in the yeast Saccharomyces cerevisiae of a native form of rat aspartyl-tRNA synthetase and of two truncated derivatives lacking 20 or 36 amino acid residues from their amino-terminal polypeptide extension. The three recombinant enzyme species were purified to homogeneity. They behave as alpha2 dimers and display catalytic parameters in the tRNA aminoacylation reaction identical to those determined for the native, complex-associated form of aspartyl-tRNA synthetase isolated from rat liver. Because the dimer dissociation constant of rat AspRS is much higher than that of its bacterial and yeast counterparts, we could establish a direct correlation between dissociation of the dimer and inactivation of the enzyme. Our results clearly show that the monomer is devoid of amino acid activation and tRNA aminoacylation activities, indicating that dimerization is essential to confer an active conformation on the catalytic site. The two NH2-terminal truncated derivatives were fully active, but proved to be more unstable than the recombinant native enzyme, suggesting that the polypeptide extension fulfills structural rather than catalytic requirements.

Polyanion-induced alpha-helical structure of a synthetic 23-residue peptide representing the lysine-rich segment of the N-terminal extension of yeast cytoplasmic aspartyl-tRNA synthetase.

Conformational studies were performed on the synthetic tricosapeptide N-acetyl-SKKALKKLQKEQEKQRKKEERAL-amide, representing the highly basic segment (residues 30-52) of the N-terminal extension of yeast cytoplasmic aspartyl-tRNA synthetase. Circular dichroism experiments show that, in aqueous solution at neutral pH, the peptide adopts a random conformation. The effects of pH, temperature, addition of trifluoroethanol (TFE), and titration with polyanions on the conformation of the peptide were studied. In TFE or in the presence of an equimolar concentration of (phosphate)18, the peptide adopts a 100% alpha-helical conformation. A partially alpha-helical conformation is induced by (phosphate)4 or d(pT)8 (respectively 40% and 35% helical content). Raising the pH in aqueous solution promotes 75% alpha-helicity, with a transition pK of 9.9 reflecting deprotonation of lysine residues. On the basis of these results, nuclear magnetic resonance studies were carried out in TFE as well as in aqueous solution in the presence of (phosphate)18, to determine the structure of the molecule. Complete 1H resonance assignments were obtained by conventional two-dimensional NMR techniques. A total of 138 interproton constraints derived from NOESY experiments were used to calculate the three-dimensional structure by a two-stage distance geometry/simulated annealing procedure. The two deduced structures were highly similar and show that nine cationic residues are segregated on one face of a helical structure, providing an ideal polycationic interface for binding to polyanionic surfaces.

Reconstitution in vitro of the valyl-tRNA synthetase-elongation factor (EF) 1 beta gamma delta complex. Essential roles of the NH2-terminal extension of valyl-tRNA synthetase and of the EF-1 delta subunit in complex formation.

Valyl-tRNA synthetase from mammalian cells is isolated exclusively as a complex with elongation factor (EF) 1H (the "heavy" form of eukaryotic EF-1, composed of subunits alpha, beta, gamma, and delta). In a previous study, the 140-kDa valyl-tRNA synthetase subunit dissociated from the purified rabbit liver complex was shown to display hydrophobic properties, unlike the corresponding yeast cytoplasmic enzyme of 125 kDa (Bec, G., and Waller, J.-P. (1989) J. Biol. Chem. 264, 21138-21143). Compared to the sequence of yeast cytoplasmic valyl-tRNA synthetase, that of the human enzyme displays an NH2-terminal extension of approximately 200 amino acid residues that bears strong sequence similarity to the NH2-terminal moiety of EF-1 gamma (Hsieh, S. L., and Campbell, R. D. (1991) Biochem. J. 278, 809-816). We now show that this NH2-terminal extension can be selectively excised by elastase treatment of the isolated rabbit valyl-tRNA synthetase, without impairing catalytic activity. To examine the role of the NH2-terminal extension of mammalian valyl-tRNA synthetase in complex formation and to identify the subunit(s) of EF-1H responsible for binding the enzyme, reconstitution experiments were undertaken. Native or truncated valyl-tRNA synthetases were incubated with the isolated EF-1 subunits beta gamma and delta, either separately or in combination, and the ensuing products were analyzed by chromatography on DEAE-Sepharose FF and Superose 6. The results demonstrate that the NH2-terminal extension of valyl-tRNA synthetase is required for complex formation and that the enzyme-binding site(s) resides on the EF-1 delta subunit. Moreover, although the EF-1 beta gamma binary complex does not bind valyl-tRNA synthetase, it is nevertheless required for assembly of a complex of defined quaternary structure by preventing the formation of high molecular weight aggregates generated in the presence of EF-1 delta alone.

Mammalian prolyl-tRNA synthetase corresponds to the approximately 150 kDa subunit of the high-M(r) aminoacyl-tRNA synthetase complex.

The high-M(r) aminoacyl-tRNA synthetase complex previously purified from sheep liver differed from those isolated from several other mammalian sources by the absence of prolyl-tRNA synthetase activity and the presence of glutamyl tRNA synthetase as a polypeptide of 85 kDa instead of 150 kDa. Using a milder extraction procedure that minimizes proteolysis, we now report the isolation of a sheep liver complex that contains both prolyl-tRNA synthetase activity and the 150-kDa polypeptide. The correspondence between prolyl-tRNA synthetase and the 150-kDa polypeptide, inferred from the results of several approaches reported in this study, was further demonstrated by showing that antibodies to a free form of sheep liver prolyl-tRNA synthetase generated by endogenous proteolysis, specifically reacted with the 150-kDa components of the complexes from sheep and rabbit, but failed to react with the previously purified complex from sheep that contained neither prolyl-tRNA synthetases activity nor the 150-kDa component. Moreover, we show that the 150-kDa polypeptide is also recognized by antibodies to the 85-kDa polypeptide previously assigned to glutamyl-tRNA synthetase. The possibility that the largest subunit of the mammalian high-M(r) complexes may be a bifunctional protein encoding both glutamyl- and prolyl-tRNA synthetase activities is considered and discussed in light of the recently published sequence of the corresponding polypeptide from HeLa cells. In accordance with this prediction, we show that the amino acid sequence of the carboxyl-terminal moiety of this bifunctional polypeptide shows significant similarity to the sequence of prolyl-tRNA synthetase from Escherichia coli.

Interaction of microtubule-associated proteins with microtubules: yeast lysyl- and valyl-tRNA synthetases and tau 218-235 synthetic peptide as model systems.

The respective contributions of electrostatic interaction and specific sequence recognition in the binding of microtubule-associated proteins (MAPs) to microtubules have been studied, using as models yeast valyl- and lysyl-tRNA synthetases (VRS, KRS) that carry an exposed basic N-terminal domain, and a synthetic peptide reproducing the sequence 218-235 on tau protein, known to be part of the microtubule-binding site of MAPs. VRS and KRS bind to microtubules with a KD in the 10(-6) M range, and tau 218-235 binds with a KD in the 10(-4) M range. Binding of KRS and tau 218-235 is accompanied by stabilization and bundling of microtubules, without the intervention of an extraneous bundling protein. tau 218-235 binds to microtubules with a stoichiometry of 2 mol/mol of assembled tubulin dimer in agreement with the proposed binding sequences alpha[430-441] and beta[422-434]. Binding stoichiometries of 2/alpha beta S tubulin and 1/alpha S beta S tubulin were observed following partial or complete removal of the tubulin C-terminal regions by subtilisin, which localizes the site of subtilisin cleavage upstream residue alpha-441 and downstream residue beta-434. Quantitative measurements show that binding of MAPs, KRS, VRS, and tau 218-235 is weakened but not abolished following subtilisin digestion of the C-terminus of tubulin, indicating that the binding site of MAPs is not restricted to the extreme C-terminus of tubulin.

Valyl-tRNA synthetase from rabbit liver. I. Purification as a heterotypic complex in association with elongation factor 1.

Valyl-tRNA synthetase occurs as a high molecular mass entity of approximately equal to 700 kDa in the crude extract from rabbit liver. The enzyme was purified as a heterotypic complex comprising four polypeptides of 140, 50, 35, and 27 kDa in the molar proportions of 1:2:1:1, respectively, as determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Co-purification of these components at each step of the purification supports the conclusion that they are physically associated within the same complex. In addition to valyl-tRNA synthetase activity, which was assigned to the 140-kDa component, the purified complex exhibits a potent Elongation Factor 1 activity, determined by its ability to sustain poly(U)-dependent polyphenylalanine synthesis in the presence of Elongation Factor 2. Our results are essentially in agreement with those from a recent report (Motorin, Y., Wolfson, A., Orlovsky, A., and Gladilin, K. (1988) FEBS Lett. 238, 262-264), according to which the polypeptides other than that assigned to valyl-tRNA synthetase correspond to the subunits of Elongation Factor 1H.

Valyl-tRNA synthetase from rabbit liver. II. The enzyme derived from the high-Mr complex displays hydrophobic as well as polyanion-binding properties.

The preceding paper (Bec, G., Kerjan, P., Zha, X.D., and Waller, J.P. (1989) J. Biol. Chem. 264, 21131-21137) described the purification to apparent homogeneity from rabbit liver, of a heterotypic complex comprising valyl-tRNA synthetase and Elongation Factor 1H. In the present study, valyl-tRNA synthetase was dissociated and separated from the other components of this complex by hydroxylapatite chromatography in the presence of 0.5 M NaSCN. The properties of the homogeneous mammalian enzyme were compared to those of the corresponding enzyme from yeast. Both behaved as monomeric entities, with apparent molecular masses of 140 and 125 kDa, respectively. Furthermore, both displayed strong affinity toward the polyanionic support heparin-Ultrogel, a property not manifested by the corresponding prokaryotic enzyme. However, unlike the yeast enzyme, that of mammalian origin additionally exhibited hydrophobic properties, as reflected by its affinity toward phenyl-Sepharose. A structural model is proposed according to which mammalian valyl-tRNA synthetase has conserved the polycationic N-terminal domain that distinguishes the corresponding lower eukaryotic enzyme from its prokaryotic counterpart, while acquiring a hydrophobic domain most likely responsible for its association to Elongation Factor 1H.

Molecular cloning and primary structure of cDNA encoding the catalytic domain of rat liver aspartyl-tRNA synthetase.

A cDNA clone encoding rat liver aspartyl-tRNA synthetase was isolated by probing a lambda gt11 recombinant cDNA expression library with antibodies directed against the corresponding polypeptide from sheep liver. The 1930-base pairs-long cDNA insert allowed the expression in Escherichia coli of an active enzyme of mammalian origin. The nucleotide sequence of that cDNA, corresponding to the DRS1 gene, was determined. The open reading frame of DRS1 corresponds to a protein of Mr = 57,061, in good agreement with the previously determined molecular weight of the purified enzyme. The deduced amino acid sequence shows extensive homologies with that of yeast cytoplasmic aspartyl-tRNA synthetase, more than 50% of the residues being identical. In rat liver, aspartyl-tRNA synthetase occurs in two distinct forms: a dimeric enzyme and a component of a multienzyme complex comprising the nine aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. The primary structure of the DRS1 gene product is discussed in relation to the occurrence of two distinct forms of that enzyme.

The yeast lysyl-tRNA synthetase gene. Evidence for general amino acid control of its expression and domain structure of the encoded protein.

The nucleotide sequence of a 3.6-kilobase pair DNA fragment containing the structural gene for yeast cytoplasmic lysyl-tRNA synthetase (KRS1) and its flanking regions was determined. The encoded protein of 67,881 kDa displays a cluster of 11 lysines within a 29-amino acid residue segment at its amino-terminal extremity. Evidence is presented that this segment is responsible for the affinity displayed by the native enzyme toward polyanionic carriers. The transcription initiation sites of the KRS1 gene were determined. Upstream from the TATA box, putative control elements corresponding to the concensus sequences for the RPG box and the general amino acid control system were identified. Evidence for transcriptional induction of the KRS1 gene via the general amino acid control system is presented.

Structure and expression of the genes encoding the alpha and beta subunits of yeast phenylalanyl-tRNA synthetase.

The two genes FRS1 and FRS2 encoding, respectively, the large (alpha) and small (beta) subunits of cytoplasmic phenylalanyl-tRNA synthetase from bakers' yeast have been cloned and sequenced. The derived protein primary structures are confirmed by peptide sequences evenly distributed along the reading frames. These predict a subunit Mr of 67,347 for alpha and 57,433 for beta, in good agreement with earlier determinations carried out on the purified protein. These subunit sequences have been compared to those of Escherichia coli phenylalanyl-tRNA synthetase as well as to the small beta subunit of the corresponding yeast mitochondrial enzyme; limited but significant homology was found between the two alpha subunits on the one hand and between the three beta subunits on the other hand. The results suggest that these three enzymes, from E. coli, yeast cytoplasm, and yeast mitochondria, have strongly diverged from one another. The initiation sites of transcription have been determined for both yeast genes. Their 5'-upstream regions show no sequence similarities that would have indicated a coordinate control of gene expression at the transcriptional level. Measurements of steady-state levels of FRS-mRNAs in overproducing strains indicate that there is no restriction in mRNA synthesis. Therefore the control of gene expression, leading to a balanced synthesis of alpha and beta subunits, is likely to occur at the translational level.

Small-scale purification of bacteriophage lambda DNA by an airfuge centrifugation step in cesium chloride gradients.

A rapid and efficient procedure for purifying bacteriophage lambda DNA is described. This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients. Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes. The method allows a 1-day purification of up to 12 independent lambda DNA (20-40 micrograms each). The recovered DNA, essentially devoid of RNA and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles.

Overexpression of mammalian phenylalanyl-tRNA synthetase upon phenylalanine restriction.

The effect of phenylalanine restriction on the level of expression of phenylalanyl-tRNA synthetase from cultured Chinese hamster ovary cells was investigated. By lowering the phenylalanine concentration from 200 to 2 microM, cell growth was arrested, tRNAPhe aminoacylation level was rapidly and specifically decreased and phenylalanyl-tRNA synthetase was derepressed. The progressive 2-fold elevation of phenylalanyl-tRNA synthetase level was determined by activity measurement and immunotitration. None of the other aminoacyl-tRNA synthetases tested were significantly affected.

Expression of the aminoacyl-tRNA synthetase complex in cultured Chinese hamster ovary cells. Specific depression of the methionyl-tRNA synthetase component upon methionine restriction.

Cultured Chinese hamster ovary cells were subjected to amino acid restriction to examine its effects on the level of expression of the nine aminoacyl-tRNA synthetase components of the multienzyme complex which was previously characterized (Mirande, M., Le Corre, D., and Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289). Lowering the methionine concentration in the medium from 100 to 1 microM led to growth arrest, rapid deacylation of tRNAMet, and progressive 2-fold elevation of the methionyl-tRNA synthetase level, as assessed by specific activity measurements and immunotitration. The levels of the other eight aminoacyl-tRNA synthetases were not affected. Total methionine deprivation led to the additional derepression of the leucyl- and isoleucyl-tRNA synthetase components, whereas the corresponding tRNAs remained fully acylated. These pleiotropic responses to total methionine restriction were abolished in the presence of 2 mM methioninol, suggesting that amino acid transport systems may play a role in the regulation of aminoacyl-tRNA synthetase expression. The effect of total deprivation of arginine, glutamine, isoleucine, leucine, lysine, or proline from the culture medium on the level of expression of the corresponding aminoacyl-tRNA synthetases was also examined. In all cases, no elevation of the level of the corresponding synthetase was observed. The behavior of methionyl-tRNA synthetase from Chinese hamster ovary cells displaying a 2-fold increased level of the enzyme due to methionine restriction was examined in detail. Failure to detect a free form of the enzyme by gel filtration, as well as the finding that the isolated complex displayed twice the amount of methionyl-tRNA synthetase relative to the other components, indicates that this multienzyme structure can accommodate at least one additional copy of one of its components.

Cloning of yeast lysyl- and phenylalanyl-tRNA synthetase genes.

Cloning of yeast lysyl- and phenylalanyl-tRNA synthetase genes was accomplished by probing a lambda gt11 recombinant DNA expression library with antibodies directed against the purified enzymes. Several DNA clones encoding either the alpha or the beta subunit of phenylalanyl-tRNA synthetase were isolated. In each case, the inserted DNA was oriented in the same direction with respect to the lambda gt11 lacZ transcription unit, giving rise to the expression of hybrid proteins. The corresponding DNA fragments constitute suitable hybridization probes for the isolation of complete nucleotide sequences encoding the alpha and beta subunits of the enzyme. Recombinant DNA lambda gt11 clones encoding lysyl-tRNA synthetase were also selected. One of these contained yeast DNA inserted with the opposite orientation with respect to lacZ. The lysogen corresponding to that recombinant DNA phage produced an active, native lysyl-tRNA synthetase. The 3.6 kbp DNA insert contained all the information necessary for the expression of yeast lysyl-tRNA synthetase in E. coli.

Purification and characterization of the isoleucyl-tRNA synthetase component from the high molecular weight complex of sheep liver: a hydrophobic metalloprotein.

Native isoleucyl-tRNA synthetase and a structurally modified form of methionyl-tRNA synthetase were purified to homogeneity following trypsinolysis of the high molecular weight complex from sheep liver containing eight aminoacyl-tRNA synthetases. The correspondence between purified isoleucyl-tRNA synthetase and the previously unassigned polypeptide component of Mr 139 000 was established. It is shown that dissociation of this enzyme from the complex has no discernible effect on its kinetic parameters. Both isoleucyl- and methionyl-tRNA synthetases contain one zinc ion per polypeptide chain. In both cases, removal of the metal ion by chelating agents leads to an inactive apoenzyme. As the trypsin-modified methionyl-tRNA synthetase has lost the ability to associate with other components of the complex [Mirande, M., Kellermann, O., & Waller, J. P. (1982) J. Biol. Chem. 257, 11049-11055], the zinc ion is unlikely to be involved in complex formation. While native purified isoleucyl-tRNA synthetase displays hydrophobic properties, trypsin-modified methionyl-tRNA synthetase does not. It is suggested that the assembly of the amino-acyl-tRNA synthetase complex is mediated by hydrophobic domains present in these enzymes.

Leucyl-tRNA and lysyl-tRNA synthetases, derived from the high-Mr complex of sheep liver, are hydrophobic proteins.

The leucyl-tRNA and lysyl-tRNA synthetase components of the multienzyme complex from sheep liver were selectively dissociated by hydrophobic interaction chromatography on hexyl-agarose and purified to homogeneity. Conservation of activities during the purification required the presence of Triton X-100. The homogeneous enzymes corresponded to a monomer of Mr 129000 and a dimer of Mr 2 X 79000, respectively. Both were strongly adsorbed to the hydrophobic support phenyl-Sepharose, in conditions where the corresponding purified enzymes from yeast and Escherichia coli were not bound. Moreover, like the corresponding enzymes from yeast but unlike those of prokaryotic origin, the purified leucyl-tRNA and lysyl-tRNA synthetases derived from the complex displayed affinity for polyanionic supports. It is shown that proteolytic conversion of lysyl-tRNA synthetase to a fully active dimer of Mr 2 X 64000, leads to loss of both the hydrophobic and the polyanion-binding properties. These results support the view that each subunit of lysyl-tRNA synthetase is composed of a major catalytic domain, similar in size to the subunit of the prokaryotic enzyme, contiguous to a chain extension which carries both cationic charges and hydrophobic residues. The implications of these findings on the structural organization of the complex are discussed in relation to its other known properties.

Do yeast aminoacyl-tRNA synthetases exist as soluble enzymes within the cytoplasm?

The aminoacyl-tRNA synthetases from a crude extract of yeast were shown to bind to heparin-Ultrogel through ionic interactions, in conditions where the corresponding enzymes from Escherichia coli did not. The behaviour of purified lysyl-tRNA synthetases from yeast and E. coli was examined in detail. The native dimeric enzyme from yeast (Mr 2 X 73000) strongly interacted with immobilized heparin or tRNA, as well as with negatively charged liposomes, in conditions where the corresponding native enzyme from E. coli (Mr 2 X 65000) displayed no affinity for these supports. Moreover, the aptitude of the native enzyme from yeast to interact with polyanionic carriers was lost on proteolytic conversion to a fully active modified dimer of Mr 2 X 65500. A structural model is proposed, according to which each subunit of yeast lysyl-tRNA synthetase is composed of a functional domain similar in size to that of the prokaryotic enzyme, contiguous to a 'binding' domain responsible for association to negatively charged carriers. The evolutionary acquisition of this property by lower eukaryotic aminoacyl-tRNA synthetases suggests that it fulfils an important function in vivo, unrelated to catalysis. We propose that it promotes the compartmentalization of these enzymes within the cytoplasm, through associations with as yet unidentified, negatively charged components, by electrostatic interactions too fragile to withstand the usual extraction conditions.