©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Importance of the N-terminal Segment for DnaJ-mediated Folding of Rhodanese While Bound to Ribosomes as Peptidyl-tRNA (*)

Wieslaw Kudlicki (1), O. W. Odom (1), Gisela Kramer (1), Boyd Hardesty (1)(§), Gerald A. Merrill (2)(¶), Paul M. Horowitz (3)

From the (1) Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, (2) Department of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, Texas 78234, and the (3) Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation. The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally incorporated at the N terminus of nascent rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of rhodanese is required for its chaperone-dependent folding on the ribosome.


INTRODUCTION

Involvement of chaperones in the steps by which proteins are synthesized on ribosomes has been inferred from a number of studies. Beckmann et al. (1990) showed that hsp 70 was bound to HeLa cell polysomes in what appeared to be a cotranslational association with the nascent proteins. Nelson et al. (1992) and more recently Craig et al. (1994) presented genetic evidence that indicated a requirement for one of the cytosolic forms of hsp 70 for protein synthesis in yeast. DnaJ was cross-linked to nascent luciferase when it had been synthesized to a length of 55 amino acids on wheat germ ribosomes (Hendrick et al., 1993). Recently, Frydman et al. (1994) released nascent luciferase from reticulocyte ribosomes by puromycin and demonstrated GroEL homologs and DnaK homologs to be present as a complex with the nascent protein. These studies indicate that at least some of the chaperone-mediated reactions by which nascent proteins are folded into their native conformation occur on ribosomes. Hardesty et al. (1993) have reviewed results indicating that nascent peptides are extended from the peptidyl transferase center into a sheltered domain or cavity of the large ribosomal subunit. Eisenstein et al. (1994) have considered evidence indicating that one or more tunnels occur within the large ribosomal subunit into which the nascent peptide is extended and in which folding reactions might take place. However, virtually nothing is known of how and where within its structure a ribosome might accommodate chaperones, of the molecular details of how individual chaperones facilitate protein folding within a ribosome, or of the apparent interplay between the recognized ribosomal reactions of protein synthesis and the chaperone-mediated folding reactions.

We have used bovine rhodanese as a model enzyme to study the synthesis and chaperone-dependent folding of proteins on ribosomes. Rhodanese is a 33-kDa thiosulfate sulfur transferase (EC 2.8.1.1) that is found in the matrix of all mitochondria (Westley, 1973). Its enzymatic activity is easily determined by a sensitive assay system, and the protein itself is well suited to study the processes by which proteins are folded into their native conformations. Refolding of rhodanese from the denatured state has been studied extensively (Horowitz, 1993). In eukaryotic cells the protein is synthesized on cytoplasmic ribosomes and then transported into the mitochondrial matrix. With the exception of the N-terminal methionine, the mature mitochondrial enzyme retains the entire encoded N-terminal amino acid sequence that is required for targeting, transport, folding, and activation. The consensus features for targeting to the mitochondria are apparent in the N-terminal 31 amino acids of the native enzyme. This peptide has seven positive charges that are embedded in what otherwise is a relatively hydrophobic sequence. The crystal structure of the protein shows that residues 11-22 form an -helix and that the N terminus is positioned on the surface of one of the two globular domains (Ploegman et al., 1978; Hol et al., 1983).

Recently, Mendoza and Horowitz (1994) reported that refolding of denatured rhodanese in the absence or presence of cpn60 and cpn10 (GroEL and GroES) is modulated by the 23-amino acid sequence at its N terminus. The renaturation process was substantially inhibited by a peptide consisting of this sequence. The N-terminal peptide reduced the extent but not the rate of the reactivation process but did not prevent the interaction of the protein with cpn60. This finding and the fact that the peptide inhibits unassisted refolding suggest that the peptide interacts directly with the partially refolded protein to inhibit its conversion to the native conformation.

We have shown that enzymatically active rhodanese can be formed during synthesis of the protein in a coupled transcription/translation system that is derived from Escherichia coli (Tsalkova et al., 1993). However, full-length rhodanese chains accumulate as peptidyl-tRNA on ribosomes during the course of synthesis in the cell-free system (Kudlicki et al., 1994a, 1994b). The accumulation of nascent polypeptides appears to be related to a failure in one or more chaperone-mediated reactions that are required for the termination and release reactions of protein synthesis. The full-length ribosome-bound polypeptides are enzymatically inactive but are in a partially folded state. They can be released from the ribosomes as fully active rhodanese by incubation with the five E. coli chaperones (DnaJ, DnaK, GrpE, GroEL, and GroES) simultaneously (Kudlicki et al., 1994b). The activation and release process is blocked by sparsomycin at concentrations that inhibit the ribosomal peptidyl transferase reaction (Cundliffe, 1980).

Using fluorescence techniques, we demonstrated that incubation of the ribosomes with DnaJ plus DnaK has an effect on the emission spectrum of the ribosome-bound nascent CPM-rhodanese that appears to reflect a DnaJ-dependent reaction in a step of folding, termination, or release of rhodanese from the ribosome (Kudlicki et al., 1994c). For these experiments, synthesis of rhodanese was initiated with CPM-SAc-[S]Met-tRNA() to incorporate coumarin at the N terminus of nascent rhodanese. The fluorescent label does not affect enzymatic activity of the in vitro synthesized rhodanese that is released from the ribosome in the presence of the five chaperones.

In this report we present data indicating that the N-terminal amino acid sequence of rhodanese is critically important for the ribosome-associated, DnaJ-dependent reaction that is required for correct folding of the nascent protein into its native form. A deletion mutant lacking the 23 N-terminal amino acids does not form the DnaJ-dependent intermediate and is not folded into an enzymatically active conformation. This lack of enzymatic activity was not anticipated from the crystal structure (Ploegman et al., 1978). A chemically synthesized peptide corresponding to the 17-amino acid N-terminal sequence of rhodanese blocks the formation of the DnaJ-dependent intermediate of the wild-type protein that was detected by fluorescence and prevents the formation of enzymatically active rhodanese. The relation of the N-terminal peptide to inhibitory leader peptides studied by Lovett and co-workers (Gu et al., 1994a, 1994b) is discussed.


EXPERIMENTAL PROCEDURES

Materials

Nucleoside triphosphates and E. coli tRNA were purchased from Boehringer Mannheim; 3-(4-maleimidophenyl)-7-diethylamino-4-methylcoumarin (CPM) was from Molecular Probes Inc. (Eugene, OR). tRNA, rifampicin, sparsomycin, and all other biochemicals were from Sigma. A mixture of ribonuclease A and TI (``RNase Mixture'') was from Ambion (Austin, TX). [C]Leucine and [S]methionine were purchased from DuPont NEN. The chaperones DnaK, DnaJ, GrpE, GroEL, and GroES were bought from Epicentre Technologies (Madison, WI). The plasmids used were pSP65 containing the rhodanese coding sequence under the SP6 promoter (Kudlicki et al., 1994b) and pET 11d containing the mutant rhodanese lacking amino acids 1-23 under the T7 promoter.()

Methods

Propagation of the plasmids, isolation of SP6 RNA polymerase, and preparation of the E. coli cell-free extract (S30) as well as the isolation of the ribosome fraction from the S30 were carried out as described previously (Kudlicki et al., 1992). T7 RNA polymerase was isolated by a procedure identical to the one used for preparation of SP6 RNA polymerase. E. coli BL21 (pAR1219) cells were used as a source for T7 RNA polymerase. The preparation of CPM-SAc-[S]Met-tRNA was as described previously (Kudlicki et al., 1994c). Anti-CPM antibodies were raised in rabbits; the IgG fraction from the serum of the immunized rabbit was prepared as described (Picking et al., 1992).

The in vitro system for coupled transcription/translation (usually a 30-µl reaction mixture) has been detailed (Kudlicki et al., 1992, 1994b). [C]Leucine was the radioactive precursor. When CPM-SAc-[S]Met-tRNA was used, folinic acid was omitted and [C]leucine was replaced by unlabeled leucine. For some of the experiments described below, reaction mixtures were enlarged to 0.9 ml. They contained salts and low molecular weight components (Kudlicki et al., 1994b) including 5 mM NaSO. About 60 A units of unwashed ribosomes were incubated with 10-15 µg of nonlinearized plasmid and the appropriate RNA polymerase. After incubation for 30 min at 37 °C, the sample was loaded over 0.6 ml of 0.5 M sucrose in 20 mM Tris-HCl, pH 7.5, 10 mM Mg(OAc), 30 mM NHOAc, 1 mM dithiothreitol (solution A) and centrifuged for 45 min at 45,000 rpm (Ti-50 rotor, Beckman). The resulting supernatant was collected and saved. The sucrose layer was removed, and the ribosomal pellet was rinsed and then resuspended in 60 µl of solution A. Immediately after centrifugation, the supernatant fraction (about 0.9 ml) was treated with ribonucleases A and T1 (0.1 mg and 2,000 units/ml, respectively) for 15 min at 37 °C to degrade the remaining CPM-SAc-[S]Met-tRNA. The resulting reaction mixture was then chromatographed on a Sephadex G-100 column (1 20 cm) equilibrated in solution A to separate newly synthesized CPM-labeled rhodanese from low molecular weight degradation products containing coumarin and [S]methionine.

In some experiments when release of ribosome-bound rhodanese was studied, the reaction mixture after incubation with chaperones or other additions was centrifuged in an airfuge (Beckman) at 150,000 g for 40 min. Then the supernatant was pipetted off, and the ribosomal pellet was resuspended in solution A.

Fluorescence measurements were carried out on a model 8000C photon-counting spectrofluorometer from SLM-Aminco Instruments Inc. (Urbana, IL). Spectra were measured at 1-nm intervals at a scanning rate of 0.5 s/wavelength increment. Unless otherwise indicated, excitation was at 390 nm. Spectra and relative quantum yields were normalized on the basis of radioactivity from [S]methionine that was present in the sample as CPM-SAc-[S]methionine incorporated into protein. Relative fluorescence quantum yields were determined from integrated emission spectra. The relative quantum yield of CPM-rhodanese in the supernatant fraction was taken as 1.00, and all other quantum yields are given relative to this value. Fluorescence anisotropy was determined as described in Odom et al. (1984) at an emission wavelength of 480 nm. For the fluorescence experiments, an aliquot of the resuspended ribosomes or of the treated supernatant fraction (see above) was incubated in the cuvette in a total volume of 420 µl containing salts and low molecular weight components as above for the coupled transcription/translation but without amino acids and without UTP and CTP (solution B). After the spectrum was taken, the components to be tested were added in a minimum volume. Additions were as follows: anti-CPM IgG, 0.1 mg; and chaperones GroES, 1.6 µg; GroEL, 6 µg; DnaK, 4 µg; DnaJ, 2 µg; GrpE, 3 µg. The cuvette was incubated for 5 min at 20 °C before the emission spectrum was taken again.

Precipitation of protein by trichloroacetic acid to quantitate the amount of rhodanese polypeptides, analysis by SDS-polyacrylamide gel electrophoresis, and autoradiography were carried out as before (Kudlicki et al., 1994a). Rhodanese activity was assayed according to Sörbo (1953) as given in Tsalkova et al. (1993) and Kudlicki et al. (1994b).

A peptide of 17 amino acids corresponding to the N-terminal sequence of bovine rhodanese was synthesized by manual solid support peptide synthesis procedures (Stewart and Young, 1984) by sequential addition of tert-butyloxycarbonyl-protected L-amino acids (Bachem, Inc., Torrance, CA, or Applied Biosystems, Foster City, CA). Similarly, a peptide was synthesized corresponding to the proline-rich interdomain tether region of rhodanese (amino acids 142-156 of the bovine rhodanese sequence). The peptides were cleaved from the resins and the protective groups of reactive side chains removed by treatment with hydrogen fluoride (Immuno-Dynamics, Inc., La Jolla, CA). The cleaved peptides were purified by reversed phase high performance liquid chromatography (HPLC) using nonlinear gradients of acetonitrile (5-85%) with 0.1% (v/v) trifluoroacetic acid (Pierce) as described previously (Merrill et al., 1992).


RESULTS

Cell-free Synthesis of Wild-type Rhodanese and Its N-terminal Deletion Mutant

A deletion mutant of the bovine rhodanese gene was constructed that lacked codons for the N-terminal 23 amino acids of the wild-type enzyme. The coding sequence of the mutant gene started with ATG followed by the GTG codon for valine 24. Protein was synthesized in the cell-free E. coli transcription/translation system from plasmids containing either the mutant or wild-type gene. Mutant rhodanese was synthesized more efficiently than wild-type protein in this system (Fig. 1). At the indicated times, aliquots were centrifuged, and then the resulting supernatant fraction and the resuspended ribosomes were analyzed separately. The initial rate of leucine incorporation into ribosome and soluble fraction was very similar for the mutant and wild-type proteins. But marked differences became apparent during the later stages of the incubation. The mutant protein that was bound to the ribosomes reached a maximum of about 23 pmol after 10 min of incubation (Fig. 1 A). This value remained essentially constant during the next 40 min of incubation, whereas total synthesis continued at a relatively high rate throughout the 50-min incubation period. In contrast, the newly formed wild-type protein accumulated on the ribosomes at a nearly linear rate for 20 min to reach a maximum of near 50 pmol (Fig. 1 B). The rate of total synthesis and release of protein into the soluble fraction declined to near zero after about 30 min of incubation. The enzyme that was released appeared to be folded into the native conformation as judged by its specific enzymatic activity. Neither the supernatant nor the ribosome-bound forms of the mutant protein were enzymatically active, whereas the specific enzymatic activity of the wild-type protein in the supernatant fraction was near that of the native enzyme. The wild-type rhodanese that was bound to the ribosomes was enzymatically inactive as previously reported (Kudlicki et al., 1994a, 1994b).


Figure 1: Synthesis of wild-type rhodanese and its 1-23 N-terminal deletion mutant in the cell-free transcription/translation system. The deletion mutant ( A) and wild-type ( B) rhodanese were synthesized in vitro in the presence of [C]leucine (40 Ci/mol). The volume of each reaction mixture was 240 µl, and a 35-µl aliquot was taken for each time point. The ribosomes and supernatants were separated by centrifugation after the time shown on the abscissa. A 15-µl aliquot of the supernatant ( A, opentriangles; B, opensquares) and the resuspended ribosome fraction ( A, filledtriangles; B, filledsquares), respectively, was taken to determine the amount of [C]leucine incorporated into protein, and an equal aliquot was used to measure rhodanese enzymatic activity. Opencircles and filledcircles represent rhodanese specific activity for supernatant and ribosome fractions, respectively.



Analysis by SDS-polyacrylamide gel electrophoresis and autoradiography indicated that nearly all of the mutant or wild-type polypeptides released into the supernatant fraction throughout the incubation were of the size anticipated for the full-length product (results not shown). About half of the ribosome-bound peptides consisted of full-length rhodanese chains (not shown). In the case of wild-type rhodanese, these were more than twice the amount compared with the ribosome-bound mutant rhodanese and constituted a high portion of the total protein synthesized. These results are consistent with the hypothesis that the wild-type protein is processed through a ribosome-dependent step in folding that limits its termination and release from the ribosome, whereas termination and release of the mutant protein is not subjected to this limiting step.

Fluorescence Analyses of the Deletion Mutant and Wild-type Rhodanese

Fluorescence techniques were used to investigate the differences indicated above in the release and folding of the N-terminal deletion mutant and wild-type rhodanese. To this end, both proteins were synthesized with CPM-SAc-[S]Met-tRNA to incorporate coumarin at their N termini. After 30 min of incubation, the reaction mixtures were centrifuged to separate the supernatant from the ribosomes. After ribonuclease treatment and gel permeation chromatography of the supernatant fraction to remove unincorporated CPM-SAc-[S]methionine (see ``Experimental Procedures''), both the supernatant fraction and the resuspended ribosomes were analyzed by fluorescence. The results are summarized in . As described previously, fluorescence intensities were normalized on the basis of [S]methionine incorporated into protein (Kudlicki et al., 1994c), and the quantum yield of wild-type enzymatically active rhodanese in the supernatant was set at 1.00. The quantum yield is higher (1.20 versus 1.00) for the enzymatically inactive mutant rhodanese in the supernatant fraction. A higher quantum yield was observed previously for wild-type rhodanese released from the ribosome by puromycin or by DnaK only (Kudlicki et al., 1994c). Both of these forms of wild-type rhodanese are enzymatically inactive. The results appear to indicate that the N-terminal conformations of the enzymatically active wild-type protein and of the wild-type protein released by puromycin or DnaK are different as is the environment of the N-terminal coumarin of the released mutant protein. The ribosome-bound forms of both the wild-type and mutant protein have lower quantum yields than their soluble counterparts as shown in . The maxima of the emission spectra of both the supernatant and ribosome-bound forms of the mutant protein are significantly higher than the corresponding values for the wild-type protein. Fluorescence anisotropy is relatively high for the ribosome-bound forms of both proteins, as expected.

The supernatant fraction of both the wild-type ( A) and the mutant rhodanese ( B) were incubated with anti-coumarin antibodies to give changes in the fluorescence emission spectra that are evident in Fig. 2 . These polyclonal antibodies combine directly with coumarin and cause a large increase in fluorescence quantum yield and a shift toward the blue in the emission spectrum that appears to be associated with binding of the coumarin into a hydrophobic environment within the antibody. An effect very similar to that shown for enzymatically active wild-type rhodanese is observed with low molecular weight model compounds containing coumarin such as the corresponding derivative of cysteine (data not shown). The anti-coumarin antibodies produce a much larger increase in fluorescence intensity (from 1.00 to 1.70) with the wild-type protein (Fig. 2, insets) than with the mutant rhodanese (from 1.11 to 1.30). These differences in quantum yield of the two proteins are associated with significant differences in fluorescence anisotropy, increases from 0.18 to 0.37 for wild-type but from 0.18 to 0.33 for the mutant protein. Our tentative interpretation of these data is that in contrast to the wild-type protein only part of the N-terminal coumarin of the mutant is accessible to the antibodies.


Figure 2: Fluorescence emission spectra of CPM wild-type and mutant rhodanese before and after incubation with anti-coumarin antibodies. Wild-type rhodanese and mutant rhodanese were synthesized in the presence of CPM-SAc-[S]Met-tRNA (4,000 Ci/mol), and then the reaction mixtures were centrifuged. The resulting supernatant fractions were treated with ribonuclease and chromatographed over Sephadex G-100, and then the emission spectra were taken in the absence of antibodies and after incubation for 5 min at room temperature with anti-coumarin IgG. A, wild-type rhodanese; B, 1-23 mutant rhodanese. Insets, quantitative fluorescence data determined in the absence ( -IgG) and in the presence ( +IgG) of anti-coumarin antibodies. RelQ, relative fluorescence quantum yield (as explained in Table I); A, fluorescence anisotropy; , wavelength of emission maximum.



Fluorescence emission spectra of the ribosome-bound rhodanese in the presence and absence of anti-coumarin antibodies were also taken. In the absence of the antibodies the wild-type and the deletion mutant gave generally similar spectra (Fig. 3, A, B, -IgG), the emission maximum of the mutant being at a somewhat longer wavelength than that of the wild-type (483 versus 471 nm, ). Addition of DnaJ plus DnaK gave only minor spectral changes (shown in Fig. 5 ). However, a pronounced difference between the spectra of the ribosome-bound wild-type and mutant protein was observed in the presence of antibodies after the ribosomes had been incubated with DnaJ plus DnaK. The ribosome-bound wild-type protein exhibited a new fluorescent species with an emission maximum near 435 nm (Fig. 3 A). This is the intermediate species that apparently is produced by a reaction promoted by DnaJ as previously reported (Kudlicki et al., 1994c). This fluorescent species was not detected with the ribosome-bound mutant protein (Fig. 3 B). The spectrum of the mutant protein that was obtained in the presence of antibodies was very similar to the corresponding spectra exhibited by both the wild-type and mutant protein that had been released into the soluble phase ( cf. Fig. 2 ). We interpret these results to indicate that the ribosome-bound mutant rhodanese is unreactive in the reactions promoted by DnaJ plus DnaK and that the N-terminal sequence of rhodanese is critically important for formation of the DnaJ- plus DnaK-dependent intermediate. This intermediate may be a prerequisite for correct folding into enzymatically active enzyme that is promoted in the presence of the other chaperones.


Figure 3: Mutant and wild-type ribosome-bound nascent rhodanese after incubation of the ribosomes with DnaJ plus DnaK and then with anti-CPM antibodies. Wild-type and N-terminal deletion mutants of rhodanese were synthesized from CPM-SAc-[S]Met-tRNA (4,000 Ci/mol) by coupled transcription/translation, and then ribosomes with bound rhodanese polypeptides were separated from the reaction mixture by centrifugation. The resuspended ribosomes were incubated with DnaJ plus DnaK and ATP for 5 min at 25 °C. The fluorescence emission spectra were taken in the absence of antibodies and after incubation with anti-coumarin IgG. A, wild-type rhodanese; B, 1-23 mutant rhodanese.




Figure 5: The effects of the N-terminal peptide and DnaJ/DnaK on the emission spectrum of ribosome-bound wild-type CPM-rhodanese in the presence and absence of anti-coumarin IgG. Ribosomes bearing wild-type CPM-rhodanese nascent peptides were incubated with the N-terminal peptide, DnaJ/DnaK, and anti-coumarin IgG as described in Table III. A, incubation first with the N-terminal peptide. Spectrum1, after incubation without additions; spectrum2, after incubation with N-terminal peptide; spectrum3, as in spectrum2 and then incubation with DnaJ/DnaK; spectrum4, as in spectrum3 and then incubation with anti-coumarin IgG. B, incubation first with DnaJ/DnaK. Spectrum1, after incubation without additions; spectrum2, after incubation with DnaJ/DnaK; spectrum3, as in spectrum2 and then then incubation with N-terminal peptide; spectrum4, as in spectrum3 and then incubation with IgG.



Effects of the N-terminal Rhodanese Peptide on Cell-free Synthesis of Rhodanese

The results with the N-terminal deletion mutant that were presented in the preceding sections prompted considerations of the effects that the N-terminal peptide itself might have on the in vitro synthesis and activation of mutant and wild-type rhodanese. Two peptides were chemically synthesized and purified by HPLC. The synthetic peptide used in the experiments described below corresponded to amino acids 1-17 of the wild-type enzyme. As a control a second peptide was synthesized that corresponded to an internal 15-amino acid sequence, amino acids 142-156 that connect the N-terminal and C-terminal globular domains of the wild-type enzyme in its native conformation (Ploegman et al., 1978). This is called the ``tether peptide'' in the description given below. Neither of these peptides had any effect on the fluorescence or enzymatic activity of wild-type rhodanese that had been released from the ribosomes or the corresponding mutant protein that is enzymatically inactive (data not shown). Interesting differences in the activity of the two peptides were observed when they were added separately to reaction mixtures in which either the wild-type or the deletion mutant of rhodanese was synthesized (Fig. 4). The N-terminal peptide caused considerably higher inhibition of synthesis of the wild-type protein than the deletion mutant. The tether peptide had no effect on the coupled transcription/translation of either the wild-type or the deletion mutant rhodanese coding sequence.


Figure 4: Effect of rhodanese N-terminal peptide on rhodanese synthesis during coupled transcription/translation. Either the N-terminal peptide ( squares) or the tether peptide ( triangles) was added in the indicated amount to the coupled transcription/translation reaction mixtures (30 µl total volume) containing either wild-type ( filledsymbols) or deletion mutant ( opensymbols) rhodanese coding sequence. The amount of protein synthesized in the whole reaction mixture (30 µl) was analyzed by incorporation of [C]leucine as described under ``Methods.'' The 100% values were 94 pmol of [C]leucine incorporated into wild-type rhodanese and 121 pmol of [C]leucine incorporated into mutant protein.



Previously we demonstrated that full-length rhodanese polypeptides accumulate on the ribosomes as peptidyl-tRNA and that this nascent rhodanese can be released in an enzymatically active form during an incubation of the ribosomes with all five chaperones under reaction conditions in which the formation of additional peptide bonds could not be detected (Kudlicki et al., 1994b). The data given in indicate that the N-terminal peptide inhibits this release and activation of ribosome-bound wild-type rhodanese. After coupled transcription/translation, the ribosomes bearing nascent rhodanese were isolated by centrifugation and then incubated with the additions shown in the table. After this incubation the ribosomes were reisolated by centrifugation. The resulting supernatant was analyzed for amount of protein released and enzymatic activity. These results are given in . Without the N-terminal or tether peptide, the chaperones (DnaJ, DnaK, GrpE, GroEL, and GroES) caused the release of more than half of the polypeptides from the ribosomes as determined by incorporated [C]leucine. Nearly all of the chaperone-released protein is full-length rhodanese; shorter peptides remain bound to the ribosomes (Kudlicki et al., 1994b). The protein that is released from the ribosomes appears to be in the native conformation as inferred from its specific enzymatic activity, which approaches the value expected for native rhodanese (Jarabak and Westley, 1974). When the N-terminal peptide was included in the reaction mixture with the chaperones, release of rhodanese from the ribosomes was reduced from 27 to 8 pmol of leucine (). The small amount of protein that was released had the specific enzymatic activity of the native enzyme. The tether peptide had no apparent effect on release or the specific enzymatic activity of the released wild-type protein.

Effect of the N-terminal Peptide on Fluorescence from Ribosome-bound CPM-rhodanese

The effect of the N-terminal peptide on the DnaJ/DnaK-dependent emission spectrum of ribosome-bound wild-type CPM-rhodanese that is seen in the absence or presence of anti-coumarin antibodies was examined, and the results are shown in Fig. 5 and I. The most striking effect is that the emission peak with a maximum at 435 nm is not observed if the ribosomes bearing CPM-rhodanese are incubated with the N-terminal peptide before they are incubated with DnaK/DnaJ (Fig. 5 A) and then with anti-coumarin IgG ( curve4). The maximum of the emission spectrum and increase in quantum yield (I) that occur with the addition of the antibodies are very similar to those seen with the N-terminal deletion mutant (Fig. 2 B). It should be noted from Fig. 5 A and I that the N-terminal peptide itself without DnaJ/DnaK or antibodies causes a significant increase in the quantum yield and a pronounced drop in fluorescence anisotropy, from 0.89 to 0.97 and 0.306 to 0.235, respectively (I). Subsequent incubation with DnaJ/DnaK causes an increase in anisotropy to 0.266 and an increase in quantum yield to 1.03. In contrast, if the CPM-rhodanese ribosomes are incubated with DnaJ/DnaK before the incubation with the N-terminal peptide, the emission peak with a maximum at 435 nm is observed after the addition of the antibodies, and the maximum of the largest peak in the emission spectrum is at about 473 nm (Fig. 5 B). Incubation of the ribosomes with DnaJ/DnaK only had little direct effect on fluorescence (Fig. 5 B, lines 1 and 2), but the subsequent incubation with the N-terminal peptide causes a relatively large increase in quantum yield (Fig. 5 B, line3; I C, quantum yield of 0.92 versus 1.29). Note that the subsequent incubation with anti-coumarin IgG after incubation with DnaJ/DnaK and then the N-terminal peptide causes a much smaller increase in the fluorescence intensity at 473 nm (Fig. 5 B, line4) than was observed in the absence of the N-terminal peptide but in an otherwise similar experiment (Fig. 3 A). The tether peptide had no detectable effects similar to those described for the N-terminal peptide (data not shown). Also, the N-terminal peptide had no effect on fluorescence from ribosome-bound mutant CPM-rhodanese comparable with those shown above for wild-type rhodanese (data not shown).

The changes in fluorescence, especially changes in anisotropy and quantum yield that are caused by the N-terminal peptide indicate that it affects the mobility and local environment of the N-terminal coumarin probe of nascent wild-type rhodanese while the latter is bound as peptidyl-tRNA to the ribosomes. Although there is a possibility that such effects might be indirect through the ribosome or mediated by a small amount of DnaJ that remains associated with the ribosomes throughout their isolation and subsequent experimental manipulation, our tentative interpretation is that the synthetic N-terminal peptide interacts directly with the ribosome-bound wild-type rhodanese chains. It is somewhat surprising that it does not appear to interact with nascent peptides of the N-terminal deletion mutant. This may reflect a requirement for the N-terminal sequence in early steps of folding that precede the state that is required for the DnaJ/DnaK reaction.


DISCUSSION

The results presented above indicate that the N-terminal segment of nascent rhodanese bound to ribosomes as peptidyl-tRNA is essential for the reaction promoted by DnaJ/DnaK and for folding of the protein into an enzymatically active conformation. Synthesis of enzymatically active wild-type rhodanese in the cell-free transcription/translation system characteristically declines to a very low rate after about 20 min of incubation with an accumulation on the ribosomes of full-length rhodanese chains as peptidyl-tRNA. This nascent protein is enzymatically inactive but can be released as fully active rhodanese by subsequent incubation of the isolated ribosomes with the five bacterial chaperones. No similar effects were seen when a deletion mutant lacking the N-terminal 23 amino acids of wild-type rhodanese was translated in the cell-free system. Efficient synthesis of the mutant protein continued at a relatively high rate for 50 min, and there was less accumulation of full-length rhodanese on the ribosomes compared with that observed with the wild-type enzyme. However, the mutant protein that was released from the ribosomes was enzymatically inactive. This was unanticipated in that examination of the crystal structure of the wild-type enzyme indicated that the N-terminal 23-amino acid segment is positioned on the surface of one of two globular domains.

A chemically synthesized peptide corresponding to the N-terminal 17 amino acids of the wild-type enzyme efficiently inhibited synthesis of the wild-type enzyme but had much less effect on the synthesis of the mutant protein. This peptide blocked the chaperone-mediated release of the wild-type enzyme from the ribosomes and prevented formation of the intermediate species that is formed in the presence of DnaJ/DnaK. A 15-amino acid peptide corresponding to an internal segment that spans the region between the two globular domains of the native enzyme had little or no effect comparable with those observed with the N-terminal peptide, indicating specificity for a specific amino acid sequence.

The recent report of Mendoza and Horowitz (1994) may provide insight into the molecular mechanisms that underlie these complex results. They found that a peptide corresponding to the N-terminal 23 amino acids of wild-type rhodanese inhibited refolding of urea-unfolded rhodanese both in the presence and absence of chaperonin 60 (GroEL). However, the peptide did not block the interaction of the partially refolded protein with the chaperone. A 13-amino acid peptide corresponding to N-terminal sequence 11-23 of the native enzyme had no comparable effects. We believe these observations are of special importance for interpretation of the results presented here because they demonstrate conclusively that the N-terminal peptide can react directly with a folding intermediate of rhodanese that is formed in the absence of chaperones or ribosomes and that this apparently specific interaction can block subsequent folding of the protein into the native conformation. We suggest that the phenomena reported here reflect a similar specific interaction of the N-terminal peptide with a partially folded state of nascent rhodanese that is bound to the ribosomes as peptidyl-tRNA in addition to possible interactions of the peptide with either DnaJ/DnaK or the ribosomes. That the N-terminal peptide has a direct effect on the local environment of the N-terminal probe on nascent rhodanese is evident from the spectra of Fig. 5 A. Binding of the peptide appears to block the subsequent reaction that is mediated by DnaJ/DnaK. However, the peptide has a much larger effect on fluorescence from the N-terminal probe if it is added subsequent to the DnaJ/DnaK reaction (Fig. 5 B). These effects appear to indicate that the DnaJ/DnaK reaction facilitates binding of the N-terminal peptide in the immediate vicinity of the N-terminal probe on the nascent protein. This may be at or near the binding site on the globular domain to which the N-terminal segment of the protein may be bound as indicated in the crystal structure.

Considered together we suggest that the results presented here and in a previous publication (Kudlicki et al., 1994c) indicate that the N-terminal segment of wild-type rhodanese facilitates early steps in the correct folding of the nascent protein within the ribosomes as it is extended vectorially from its N terminus. Without the N-terminal segment the truncated mutant protein does not properly undergo these early folding reactions and is released, apparently through the standard termination reactions. The reaction that is mediated by DnaJ/DnaK, which is required for wild-type rhodanese to fold into the native conformation, is dependent upon the correct form of the partially folded species. The primary effect of the DnaJ/DnaK reaction may be to position the nascent protein on the ribosome so that it can undergo subsequent folding reactions such as those mediated by GroEL/GroES. These chaperones are critically required in the same reaction mixture with DnaJ/DnaK to form enzymatically active protein, but it is not known whether or not the reactions they promote take place while the nascent protein is associated with the ribosome.

Lovett and his co-workers (Gu et al., 1994a, 1994b) have described a very provocative series of studies involving the effect of peptides corresponding to the N-terminal sequences of certain chloramphenicol-inducible genes. These peptides inhibited the ribosomal peptidyl transferase reaction. Inhibitory peptides appeared to bind directly to the ribosomes in that their binding was competitive with erythromycin. Both erythromycin and the peptide footprinted to overlapping sites in the central loop of domain V of 23 S rRNA (Gu et al., 1994a). Recently, data were reported indicating that the inhibitory peptides can block the release factor-dependent hydrolysis of ribosome-bound Met-tRNA or reaction of this tRNA species with puromycin (Moffat et al., 1994). These results clearly indicate that the binding site for the inhibitory peptide is on the ribosome. The observations have intriguing similarities to the results reported here. However, as yet we have been unable to show that the 17-amino acid N-terminal rhodanese peptide at concentrations similar to those used in the experiments described above has a direct inhibitory effect on the peptidyl transferase reaction. Despite this failure to detect direct inhibition of the peptidyl transferase reaction, our results indicate that some step of the normal release mechanism fails during the translation of wild-type rhodanese in the cell-free system. We suggest that the observations reported by Lovett and his co-workers (Gu et al. 1994a, 1994b) and those reported here reflect different aspects of the same steps in the sequence of reactions by which nascent proteins are folded on the ribosomes and released by the termination reactions. We also suggest that the nascent wild-type rhodanese folds into a conformation in which its N terminus is brought into a position within the ribosome in which it can interact with a peptide-binding site in the 23 S rRNA that was observed by Lovett and his co-workers.

  
Table: Fluorescence characteristics of CPM-SAc-Met-rhodanese (free in solution and bound to ribosomes)

Fluorescence parameters for wild-type rhodanese and deletion mutant rhodanese were determined after their synthesis by coupled transcription/translation with CPM-SAc-[S]Met-tRNA as the initiator tRNA. After a 30-min incubation at 37 °C, the reaction mixtures were centrifuged to obtain the ribosomal fraction and supernatant and analyzed as described under ``Methods.''


  
Table: The effects of the N-terminal peptide on the chaperone-dependent activation and release of ribosome-bound wild-type rhodanese

An aliquot of the ribosomal fraction (50 pmol of [C]leucine in protein) isolated after coupled transcription/translation (see legend of Table I and ``Methods'') was incubated in the absence or presence of the N-terminal peptide (4 µg), tether peptide (5 µg), chaperones (amounts: 3.5 µg of GroEL, 0.8 µg of GroES, 2 µg of DnaK, 1 µg of DnaJ, 1 µg of GrpE), plus low molecular weight components as specified under ``Experimental Procedures.'' The volume of reaction mixture was 30 µl. After the 30-min incubation, the ribosomes were separated from the supernatant by a second centrifugation. The resulting supernatant fractions were analyzed for the amount of protein that had been released from the ribosomes and its enzymatic activity.


  
Table: Effect of the N-terminal peptide on DnaJ/DnaK-mediated changes in fluorescence from wild-type CPM-rhodanese bound to ribosomes

Ribosomes containing 4 pmol of CPM-[S]methionine in nascent rhodanese peptides were incubated for 5 min at room temperature under nontranslating conditions with the indicated additions. For B and C the ribosomes were incubated first with either the N-terminal peptide or with DnaJ/DnaK, as indicated, and then the missing components, either the N-terminal peptide or DnaJ/DnaK, were added, and the incubation continued for an additional 5 min. Where indicated, anti-coumarin antibodies were then added, and the incubation continued for an additional 5 min. The fluorescence characteristics of the reaction mixtures were determined as described for Table I. Amounts of antibodies, DnaJ, DnaK, and N-terminal peptide added were 0.1 mg, 2.5 µg, 5 µg, and 4 µg, respectively. Abbreviations are as defined in Table I.



FOOTNOTES

*
This work was supported by a grant from The Foundation for Research, by National Science Foundation Grant MCB-9315034 (to B. H.), by National Institutes of Health Grants GM25177 and ES05729 (to P. M. H.), and by Welch Foundation Grant AQ 723 (to P. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 512-471-5874; Fax: 512-471-8696.

Recipient of Brook Army Medical Center Grants C-18-88 and A-18-90.

The abbreviations used are: CPM-[S]SAc-Met-tRNA, initiator tRNA that has been aminoacylated with methionine and then labeled on the methionine amino group via mercaptoacetic acid by CPM; CPM, 3-(4-maleimidophenyl)-4-methyl-7-(diethylamino)coumarin; HPLC, high performance liquid chromatography; CPM-rhodanese, rhodanese labeled at its N-terminal methionine with CPM; anti-CPM IgG, anti-coumarin polyclonal antibodies produced in rabbits.

P. M. Horowitz, D. M. Miller-Martini, and J. M. Chirgwin, unpublished results.


ACKNOWLEDGEMENTS

We thank Ronda Barnett for preparing the typescript.


REFERENCES
  1. Beckmann, R. P., Mizzen, L. A., and Welch, W. J. (1990) Science 240, 850-854
  2. Craig, E., Baxter, B., Becker, J., Halladay, J., and Ziegelhoffer, T. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R., Tissières, A., and Georgopoulos, C., eds) pp. 31-52, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  3. Cundliffe, E. (1980) in Ribosomes: Structure, Function and Genetics (Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L., and Nomura, M., eds) pp. 555-581, University Park Press, Baltimore
  4. Eisenstein, M., Hardesty, B., Odom, O. W., Kudlicki, W., Kramer, G., Arad, T., Franceschi, R., and Yonath, A. (1994) in Supromolecular Structure and Function (Pifat, G., ed) pp. 231-246, Balaban Publishers, Rehovot
  5. Frydman, J., Nimmesgern, E., Ohtsuka, K., and Hartl, F. U. (1994) Nature 370, 111-117 [CrossRef][Medline] [Order article via Infotrieve]
  6. Gu, Z., Harrod, R., Rogers, E. J., and Lovett, P. S. (1994a) Proc. Natl. Acad. Sci. U. S. A. 91, 5612-5616 [Abstract]
  7. Gu, Z., Harrod, R., Rogers, E. J., and Lovett, P. S. (1994b) J. Bacteriol. 176, 6238-6244 [Abstract]
  8. Hardesty, B., Odom, O. W., Kudlicki, W., and Kramer, G. (1993) in The Translational Apparatus (Nierhaus, K. H., Subramanian, A. R., Erdmann, V. A., Franceschi, F., and Wittmann-Liebold, B. eds) pp. 347-358, Plenum Press, Berlin
  9. Hendrick, J. P., Langer, T., Davis, T. A., Hartl, F. U., and Wiedmann, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10216-10220 [Abstract]
  10. Hol, W. G. J., Lijk, L. J., and Kalk, K. H. (1983) Fundam. Appl. Toxicol. 3, 370-376
  11. Horowitz, P. M. (1993) in Biocatalyst Design for Stability and Specificity (Himmel, M. E., and Georgiou, G., eds) pp. 167-172, American Chemical Society, Washington, D. C.
  12. Jarabak, R., and Westley, J. (1974) Biochemistry 13, 3233-3236 [Medline] [Order article via Infotrieve]
  13. Kudlicki, W., Kramer, G., and Hardesty, B. (1992) Anal. Biochem. 206, 389-393 [Medline] [Order article via Infotrieve]
  14. Kudlicki, W., Mouat, M., Walterscheid, J. P., Kramer, G., and Hardesty, B. (1994a) Anal. Biochem. 217, 12-19 [CrossRef][Medline] [Order article via Infotrieve]
  15. Kudlicki, W., Odom, O. W., Kramer, G., and Hardesty, B. (1994b) J. Biol. Chem. 269, 16549-16553 [Abstract/Free Full Text]
  16. Kudlicki, W., Odom, O. W., Kramer, G., and Hardesty, B. (1994c) J. Mol. Biol. 244, 319-331 [CrossRef][Medline] [Order article via Infotrieve]
  17. Mendoza, J. A., and Horowitz, P. M. (1994) J. Protein Chem. 13, 15-22 [Medline] [Order article via Infotrieve]
  18. Merrill, G. A., Miller, D., Chirgwin, J., and Horowitz, P. M. (1992) J. Protein Chem., 11, 193-199 [Medline] [Order article via Infotrieve]
  19. Moffat, J. G., Tate, W. P., and Lovett, P. S. (1994) J. Bacteriol. 176, 7115-7117 [Abstract]
  20. Nelson, R. J., Ziegelhoffer, T., Nicolet, C., Werner-Washburne, M., and Craig, E. A. (1992) Cell 71, 97-105 [Medline] [Order article via Infotrieve]
  21. Odom, O. W., Deng, H.-Y., Dabbs, E., and Hardesty, B. (1984) Biochemistry 23, 5069-5076 [Medline] [Order article via Infotrieve]
  22. Picking, W. D., Picking, W. L., Odom, O. W., and Hardesty, B. (1992) Biochemistry 31, 2368-2375 [Medline] [Order article via Infotrieve]
  23. Ploegman, J. H., Drent, G., Kalk, K. H., Hol, W. G. J., Heinrikson, R. L., Keim, P., Weng, L., and Russell, J. (1978) Nature 273, 124-129 [Medline] [Order article via Infotrieve]
  24. Sörbo, B. H. (1953) Acta Chem. Scand. 7, 1129-1136
  25. Stewart, J. M., and Young, J. D. (1984) Solid Phase Peptide Synthesis, Pierce, Rockford, IL
  26. Tsalkova, T., Zardeneta, G., Kudlicki, W., Kramer, G., Horowitz, P. M., and Hardesty, B. (1993) Biochemistry 32, 3377-3380 [Medline] [Order article via Infotrieve]
  27. Westley, J. (1973) Adv. Enzymol. Relat. Areas Mol. Biol. 39, 327-368 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.