©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of the N-terminal 118 Amino Acids in the Processing of the Rat Renal Mitochondrial Glutaminase Precursor (*)

(Received for publication, October 20, 1994)

Maithreyan Srinivasan (1) Frantisek Kalousek (2) Lynn Farrell (1) Norman P. Curthoys (1)(§)

From the  (1)Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523 and the (2)Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06520

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Rat renal mitochondrial glutaminase (GA) is synthesized as a 74-kDa cytosolic precursor that is translocated into mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of mature 66- and 68-kDa subunits, respectively. The 66-kDa subunit is derived by removal of a 72-amino-acid presequence. The structural determinants necessary for translocation and proteolytic processing were further delineated by characterizing the processing of different chimeric constructs formed by fusing various segments of the N-terminal sequence of the GA precursor to chloramphenicol acetyl transferase (CAT). GACAT is translocated and processed in isolated rat liver mitochondria or cleaved by purified mitochondrial processing peptidase (MPP) to yield an intermediate peptide and two mature subunits that are analogous to the products of processing of the GA precursor. The two reactions also occur with kinetics which are similar to those observed for processing of the GA precursor. Thus, all of the information required for the translocation and synthesis of the mature subunits of GA reside in the N-terminal 118 amino acids of the GA precursor. In contrast, GACAT, a construct that contains the GA presequence fused to CAT, is apparently translocated and processed less efficiently. It yields only two peptides that are analogous to the intermediate and 68 kDa forms of GA. In addition, GACAT associates with mitochondria but is not proteolytically processed and GACAT is slowly translocated and processed to a single peptide that is analogous to the 66 kDa form of GA. The latter results suggest that the MPP cleavage reactions which yield the GA intermediate and the 66-kDa subunit depend primarily on information that is present C-terminal to the respective sites of cleavage.


INTRODUCTION

Renal glutaminase (GA) (^1)is a mitochondrial enzyme that plays a pivotal role in pH homeostasis during metabolic acidosis (Curthoys, 1988). The active GA is a heterotetramer that is composed of three 66-kDa (^2)and one 68-kDa subunits (Haser et al., 1985). The mature peptides are derived from a single 74-kDa precursor that is processed through a 72-kDa intermediate (Perera et al., 1990; Shapiro et al., 1991). The N-terminal sequence of the 66-kDa subunit was previously determined by automated Edman degradation and compared to the sequence deduced from the GA cDNA (Shapiro et al., 1991). This information established the length of the presequence of the 66-kDa subunit of GA to be 72 amino acids. Mitochondrial presequences are enriched in arginines, leucines, and serines and usually do not contain valines, isoleucines, and negatively charged amino acids (von Heijne, 1986). Stretches of amino acids within presequences also possess the ability to fold into amphipathic alpha-helices which putatively aid in the interaction of the presequence with the receptors on the mitochondrial membrane (Roise, 1993; von Heijne, 1986). Another characteristic feature of the presequences is the presence of an arginine residue at position -2 from the mitochondrial processing peptidase (MPP) cleavage site. This arginine is conserved in many mitochondrial precursors from various species (Hartl et al., 1989). Mutation of the -2 arginine to a glycine in the presequence of ornithine transcarbamylase inhibits import and processing of the precursor in mitochondria (Horwich et al., 1986). Thus, this arginine residue is an important recognition element for MPP.

The GA presequence also contains a high content of arginines, leucines, and serines. Based upon calculated propensities (Schiffer and Edmundson, 1967), the N-terminal 16 amino acids of the GA precursor are likely to form an amphipathic alpha-helix (Shapiro et al., 1991). In addition, the GA presequence appears to exhibit a few unique features. Very few cytosolically synthesized mitochondrial proteins have presequences that are as long as the GA presequence (Hartl et al., 1989). The GA presequence lacks an arginine residue at position -2 from the site of MPP cleavage that generates the 66-kDa GA subunit. This site of cleavage also lacks the consensus amino acid residues (hydrophobic residues at position -8 and a serine, threonine, or glycine residue at position -5) for mitochondrial intermediate peptidase recognition (Isaya et al., 1991). Therefore, the role of the presequence in the translocation and processing of GA and the features of the presequence that are required for MPP recognition and cleavage were investigated by fusing different lengths of the presequence and the mature protein sequence to a passenger protein, chloramphenicol acetyltransferase (CAT). The reported data demonstrate that 1) the presequence aids in the translocation of CAT into mitochondria, 2) the information required for the synthesis of the two mature subunits of GA resides in the N-terminal 118 amino acids of the GA precursor, 3) purified MPP can catalyze formation of the mature peptides of the various fusion proteins, and 4) MPP recognition and processing of the GA presequence appears to depend primarily on information present C-terminal to the site of cleavage.


MATERIALS AND METHODS

Male Sprague-Dawley rats weighing 200-250 g were obtained from The Charles River Laboratory and maintained on Purina Rat Chow. Restriction enzymes were purchased from Boehringer Mannheim. RNAsin and T7 RNA polymerase were obtained from Promega and United States Biochemical Corp., respectively. L-[S]Methionine (specific activity >1000 Ci/mmol) was purchased from DuPont NEN. Sequenase version 2.0 kit for dideoxy nucleotide sequencing was obtained from United States Biochemical Corp. Cell-free protein synthesis was carried out using a rabbit reticulocyte lysate (Pelham and Jackson, 1976). Chemicals for SDS-PAGE were purchased from Bio-Rad. CAT antibodies were obtained from 5 Prime 3 Prime, Inc. All other chemicals were obtained from Sigma.

Synthesis of CAT Constructs

A 1.6-kilobase HindIII/BamHI fragment from pSVOCAT which contains the full coding sequence of the CAT cDNA followed by an SV40 intron and polyadenylation site was subcloned into the pBluescriptII SK plasmid (Stratagene). The ATG start codon of the CAT gene was mutated to an MluI site using the method of Kunkel et al. (Ausubel et al., 1993; Kunkel et al., 1987), and a 413-base pair KpnI-MluI fragment of pGA (Shapiro et al., 1991) which encodes the N-terminal 118 amino acids of GA was cloned upstream of the CAT sequence to generate pGACAT (Fig. 1). Another MluI site was created at position 280 base pair from the 5` end of pGACAT. MluI digestion released a 138-base pair fragment, and subsequent closing of the vector resulted in pGACAT which encodes the 72-amino acid mitochondrial presequence of GA attached to CAT. Another construct, pGACAT was created in a similar fashion. pGACAT was linearized with MluI, and the 138-base pair MluI fragment which encodes residues 73-118 of GA was obtained as described above and cloned into the linearized pGACAT to yield pGACAT. All the chimeric constructs were confirmed by dideoxynucleotide sequencing (Sanger et al., 1977). The scheme used for synthesis of the various constructs is illustrated in Fig. 1.


Figure 1: Creation of the chimeric constructs. The method for synthesis of the various chimeric cDNA constructs is illustrated. The intermediate constructs that contain two MluI restriction sites are indicated as GACAT(M) and GACAT(M). The abbreviations used are: MCS, multicloning site of the vector pBluescriptII (SK); MTS, mitochondrial targeting signal or presequence.



In Vitro Synthesis and Processing

In vitro transcriptions were carried out using T7 RNA polymerase (Krieg and Melton, 1987; Maniatis et al., 1989). The various plasmids were digested with BssHII to release the cDNA encoding the GA-CAT construct with the T7 promotor immediately upstream. In vitro transcriptions and translations were performed as described in the preceding article (Srinivasan et al., 1994).

In Vitro processing of the labeled precursors was carried out using rat liver mitochondria isolated as described previously (Cohen et al., 1985; Perera et al., 1990). The translocation reactions were performed as described in the preceding article (Srinivasan et al., 1994). The mitochondria were then pelleted, washed, solubilized in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.2% sodium dodecyl sulfate, and 0.5 mM phenylmethylsulfonyl fluoride) and incubated on ice for 10 min. The samples were centrifuged at 210,000 times g for 30 min at 4 °C, and the supernatants were incubated overnight at 4 °C with CAT antibodies. A 100-µl aliquot of a 10% suspension of protein A-Sepharose in RIPA buffer containing 0.1% bovine serum albumin was added to the samples and incubated on ice for 1 h. The protein A-Sepharose beads were pelleted by centrifugation at 10,000 times g for 1 min, washed three times in RIPA buffer, and the immunoprecipitated products were solubilized in SDS-PAGE sample buffer or two-dimensional gel electrophoresis buffer before being subjected to electrophoresis (Laemmili, 1970; O'Farrell, 1975).


RESULTS

Description of the Fusion Protein Constructs

To further characterize the differential processing reactions and to determine regions of the targeting signal that may be important in alternative processing, various fusion protein constructs were created (Fig. 2). These constructs have different segments of the GA presequence fused in frame to the N terminus of a non-mitochondrial passenger protein, CAT.


Figure 2: Chimeric constructs. The different chimeric constructs are illustrated along with a description of their properties. The dark arrows represent putative processing sites. The open arrow refers to the authentic processing site that gives rise to the 66-kDa subunit of GA. The shaded box represents the portion of the presequence that may form an amphipathic alpha-helical structure.



The first construct, pGACAT, encodes the N-terminal 118 amino acids of the GA precursor fused in frame to CAT cDNA. The N-terminal 118 amino acid sequence includes the 72 amino acid presequence and 46 amino acids from the N terminus of the 66-kDa subunit. pGACAT and pGACAT code for 72 amino acids (the entire presequence) and 31 amino acids from the N terminus of the GA precursor fused to CAT cDNA, respectively. pGACAT lacks the cDNA encoding amino acids 32 thru 72 of the presequence. The minimal changes in amino acid sequence introduced at the sites of fusion by the recombinant DNA procedures are illustrated in Fig. 3.


Figure 3: N-terminal amino acid sequence of the GA precursor and the various fusion proteins. A, GA precursor: the N-terminal 16 amino acids of the GA precursor which have the calculated propensity to fold into an amphipathic alpha-helix are represented in bold letters. The positively (+) and negatively charged(-) amino acids are indicated. The pair of tryptophans whose function is not known are indicated in lower case bold letters. The last amino acid of the presequence is indicated by an arrowhead. The N-terminal amino acids of the 66-kDa subunit that were confirmed by automated Edman degradation are double underlined, and the stretch of glutamines of unknown function are underlined. B, fusion proteins: the amino acids of GA are shown and numbered in normal letters and are underlined. The amino acids of CAT are displayed and enumerated in bold letters. The amino acid residues that were changed are indicated in bold letters and are double underlined.



Import and Processing of pGACAT into Isolated Mitochondria

A BssHII fragment of pGACAT was transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. This procedure yields a protein of the expected mass (40 kDa) which is immunoprecipitated with CAT antibodies (Fig. 4, lane 1). Incubation of the GACAT protein with isolated rat liver mitochondria yields three additional peptides of relative molecular masses of 37, 32, and 30 kDa (Fig. 4, lane 2). Based upon the mass differences and relative abundances, the lower molecular weight form, m(1), appears to correspond to the 66-kDa subunit of GA and the intermediate molecular weight form, m(2), appears to correspond to the 68-kDa subunit of GA. No translocation or processing was observed when CAT synthesized in vitro was incubated with isolated rat liver mitochondria (data not shown). Therefore, the mitochondrial uptake and processing of the chimeric protein is directed by the presequence of GA.


Figure 4: Translocation of GACAT into isolated mitochondria. [S]Methionine-labeled GACAT precursor was synthesized by in vitro translation and incubated either in the absence (lane 1) or presence (lane 2) of isolated rat liver mitochondria at 28 °C for 60 min. The samples were centrifuged and the resulting pellets were solubilized, immunoprecipitated with CAT-specific antibodies, and subjected to 10% SDS-PAGE and fluorography. The abbreviations used are: p, precursor; i, intermediate; m(1), mature form 1; m(2), mature form 2.



To determine whether the 37-kDa peptide was a true intermediate, GACAT precursor was incubated with isolated rat liver mitochondria for different periods of time (Fig. 5, panel A). Much of the precursor was converted to the 37-kDa peptide within 5 min. The levels of this peptide peaked at 10-15 min and then decreased consistent with it being an intermediate. After 45-60 min most of the precursor and intermediate were converted to the mature forms, m(1) and m(2). Interestingly, m(1) is synthesized at a faster rate than m(2) (Fig. 5, panel B).


Figure 5: Kinetics of import of GACAT into isolated mitochondria. A, labeled GACAT precursor was synthesized by in vitro translation and incubated with isolated rat liver mitochondria at 28 °C for the indicated periods of time. The samples were centrifuged, and the resulting pellets were solubilized and immunoprecipitated with CAT antibodies. B, the fluorograph obtained from a duplicate experiment was scanned using a Microscan 2000-HP Laserjet scanner (Technology Research Inc.) and the relative percentages of the intermediate and mature processed forms at each time point were plotted against the time of incubation. The abbreviations used are: p, precursor; i, intermediate; m(1), mature form 1; m(2), mature form 2.



To investigate whether m(1) and m(2) arise by differential proteolytic processing of the presequence by MPP, radiolabeled precursor of GACAT was incubated for various times with purified rat liver MPP (Fig. 6). The action of MPP yields form i and the mature subunits m(1) and m(2). MPP processing results in the generation of form i within the initial 3 min followed by the simultaneous appearance of m(1) and m(2) after approximately 10 min of incubation. The generation of the two mature subunits continues to increase at similar rates during the 1-h incubation.


Figure 6: Kinetics of MPP cleavage of GACAT precursor. S-Labeled precursor was incubated with 1 µg of purified MPP at 28 °C for the indicated time intervals (min). The different samples were analyzed by 10% SDS-PAGE and visualized by fluorography. The abbreviations used are: p, precursor; i, intermediate; m1, mature form 1; m(2), mature form 2.



When the mature forms of GACAT were subjected to two-dimensional gel electrophoresis and fluorography (Fig. 7), m(2) (pI 6.1) was observed to be more basic than m(1) (pI 5.9). The 37-kDa intermediate peptide (i) displayed the most basic pI (6.5) of the three peptides, consistent with the basic nature of the presequence. Thus, the processing of GACAT closely parallels that observed for native GA (Srinivasan et al., 1994). Therefore, different deletions of this construct should be useful to further characterize the functional significance of the processing reactions and the specificity of the MPP.


Figure 7: Two-dimensional gel electrophoresis of the processed peptides of GACAT. The processed forms of GACAT were subjected to two-dimensional PAGE analysis using a gradient of pH 7 to 4 and detected by fluorography. The more basic end of the pH gradient (+) is to the left. The radioactive samples in a vertical line on the left side of the figure are peptides that were separated only by SDS-PAGE. The abbreviations used are: p, precursor; i, intermediate; m(1), mature form 1; m(2), mature form 2.



Translocation of GACAT into Mitochondria

The GACAT precursor was synthesized in vitro, immunoprecipitated with CAT-specific antibodies, and subjected to SDS-PAGE. The precursor was observed to migrate with the expected mass of 33 kDa (Fig. 8, lane 1). Incubation of the precursor with isolated rat liver mitochondria results in translocation and processing to an apparent intermediate and a single apparent mature form (Fig. 8, lane 2). Treatment of the GACAT precursor with purified rat liver MPP gives rise to peptides that are identical in apparent mass to the intermediate and the single mature form produced by mitochondrial processing (Fig. 8, lanes 3 and 4). When GACAT was incubated with mitochondria for different times, the kinetics of import and processing appear to be slower than those of GA CAT (Fig. 9).


Figure 8: Processing of GACAT in isolated mitochondria or by purified MPP. [S]Methionine-labeled GACAT precursor was synthesized by in vitro translation and incubated at 28 °C for 60 min either in the absence (lane 1) or presence (lane 2) of isolated rat liver mitochondria or in the absence (lane 3) or presence (lane 4) of 1 µg of purified MPP. The samples were immunoprecipitated with CAT-specific antibodies and subjected to 10% SDS-PAGE and fluorography. The abbreviations used are: p, precursor; i, intermediate; m, mature form.




Figure 9: Kinetics of import of GACAT into isolated mitochondria. Labeled GACAT precursor was synthesized by in vitro translation and incubated with isolated rat liver mitochondria at 28 °C for the indicated periods of time. The samples were centrifuged and the resulting pellets were solubilized and immunoprecipitated with CAT-specific antibodies. The different samples were analyzed by 10% SDS-PAGE and visualized by fluorography. The fluorograph was scanned using a Microscan 2000 HP Laserjet scanner, and the relative percentages of the intermediate and mature processed forms at each time point were plotted against the time of incubation.



When the processed forms of GACAT were subjected to two-dimensional gel electrophoresis and fluorography, the mature subunit was observed to migrate with a pI of 6 (Fig. 10). This pI is slightly greater than the pI of CAT (pI = 5.8). When fully processed GACAT and CAT synthesized in vitro were subjected to two-dimensional gel electrophoresis and fluorography, the mature form of GACAT was observed to be larger and more basic than CAT (Fig. 10). These data indicate that the observed difference in mobility is due to the presence of basic amino acids derived from presequence of the GACAT. Thus, the observed processing reaction appears to be similar to the processing reaction that gives rise to the 68-kDa subunit of GA.


Figure 10: Two-dimensional gel electrophoresis of the processed peptides of GACAT and a combination of the processed peptides of GACAT and CAT synthesized in vitro. The processed forms of GACAT were subjected to two-dimensional gel analysis and fluorography in the absence (left) and presence (right) of CAT. The more basic end (+) of the gradient (pH 7 to 4) is to the left. The radioactive samples in a vertical line on the left side of the figure are peptides that were separated only by SDS-PAGE. The abbreviations used are: i, intermediate; m, mature form.



Mitochondrial Processing of GACAT

GACAT was synthesized by in vitro translation (Fig. 11, lane 1) and incubated with isolated rat liver mitochondria. The precursor was observed to be associated with the mitochondria but was not proteolytically processed (Fig. 11, lane 2). Thus, the initial 16 amino acids, which have the calculated propensity to form an amphipathic alpha-helix, may function as a mitochondrial targeting signal but are not sufficient for interaction with MPP required to generate the intermediate form of GA. This suggests that the mitochondrial import apparatus and the MPP recognize distinct structural elements of the presequence of GA.


Figure 11: Processing of GACAT and GACAT precursor in isolated mitochondria. The GACAT (lanes 1 and 2) and GACAT (lanes 3 and 4) precursors were generated by in vitro translation in the presence of [S]methionine. The radiolabeled precursors were incubated either in the absence (lanes 1 and 3) or in the presence (lanes 2 and 4) of isolated rat liver mitochondria. The samples were centrifuged and the resulting pellets were solubilized, immunoprecipitated with CAT-specific antibodies, and subjected to 10% SDS-PAGE and fluorography. The processed forms of GACAT are shown in lane 5 for comparison. The abbreviations used are: BI, before import; AI, after import; p, precursor; i, intermediate; m, mature form; m(1), mature form 1; m(2), mature form 2.



In Vitro Processing of GACAT

In vitro transcription and translation of pGACAT yields a peptide of the expected mass (Fig. 11, lane 3). Incubation of this precursor with isolated rat liver mitochondria yields a single mature form (Fig. 11, lane 4). This mature subunit has the same apparent molecular weight as that of the m(1) subunit produced from GACAT (Fig. 11, lane 5). The m(1) subunit corresponds to the 66-kDa peptide of mature GA. An interesting observation in this experiment is the conspicuous absence of an intermediate form which is consistent with the lack of processing of GACAT.


DISCUSSION

Presequences function to target and import precursors into mitochondria. The presequence of GA is unique in that it is processed alternatively to yield two different peptides. In order to elucidate the role played by different portions of the presequence and to delineate the structural features that are recognized by the MPP complex, various fusion protein constructs were created. The GACAT preprotein (40 kDa) behaved in a manner very similar to that of GA precursor. It was also processed in the presence of isolated rat liver mitochondria to two mature subunits, m(1) and m(2) (30 and 32 kDa, respectively) via an intermediate (37 kDa) ( Fig. 4and Fig. 5). The relative differences in size between these subunits closely corresponds to the differences in apparent molecular mass between the processed forms of GA. This implies that the processing of GACAT precursor and the GA precursor are essentially the same. The kinetics of GACAT processing (Fig. 5) essentially mimicked those of the GA precursor (Srinivasan et al., 1994). However, the final ratio of the subunits m(1) (analogous to the 66-kDa form of GA) and m(2) (analogous to the 68-kDa subunit of GA) were 2:1 and not 3:1 as observed with native GA. This might be due to inefficient processing of the fusion protein precursor owing to the propensity of the CAT domain to fold into a globular moiety (Robben et al., 1993). Alternatively, only the presequence may enter the matrix and undergo proteolytic processing while the folded CAT domain may remain outside the mitochondria. However, the latter possibility is unlikely due to the following observation. When mitochondria are treated with protease after import, the processed products are protected from proteolytic degradation (data not shown). This suggests that the processed products are inside the mitochondria. Thus, the altered ratio of products obtained from GACAT precursor may be due to either an altered conformation or reduced accessibility of the GA presequence.

Action of the purified MPP complex on GACAT precursor generated an intermediate and the two mature forms in the ratio of 1:1 (Fig. 6). Proteolytic processing of the GA precursor by the purified MPP also yielded equivalent amounts of the 68- and 66-kDa GA subunits (Srinivasan et al., 1995). Thus, additional factor(s) residing in the mitochondria may be essential to ensure the fidelity of processing. The observed kinetics of MPP processing suggest that the peptide designated as i functions as an intermediate. It appears before either of the mature peptides and then reaches an apparent steady state level. The two mature peptides appear to be synthesized at nearly equivalent rates, suggesting that they may be produced by alternative processing of the intermediate. However, the in vitro processing reaction by MPP was not very efficient as large amounts of precursor molecules were not processed, and the intermediate does not show a decrease. This problem may be alleviated by developing in vitro conditions which would further stabilize the protease. On the other hand, the GA precursor may behave like the ornithine transcarbamylase precursor which is never fully cleaved in vitro (Isaya et al., 1991). This and other experiments are currently being performed to better characterize the action of MPP on the precursors of GA and the GA chimeras. When the mature forms of GACAT were subjected to two-dimensional gel electrophoresis, m(2) was observed to be more basic than m(1) (Fig. 7). This finding is consistent with the conclusion that the two mature peptides are synthesized by alternative processing of the basic presequence. It also implies that all the information necessary for the synthesis of mature GA resides in the N-terminal 118 amino acids of the GA precursor.

When the precursor of GACAT synthesized in vitro was incubated either with mitochondria or with purified rat liver MPP, it was processed via an intermediate to a single mature subunit (Fig. 8). The translocation and processing in the presence of mitochondria were apparently less efficient than that observed with GACAT. The chimeric precursors were not synthesized in sufficient amounts in vitro to assay for CAT activity. The low yields of the fusion proteins may be due to poor translation of the extremely GC-rich GA presequence. However, significant levels of CAT activity were observed when the native CAT protein was synthesized in vitro and assayed (data not shown). This indicates that CAT synthesized de novo can fold into an active enzyme in a rabbit reticulocyte lysate. Hence the observed inefficiency of import may be due to the folding of the CAT domain. Alternatively, it may be due to cooperative folding interactions within the CAT peptide which could be initiated during progressive translocation of the presequence into mitochondria. This might block the import sites and thus prevent further uptake of precursor. A low efficiency of import could also be caused by reduced exposure of the presequence on the surface of the precursor protein. This would result in an improper or reduced association of the precursor with the mitochondria and therefore insufficient perturbation of the membrane (Roise, 1993). However, the latter possibility seems less likely since the proteolytic cleavage of GACAT by the purified MPP occurred at a rate equal to that observed for GA or GACAT. Thus, the presequences of the chimeric constructs must be readily accessible to the action of MPP in the absence of other factors that may be present in mitochondria.

The apparent molecular mass of the mature subunit derived from GACAT was greater than that of the CAT protein synthesized in vitro. This suggests that MPP identifies recognition elements of the presequence that would give rise to products that are analogous to the intermediate and the 68-kDa form of GA. This conclusion is further supported by the more basic nature of processed GACAT than CAT protein (Fig. 10). The combined data suggest that the processing reactions which yield the intermediate and the 68-kDa subunit of GA do not require the amino acids that form the N terminus of the 66-kDa subunit. However, the absence of a second mature subunit suggests that the processing reaction required for formation of the 66-kDa subunit is inhibited. Thus, the latter reaction may require amino acid determinates that are located in the N terminus of the 66-kDa subunit of GA. Hence, MPP activity may require recognition of sequence and/or structural elements that are C-terminal to the site of cleavage. In addition, the data support the hypothesis that the 68-kDa subunit is generated by MPP recognition of specific sequence and/or structural elements that are different from those required for the production of the 66-kDa subunit.

The conclusion that MPP may recognize sequence and/or structural determinants C-terminal to the site of cleavage that gives rise to the 72-kDa intermediate is supported by the finding that incubation of GACAT preprotein with isolated mitochondria resulted in the association of the precursor with mitochondria but no processing (Fig. 11). The lack of proteolytic processing of GACAT indicates that this preprotein lacks the determinants necessary for the synthesis of a peptide akin to the 72-kDa intermediate form of GA. Thus, information present C-terminal to the site of proteolysis which produces the 72-kDa intermediate may be the deciding factor for MPP recognition and cleavage. This conclusion is consistent with the characterization of the specificity of the MPP isolated from Neurospora crassa (Arretz et al., 1994). In this paper, the authors have demonstrated that MPP interacts with the C-terminal, non-amphipathic half of the presequence of cytochrome b(2) and that amino acid substitutions at positions -2 and +1 from the MPP cleavage site affect processing but not import of a chimeric protein containing the presequence of cytochrome b(2) fused to dihydrofolate reductase. In the absence of an arginine at position -2, as in the case of the GA presequence, the amino acid(s) C-terminal to the proteolytic processing site may determine the fidelity of MPP action.

Interestingly, when mitochondria were added to GACAT preprotein, the precursor was processed to a single subunit which had an apparent size equal to that of the m(1) subunit obtained from GACAT. In contrast, GACAT precursor was processed via an intermediate to a peptide corresponding to the 68-kDa subunit of GA. This suggests that the proteolytic processing of the presequence of GA by MPP to give rise to the two mature subunits of GA are independent events. The bias in the generation of the mature subunits of GA may be due to other factors that are present in the mitochondria. It is conceivable that hsp60 binding of the precursor inside mitochondria might predispose MPP to recognize and cleave one of the two sites more rapidly than the other. The available data do not establish that the proteolytic processing site of GACAT is identical with that of the GACAT which generates m(1). Nevertheless, the non-identity of the apparent m(1) forms derived from the two chimeric constructs would not affect the above conclusion.

The deletion of amino acids 32 through 72 of the presequence also decreased the apparent efficiency of the import and processing of the above precursors. This suggests that recognition elements present on the N-terminal side of the site of cleavage which yields the 66-kDa GA subunit may be necessary for efficient processing by MPP. This observation is consistent with the previously reported experiments (Isaya et al., 1991) which have characterized the structural requirements for MPP processing of other mitochondrial precursor proteins. In summary, MPP recognition and processing of the three cleavage sites in the GA presequence appear to be independent events which primarily depend upon the information present C-terminal to the sites of cleavage. However, the efficiency of processing may be effected by determinants present N-terminal to the site of proteolytic action.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK-37124 (to N. P. C.) and by DK-09527 (to F. K.). 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: Dept. of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523. Tel.: 303-491-5566; Fax: 303-491-0494.

(^1)
The abbreviations used are: GA, glutaminase; MPP, mitochondrial processing peptidase; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis.

(^2)
The relative molecular masses of the GA subunits differ slightly from previously published values. The estimates used in this study are based on the amino acid sequence deduced from the GA cDNA sequence (Shapiro et al., 1991).


REFERENCES

  1. Arretz, M., Schneider, H., Guiard, B., Brunner. M., and Neupert, W. (1994) J. Biol. Chem. 269, 4959-4967 [Abstract/Free Full Text]
  2. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Strahl, K. (1993) Current Protocols in Molecular Biology , pp. 8.1.3-8.1.6, John Wiley and Sons, Inc., New York
  3. Cohen N. S., Kyan, F. S., Kyan, S. S., Cheung, C.-W., and Raijman, L. (1985) Biochem. J. 229, 205-211 [Medline] [Order article via Infotrieve]
  4. Curthoys, N. P. (1988) in pH Homeostasis-Mechanisms and Control (H ä ussinger, D., ed) pp. 323-336, Academic Press, New York
  5. Hartl, F.-U., Pfanner, N., Nicholson, D. W., and Neupert, W (1989) Biochim. Biophys. Acta 988, 1-45 [Medline] [Order article via Infotrieve]
  6. Haser, W. G., Shapiro, R. A., and Curthoys, N. P. (1985) Biochem. J. 229, 399-408 [Medline] [Order article via Infotrieve]
  7. Horwich, A. L., Kalousek, F., Fenton, W. A., Pollock, R. A., and Rosenberg, L. E. (1986) Cell 44, 451-459 [Medline] [Order article via Infotrieve]
  8. Isaya, G., Kalousek, F., Fenton, W. A., and Rosenberg, L. E. (1991) J. Cell Biol. 113, 65-76 [Abstract]
  9. Jagus, R. (1987) Methods Enzymol. 152, 267-276 [Medline] [Order article via Infotrieve]
  10. Krieg, P. A., and Melton, D. A. (1987) Methods Enzymol. 155, 397-415 [Medline] [Order article via Infotrieve]
  11. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367 [Medline] [Order article via Infotrieve]
  12. Laemmili, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  13. Maniatis, T. Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  14. O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021 [Abstract]
  15. Pelham, H. R. B., and Jackson, R. J. (1976) Eur. J. Biochem. 67, 247-256 [Abstract]
  16. Perera, S. Y., Chen, T. C., and Curthoys, N. P. (1990) J. Biol. Chem. 265, (29) 17764-17770
  17. Robben, J., Van der Schueren, J., and Volckaert, G. (1993) J. Biol. Chem. 268, (33) 24555-24558
  18. Roise, D. (1993) in The Amphipathic Helix. (Epand, R. M., ed) pp. 257-283, CRC Press, Inc., Boca Roton, FL
  19. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  20. Schiffer, M., and Edmundson, A. B. (1967) Biophys. J. 7, (2) 121-135
  21. Shapiro, R. A., Farrell, L., Srinivasan, M., and Curthoys, N. P. (1991) J. Biol. Chem. 266, 18792-18796 [Abstract/Free Full Text]
  22. Srinivasan, M., Kalousek, F., and Curthoys, N. P. (1995) J. Biol. Chem. 270, 1185-1190 [Abstract/Free Full Text]
  23. von Heijne, G. (1986) EMBO J. 5, 1335-1342 [Abstract]

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