©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vitro Characterization of the Mitochondrial Processing and the Potential Function of the 68-kDa Subunit of Renal Glutaminase (*)

(Received for publication, October 20, 1994)

Maithreyan Srinivasan (1) Frantisek Kalousek (2) 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
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Rat renal mitochondrial glutaminase (GA) is initially synthesized in primary cultures of proximal tubule cells as a 74-kDa precursor and is processed via a 72-kDa intermediate to generate a heterotetrameric enzyme which contains three 66-kDa subunits and one 68-kDa subunit (Perera, S. Y., Chen, T. C., and Curthoys, N. P.(1990) J. Biol. Chem. 265,17764-17770). The two mature subunits may be derived by either of two possible mechanisms: 1) alternative proteolytic processing or 2) initial synthesis of the 66-kDa subunit followed by its covalent modification to generate the 68-kDa subunit. An in vitro system was utilized to further characterize this unique processing pathway and to investigate the potential function of the 68-kDa subunit. In vitro transcription and translation of the GA cDNA yields a single 74-kDa precursor. Upon incubation with isolated rat liver mitochondria, the precursor is translocated into the mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of the 66- and 68-kDa subunits, respectively. The kinetics of the in vitro processing reaction also closely approximate the kinetics observed in cultured cells. Mitochondrial processing is blocked by o-phenanthroline, an inhibitor of the matrix processing peptidase (MPP). The 72-amino acid presequence of the 66-kDa subunit contains a large proportion of basic amino acids. Two-dimensional gel electrophoresis of mature GA established that the 68-kDa subunit is slightly more basic than the 66-kDa subunit. In addition, incubation of the 74-kDa precursor with purified MPP yields equimolar amounts of the two mature peptides. A cDNA construct, pDeltaGA, was created which lacks the nucleotides that encode the amino acid residues 32 through 72 of GA. When transcribed and translated in vitro, pDeltaGA yields a 70-kDa precursor. This precursor is processed by mitochondria to a single mature subunit with a M of 66 kDa. This observation suggests that the 68-kDa subunit is not produced by covalent modification of the 66-kDa subunit and further supports the conclusion that the two mature subunits of GA are produced by alternative processing reactions which can be catalyzed by MPP. However, the yield of products obtained in intact mitochondria may be determined by some unidentified accessory factor. Submitochondrial fractionation of imported GA and DeltaGA precursors suggest that the 68-kDa subunit may function to retain the mature GA within the mitochondrial matrix.


INTRODUCTION

Proteins destined for the mitochondrial matrix are usually synthesized as cytosolic precursors. These precursors possess N-terminal extensions called presequences (Hartl and Neupert, 1990). It has been reported that the precursors are maintained in a loosely folded conformation by cytosolic heat shock proteins (Chen and Douglas, 1987; Eilers and Schatz, 1986; Eilers et al., 1988). The loosely folded conformation facilitates the interaction of the presequence with receptors present on the outer membrane of mitochondria. The precursor is translocated into the matrix of mitochondria with the help of the dynamic import machinery of the outer membrane and inner membrane (Hannavy et al., 1993). After the precursor enters the matrix, it is bound by mitochondrial heat shock proteins before the presequence is cleaved by resident matrix proteases. There are two proteases in the matrix of rat liver mitochondria, the matrix processing peptidase (MPP) (^1)(Kleiber et al., 1990; Paces et al., 1993) and the matrix intermediate peptidase (Kalousek et al., 1992). MPP is composed of two subunits, alpha and beta (Kalousek et al., 1993) and cleaves most mitochondrial precursors in a single step to yield the mature protein. Such precursors often contain an arginine residue at position -2 from the site of MPP cleavage. MPP acts on other mitochondrial precursors to yield an N-terminal octapeptide intermediate, and matrix intermediate peptidase removes 8 amino acids from the N terminus of this intermediate to generate the mature form. The octapeptide removed by matrix intermediate peptidase frequently contains a hydrophobic residue at position -8 and a serine, threonine, or glycine residue at position -5, relative to the amino terminus of the mature protein (Isaya et al., 1991).

The rat renal mitochondrial glutaminase (GA) initiates the catabolism of glutamine and participates in the increased renal ammoniagenesis and gluconeogenesis which occur in response to metabolic acidosis (Curthoys, 1988). It is a heterotetramer and is composed of three 66-kDa subunits (^2)and one 68-kDa subunit (Haser et al.,1985). The renal isozyme of GA is also expressed in the brain, small intestine, and fetal liver. GA-specific antibodies which were affinity purified versus the isolated 66-kDa subunit react with both the 66- and 68-kDa subunits of GA (Shapiro et al., 1987). The 66- and 68-kDa subunits purified from brain GA also generate nearly identical peptide maps when subjected to partial proteolysis with Staphylococcus aureus V8 protease (Shapiro et al., 1987). Therefore, the two subunits of GA share common immunological determinants and amino acid sequences. Thus, the two GA subunits may result from alternative processing of the presequence, or one subunit may be the product of a covalent modification reaction.

Pulse-chase experiments performed on primary cultures of proximal convoluted tubular epithelial cells (Perera et al., 1990) and HTC-hepatoma cells (Perera et al., 1991) have suggested that GA is synthesized as a 74-kDa precursor and is processed via a 72-kDa intermediate to its mature forms. The reported data also indicate that the synthesis of the 66-kDa subunit occurs more rapidly and is initiated before synthesis of the 68-kDa subunit. Thus, the observed kinetics account for the final 3:1 ratio of peptides and suggest that the 68-kDa peptide may be produced by covalent modification of the 66-kDa subunit. A cDNA for GA has been isolated, cloned, and sequenced (Shapiro et al., 1991). In vitro transcription and translation of the GA cDNA yielded a single precursor of the correct molecular mass. Incubation of the precursor with isolated rat liver mitochondria results in the synthesis of the mature subunits of GA (Shapiro et al., 1991). These observations indicate that the mature form of GA arises from a single precursor protein which is generated from a single mRNA. A mitochondrial precursor giving rise to two different mature peptides is a novel discovery. Thus, investigation of GA synthesis should add a new dimension to the various known maturation pathways that nature has adopted as part of the biogenesis of mitochondria. In addition, characterization of the associated processing reaction may also shed light on the specificity of MPP and matrix intermediate peptidase action.

The processing of the GA precursor and a modified GA precursor (DeltaGA) which lacks amino acid residues 32 through 72 were further characterized in vitro using isolated mitochondria and purified processing peptidases. The resulting data suggest that 1) the 68-kDa subunit of mature GA arises by alternative processing of the presequence and not by a covalent modification of the 66-kDa subunit, 2) the entire processing reaction is catalyzed in vitro by purified MPP and is unaffected by purified matrix intermediate peptidase, and 3) the 68-kDa subunit of GA may function to retain the mature GA within the mitochondrial matrix.


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. Cell-free protein synthesis was carried out using a rabbit reticulocyte lysate (Pelham and Jackson, 1976; Jagus, 1987). Rat liver MPP was prepared as described previously (Isaya et al., 1991). The MPP preparation lacks any detectable matrix intermediate peptidase activity. GA antibodies were purified using DEAE Affi-Gel Blue (Bio-Rad) according to a previously described method (Shapiro et al., 1987). 3,3`,5,5`-Tetramethyl benzidene stabilized substrate was obtained from Promega Corporation. Chemicals for sodium dodecyl sulfate-polyacrylamide gel electrophoresis were purchased from Bio-Rad. All other chemicals were obtained from Sigma. Antibodies for cytochrome oxidase complex III and monoamine oxidase were obtained from Dr. E. Bonilla (Columbia University) and Dr. Akio Ito (Kyushu University), respectively.

Synthesis of pDeltaGA

A 300-base pair KpnI-MluI fragment was isolated by performing a KpnI digestion and a partial digestion with MluI on pGACAT (Srinivasan et al., 1995). This fragment was cloned into KpnI-MluI-digested pGA to obtain pDeltaGA.

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 GA or DeltaGA with the T7 promotor immediately upstream. The fragments were purified from a low melting point agarose gel. A typical transcription reaction contained a final concentration of 40 mM Tris-HCl, pH 7.5, 2 mM spermidine, 500 units/ml RNAsin, 10 mM NaCl, 10 mM dithiothreitol, 80 µg/ml bovine serum albumin, 22 mM MgCl(2), 4 mM each of ATP, CTP, GTP, and UTP, 0.1% Triton X-100, 0.5 µg of template, and 200 units/ml of T7 RNA polymerase. The reaction was allowed to proceed for 90 min at 30 °C. A fraction of the reaction was analyzed by agarose gel electrophoresis to determine the integrity of the RNA. The reaction mixture containing the transcribed RNA was stored frozen at -70 °C, and aliquots were used for in vitro translations.

The synthesis of proteins in vitro was carried out using a rabbit reticulocyte lysate (Pelham and Jackson, 1976; Jagus, 1987). A 100-µl translation reaction included a final concentration of 100 mM KCl, 0.5 mM magnesium acetate, 0.15-0.25 mM amino acid mixture minus methionine, 10 mM phosphocreatine, 0.2 units of creatine phosphokinase, 40 units of RNAsin, 10 µM hemin hydrochloride, 6 µl of [S]methionine (30 µCi), and 75 µl of mRNA-dependent lysate. A 1-3-µl aliquot of the reaction mixture containing approximately 2 µg/µl of the transcribed RNA was pretreated with 3 mM methyl mercury hydroxide for 5 min at 25 °C. The RNA sample was then added to 100 µl of translation reaction mixture and incubated at 30-32 °C for 60 min.

In vitro processing of the labeled precursors were carried out using rat liver mitochondria isolated as described previously (Cohen et al., 1985; Perera et al., 1991). The translocation reaction mixture containing 39 µl of mRNA-dependent reticulocyte lysate, 4 µl of 1 M phosphocreatine, 2 µl of creatine phosphokinase (200 units/ml), 5 µl of 30 mM ATP, 50 µl of freshly isolated mitochondria, and 50 µl of in vitro translate containing the labeled precursor protein was incubated at 28 °C for 5-60 min. Following translocation, the mitochondria were pelleted by centrifugation for 5 min at 10,000 times g, washed, and solubilized in Triton X-100 containing rat brain extract (Haser et al., 1985). Rat brain extract contains brain GA which functions as a carrier and facilitates the immunoprecipitation of GA from in vitro translation and processing reactions (Perera et al., 1990). The samples were centrifuged at 210,000 times g for 30 min to remove any particulate material and incubated with GA-specific antibodies. The samples were shaken on a rotary shaker at 4 °C for 120 min or left overnight at 4 °C. The samples were gently overlaid on a 0.5 M sucrose cushion (500 µl) and centrifuged at 16,000 times g for 10 min at 4 °C or room temperature. The pellets were washed twice with 1% Triton X-100 in 10 mM Tris acetate, pH 8.0, and once with 0.1% lithium dodecyl sulfate in homogenate buffer (0.33 M sucrose, 0.02 M Tris, 0.2 mM EDTA, pH 7.5) before the addition of sample buffer. The samples were then subjected to SDS-PAGE and fluorography.

Two-dimensional Gel Electrophoresis of Brain GA

Brain GA was immunoprecipitated with GA antibodies from a solubilized rat brain extract (Haser et al., 1985) containing 0.7 units of GA. The immunoprecipitates were collected by layering the solution over 0.5 M sucrose and centrifuging the samples for 10 min at 16,000 times g in a microfuge. The pellets were washed twice with 1% Triton X-100 in 10 mM Tris acetate, pH 8, and once with 0.1% lithium dodecyl sulfate in homogenate buffer. The washed pellets were dissolved in sample buffer and subjected to two-dimensional gel electrophoresis (O' Farrell, 1975).

In Vitro Processing of the GA Precursor with MPP

A 5-µl aliquot of the in vitro translation reaction was incubated with 1 µg of purified MPP at 28 °C for 1 h (Isaya et al., 1991). The processed products were subjected to 10% SDS-PAGE (Laemmili, 1970) and fluorography.

Submitochondrial Fractionation

Mitochondria were prepared as described above and suspended in SEOMK buffer (Glick et al., 1992). A 200-µl volume of 0.375% digitonin in SEOMK buffer was added to 50 µl of mitochondria and mixed gently. The suspension was incubated on ice for 15 min and mixed once during the incubation. After the incubation the mitochondria were centrifuged at 10,000 times g in an SS-34 rotor. The pellet and supernatant obtained at this step were processed differently. The supernatant was transferred to another tube and centrifuged at 370,000 times g (83,000 revolutions/min) in a Beckman TLA-100 rotor for 30 min. The supernatant obtained after this step is the intramembrane space (IMS) fraction, and the pellet is the outer membrane fraction. Aliquots of both fractions were suspended in appropriate volumes of SDS sample buffer.

The pellet obtained in the first step contains mitoplasts which contain about 85% of the total mitochondrial protein. The mitoplasts were suspended in SEOMK buffer to a protein concentration of 40 mg/ml and were treated with Lubrol to a final concentration of 0.18 mg/mg of mitochondrial protein. The mitoplasts were incubated on ice for 30 min and centrifuged at 370,000 times g for 30 min in a Beckman TLA-100 rotor. The supernatant is the matrix fraction and the pellet is the inner membrane fraction. Both fractions were dissolved in suitable volumes of SDS sample buffer. Samples representing equivalent proportions of each of the submitochondrial fractions were subjected to SDS-PAGE and probed with antibodies for monoamine oxidase (outer membrane marker), cytochrome oxidase complex III (inner membrane marker) and ornithine transcarbamylase (matrix marker).

Western Blotting

Immunoblotting of proteins were performed essentially as described (Towbin et al., 1979; Haser et al., 1985; Shapiro et al., 1987). Briefly, the mitochondrial proteins corresponding to the various fractions were resolved on 10% SDS-PAGE and electroblotted onto a nitrocellulose or Immobilon-P membrane. The membrane was blocked with 3% gelatin and probed with the appropriate primary antibodies. The dilutions used were 1/1000 for monoamine oxidase and ornithine transcarbamylase and 1/5000 for cytochrome oxidase complex III. Goat anti-rabbit antibodies coupled to horseraddish peroxidase were used to bind to the primary antibodies, and the various mitochondrial markers were detected by color development due to horseradish peroxidase action on a chromogenic substrate (3,3`,5,5`-tetramethyl benzidene stabilized substrate).


RESULTS

Import Competence of Mitochondria

An in vitro study of the different processing reactions of GA requires that the translocation into mitochondria and the processing reactions conform to certain established criteria. Therefore, the GA precursor was incubated with isolated rat liver mitochondria under a variety of conditions that would determine the validity and reproducibility of the import process. We have previously shown that rat kidney mitochondria are unsuitable for in vitro processing studies owing to the presence of an uncharacterized protease (Perera et al., 1990).

The radiolabeled GA precursor was synthesized in vitro, and 50 µl of the translation reaction mixture was immunoprecipitated and subjected to SDS-PAGE (Fig. 1, lane 1). Equivalent volumes of the translation reaction were incubated with freshly thawed reticulocyte lysate and isolated rat liver mitochondria at 28 °C. Import and processing of the GA precursor requires the addition of fresh reticulocyte lysate (Perera et al., 1990). After incubation, the mitochondria were sedimented, and both the supernatant and pellet fractions were individually immunoprecipitated with GA antibodies. When the 74-kDa precursor was incubated with mitochondria for 5 min (Fig. 1, lanes 2 and 3), the pellet contained predominantly the 72 kDa intermediate form of GA while the precursor remained in the supernatant, suggesting that the translocated precursor is rapidly processed to the intermediate. The 66 kDa form was also present in small amounts in the pellet but the 68 kDa form was not detected. These data corroborate the previous observation that the 66-kDa subunit appears to be synthesized at a faster rate than the 68-kDa subunit (Perera et al., 1990). When the incubation was allowed to continue for 90 min (Fig. 1, lanes 4 and 5), the 68- and 66-kDa mature subunits of GA and a small amount of the intermediate were observed in the mitochondrial pellet. In addition, very little of the precursor remained in the supernatant. These observations support the validity of the precursor-product relationship and indicate that the in vitro import reaction reproduces the processing reactions observed in vivo.


Figure 1: In vitro processing of mitochondrial GA. The GA precursor was obtained by in vitro translation in the presence of [S]methionine. The different samples were centrifuged and the pellets (P) and supernatants (S) were solubilized and immunoprecipitated separately with GA-specific antibodies. The immunoprecipitates were subjected to SDS-PAGE and fluorography. Lane 1, precursor before import (BI). Lanes 2 and 3, precursor incubated with isolated mitochondria for 5 min. Lanes 4 and 5, precursor incubated with isolated mitochondria for 90 min. The relative molecular masses of the different processed forms are indicated in kilodaltons.



Various controls established that the in vitro processing reaction requires a membrane potential, the presence of external ATP, and the involvement of proteinaceous receptors. The GA precursor is also not processed when the mitochondria were pretreated with 2 mMo-phenanthroline, an inhibitor of the MPP (Kleiber et al., 1990).

Kinetics of Translocation of GA Precursor into Isolated Mitochondria

To further establish the validity of the in vitro processing system, equal volumes of the translation reaction containing the radiolabeled GA precursor were incubated with isolated rat liver mitochondria for different periods of time. The processed products present in the mitochondrial pellet were immunoprecipitated with GA antibodies and subjected to SDS-PAGE and fluorography (Fig. 2A). The intensities of the various products in the fluorogram were quantitated, expressed as a percentage of the total amount of precursor utilized per import reaction and plotted versus time of incubation (Fig. 2B). Very little of the 74-kDa precursor was recovered from the pelleted mitochondria. However, with increasing time the amount of the 72-kDa peptide increases, peaks at about 10 min, and then decreases. This behavior is characteristic of an intermediate. The levels of the 66- and 68-kDa peptides increase with time and plateau. However, the 66-kDa subunit is synthesized at a faster rate than the 68-kDa subunit. The difference in rates of synthesis accounts for the 3:1 difference in the relative levels of the 66- and 68-kDa mature peptides.


Figure 2: Kinetics of translocation of GA precursor. A, labeled GA precursor was synthesized by in vitro translation and incubated with isolated rat liver mitochondria at 28 °C for the indicated periods of time, as described under ``Materials and Methods.'' The mitochondria were centrifuged, and the processed products in the mitochondrial pellet were immunoprecipitated with GA antibodies. The samples were analyzed by SDS-PAGE and visualized by fluorography. The relative molecular masses of the different processed forms are indicated in kilodaltons. B, the fluorograph in A was analyzed using a Microscan 2000 scanner, and the intensities of the various processed forms relative to the amount of precursor added were plotted against the time of incubation.



Relationship between the 66- and the 68-kDa Subunits of GA

To determine whether the two subunits differ with respect to net charge, GA was immunoprecipitated from a solubilized rat brain extract and subjected to two-dimensional-PAGE (O'Farrell, 1975) with isoelectric focusing in one dimension and SDS-PAGE in the second dimension. After silver staining, the 68-kDa subunit was observed to be more basic than the 66-kDa subunit. The pI of each subunit was estimated from a plot of pH versus distance migrated (Fig. 3). The experimentally determined pI of 6.1 for the 66-kDa subunit agrees well with the pI of 6.0 calculated from the amino acid sequence. The experimentally obtained pI for the 68-kDa subunit was 6.4. A mature GA peptide which retained 20 or 30 additional amino acid residues from the C-terminal portion of the presequence would manifest a calculated pI of 6.3 or 6.4, respectively. Thus, the observed pI suggests that the 68-kDa subunit is produced by alternative processing of the GA precursor.


Figure 3: Two-dimensional gel analysis of mature GA. GA was immunoprecipitated from solubilized rat brain extract with GA-specific antibodies and subjected to two-dimensional gel electrophoresis with isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. The bands were visualized by silver staining. The distances migrated by the mature subunits was determined. The pI of the 66- and 68-kDa subunits were extrapolated from the pH gradient.



MPP Action on GA Precursor

MPP is the matrix protease that removes the N-terminal targeting signal from most cytosolically synthesized precursors of mitochondrial proteins. To further investigate the alternative processing hypothesis, radiolabeled GA precursor was synthesized in vitro and incubated either in the absence or presence of rat liver MPP (Fig. 4). Addition of MPP generates the two mature subunits of GA. The 72-kDa intermediate is masked by the intensity of the precursor and hence is not discernible on the gel. This observation confirms that the 66- and 68-kDa subunits arise by alternative processing of the N-terminal presequence. The addition of matrix intermediate peptidase neither cleaves the GA precursor nor alters the products generated by MPP (data not shown).


Figure 4: MPP action on the GA precursor. S-Labeled GA precursor was synthesized by in vitro translation as described under ``Materials and Methods'' and incubated either in the absence (lane 1) or presence (lane 2) of 1 µg of purified rat liver MPP at 28 °C for 1 h. The products were subjected to 10% SDS-PAGE and fluorography. The relative molecular masses are indicated in kilodaltons.



Processing of DeltaGA

In vitro transcription and translation of the DeltaGA cDNA yields a 70-kDa precursor. To investigate its rate of translocation and processing, a kinetic analysis of DeltaGA uptake and cleavage was performed. S-Labeled precursor was synthesized by in vitro translation and was incubated with isolated rat liver mitochondria for the indicated periods of time. The mitochondria were pelleted by centrifugation, and the resuspended samples were immunoprecipitated and processed for SDS-PAGE and fluorography (Fig. 5). A single processed product is observed within 5 min which continues to accumulate through 45 min of incubation. This single product exhibits an electrophoretic mobility identical to that of the 66-kDa subunit of GA. A notable feature of this experiment is that an intermediate form is not synthesized. This implies that the synthesis of the intermediate form depends on the deleted stretch of amino acids. This also suggests that the synthesis of the intermediate form is not obligatory for the synthesis of the 66-kDa subunit.


Figure 5: Kinetics of import of DeltaGA into isolated rat liver mitochondria. Radiolabeled DeltaGA precursor was obtained by in vitro translation and was incubated with isolated rat liver mitochondria for the indicated periods of time. The different samples were centrifuged, and the resulting pellets were solubilized and immunoprecipitated with GA antibodies and subjected to 10% SDS-PAGE and fluorography. The processed products of GA are also shown for comparison.



Intramitochondrial Location of Mature GA

Earlier experiments have indicated that rat renal GA is located either in the matrix or is associated with the inner leaflet of the inner mitochondrial membrane (Curthoys and Weiss, 1974). The current experiments were performed to characterize the mitochondrial distribution of the in vitro processed products of GA and DeltaGA precursors. Isolated mitochondria were subfractionated into four compartments with digitonin/Lubrol-WX. Immunoblot analysis using antibodies for monoamine oxidase (outer membrane marker), cytochrome oxidase complex III (inner membrane marker), and ornithine transcarbamylase (matrix marker) showed a preferential enrichment of the various marker enzymes in their respective compartments (data not shown).

S-Labeled GA precursor was synthesized by in vitro translation and imported into isolated rat liver mitochondria. The mitochondria were then subfractionated with digitonin/Lubrol-WX. Aliquots of the outer membrane, IMS, inner membrane, and matrix fractions were immunoprecipitated with GA antibodies before performing SDS-PAGE and fluorography (Fig. 6). The processed GA peptides were found preferentially enriched in the matrix fraction suggesting that newly synthesized GA is initially localized in the matrix. When radiolabeled DeltaGA precursor was synthesized in vitro and subjected to the same treatment, processed DeltaGA was observed to be preferentially recovered from the IMS fraction. Thus, DeltaGA is not retained in the matrix and leaks into the IMS. Hence the 68-kDa subunit may function to retain the GA inside the matrix either by participating in the assembly of the active tetrameric enzyme or by facilitating its association with other proteins.


Figure 6: Intramitochondrial location of mature GA and DeltaGA. Radiolabeled GA and DeltaGA precursors were obtained by in vitro translation and were incubated separately with isolated rat liver mitochondria for 90 min. The mitochondria were pelleted and subjected to submitochondrial fractionation with digitonin/Lubrol. The various fractions were immunoprecipitated with GA antibodies and subjected to 10% SDS-PAGE and fluorography. The abbreviations used are: OM, outer membrane; IMS, intramembrane space; IM, inner membrane; and M, matrix. The size of the GA and DeltaGA peptides are indicated in kilodaltons.




DISCUSSION

Translocation and Processing of GA Precursor in Mitochondria

When the GA precursor was incubated with freshly thawed mRNA-dependent reticulocyte lysate and freshly isolated rat liver mitochondria, the precursor was translocated into the mitochondria and processed to its mature subunits (Fig. 1, lanes 4 and 5). In the absence of added reticulocyte lysate, the import and processing reactions are significantly inhibited (data not shown). Thus, the reticulocyte lysate must possess factors that are required for GA import. When the precursor of GA was incubated with isolated mitochondria for increasing periods of time, the amount of precursor decreased to less than half its initial level within 5 min and to an undetectable level after 90 min of incubation. The 72-kDa intermediate rose sharply relative to the GA precursor, peaked at about 10 min, and then declined. This decrease in the level of the intermediate was matched by a commensurate increase in the amounts of the 66- and 68-kDa subunits of mature GA. The 66-kDa subunit accumulates at a faster rate than the 68-kDa subunit which accounts for the final 3:1 distribution of the 66- and 68-kDa subunits (Fig. 2). It is not clear at present how or why the 66-kDa subunit is synthesized in greater amounts. However, the observed kinetic profile confirms previous experiments and further establishes the origin of the two mature subunits as being produced from one precursor derived from a single mRNA (Shapiro et al., 1991).

When mitochondria were treated with protease after translocation, the mature subunits were protected from degradation. Thus, the processed forms were present only inside the mitochondria. This observation also indicates that all of the processing reactions occur within mitochondria and are not due to leakage of the MPP into the external medium. Hence the mitochondrial preparation was intact and could be used in translocation studies.

Intersubunit Relationship of GA

Investigation into the requirement of the 68-kDa subunit in the structure-function of GA necessitates a thorough understanding of the possible relationship between the 66- and 68-kDa subunits of GA. It is known from prior studies that the two peptides share common immunological determinants and contain large portions of similar amino acid sequences (Shapiro et al., 1987). Therefore, the possibility of the 68 kDa form being a covalently modified version of the 66 kDa form was considered (Perera et al., 1990). This hypothesis was proposed as a short term regulatory pathway to explain the observed increase in flux through renal GA upon acute acidosis and was supported by the observation that many mitochondrial proteins are fatty acylated (Vijayasarathy et al., 1989). Two potential covalent modifications, fatty acylation and phosphorylation were investigated. In vitro labeling experiments suggested that the 68-kDa peptide is neither derived by fatty acylation nor by phosphorylation of the 66-kDa subunit (data not shown). These observations are consistent with the previous conclusion that the increase in activity of GA during chronic metabolic acidosis is due to increased synthesis of the enzyme and not due to a modification of the existing enzyme (Curthoys et al., 1976).

When the mature subunits of GA were subjected to two-dimensional electrophoresis, the two subunits were observed to possess a different net charge (Fig. 3). The 68-kDa subunit was more basic. This also rules out the possibility that the 68-kDa peptide arises from phosphorylation of the 66-kDa peptide since this process would decrease the pI of the peptide. Given the high content of basic amino acids contained in the GA presequence (Shapiro et al., 1991), the observed difference in pI supports the hypothesis that the two subunits arise by alternative processing of the presequence.

Incubation of the GA precursor with purified rat liver MPP yields both of the mature subunits of GA (Fig. 4). Furthermore, mitochondrial processing of the GA precursor is blocked by o-phenanthroline, a known inhibitor of MPP (Kleiber et al., 1990). Matrix intermediate peptidase does not cleave the presequence of GA (data not shown). It is unclear why MPP does not cleave all the precursor molecules to the 66-kDa subunit. A way to test this would be to treat the 72-kDa intermediate or the 68-kDa subunit with MPP individually. However, it was not possible to obtain sufficient amounts of the 72-kDa intermediate and the 68-kDa subunit from the in vitro processing reaction to perform the in vitro analysis with MPP. Regardless of the mechanism which results in the synthesis of the 66- and 68-kDa subunits, the current studies strongly support the conclusion that the two subunits result from alternative processing catalyzed by MPP.

Alternative processing of the GA precursor by MPP invokes a critical role for the 68-kDa subunit in the structure and function of GA. In addition, elucidation of the role of the 68-kDa subunit may shed light on the structural features of the presequence that are recognized by the MPP complex. Furthermore, the formation of the 68-kDa subunit per se is intriguing because, as the precursor traverses the mitochondrial membranes, the site of cleavage that would give rise to the 68 kDa form would be exposed to the action of MPP first and yet the rapid synthesis of the 66-kDa subunit occurs before the synthesis of the 68-kDa subunit. This observation can be interpreted as follows. The preprotein is completely taken into the mitochondria before MPP can proteolytically process the presequence. Once inside the mitochondria, MPP may be biased to recognize elements of the presequence that are essential for the synthesis of the 66 kDa form and/or the processing reaction that gives rise to the 68-kDa subunit is made rate-limiting due to other accessory factors. Such effects result in increased synthesis of the 66-kDa subunit and hence the synthesis of the 68 kDa form is not observed during early time periods. The processing of the GA presequence by MPP may be regarded as unbiased because in vitro incubation of the precursor with MPP yields both subunits in approximately equal amounts. Therefore, synthesis of the 68-kDa subunit might be a crucial step in the assembly or localization of GA.

The function of the 68-kDa subunit was investigated by using a deletion construct of the GA cDNA which encodes a precursor that is processed to yield only the 66-kDa subunit (Fig. 5). The processing reaction was extremely slow suggesting that the deleted amino acid sequence is required for efficient MPP processing in the mitochondria. However, the synthesis of the 68-kDa subunit was completely obliterated. This suggests that the larger subunit is not produced by covalent modification of the 66-kDa subunit and further supports the alternative processing hypothesis. It is not known whether the N terminus of the putative 66-kDa subunit is identical to that of the 66-kDa subunit of mature GA. However, this construct was used to investigate a possible function of the 68-kDa subunit.

Submitochondrial fractionation experiments have suggested that GA is loosely associated with the inner leaflet of the IM. However, GA translocated in vitro was largely recovered from the matrix fraction (Fig. 6). This suggests that GA synthesized de novo is retained in the matrix of the mitochondria. In contrast, the processed form of DeltaGA was recovered primarily from the IMS with some present in the matrix (Fig. 6). This suggests that the processed DeltaGA had leaked from the matrix space. Hence the 68-kDa subunit might be involved in retaining GA in the matrix by helping GA to fold into the appropriate quaternary structure. This would imply that pre-DeltaGA is still targeted to the matrix and that the deletion does not affect translocation of the preprotein. The alternative interpretation that processed DeltaGA was directly translocated to the IMS without going through the matrix seems unlikely because of three reasons: 1) such a premise would predict the complete absence of the processed forms in the matrix, 2) the required MPP is present only in the matrix, and 3) proteins that are processed by MPP and eventually accumulate in the IMS require the presence of hydrophobic stop-transfer sequence (Glick et al., 1992) and an additional cleavage by an intramembrane peptidase (Nunnari et al., 1993). The amino acid sequence of DeltaGA lacks such membrane spanning regions. Thus, the formation of the 68-kDa subunit may not only be required for efficient processing of the precursor by the MPP complex but also for retention of the mature enzyme at the correct intramitochondrial location. The large amount of DeltaGA precursor associated with each submitochondrial fraction may reflect the slow rate of processing.

GA may be compartmentalized with other enzymes in the matrix or peripherally associated with other enzymes of the inner membrane (Fahien et al., 1989; Schoolwerth and LaNoue, 1980). Such interactions could be facilitated if the N terminus of the 68-kDa subunit contains the 2 tryptophan residues (residues 57 and 58) of the presequence of GA (Shapiro et al., 1991). Tryptophan residues are present in low abundance in soluble proteins, and the occurrence of two adjacent tryptophan residues implies a critical function (Schiffer et al., 1992). A recent study suggests that tryptophan residues present in mitochondrial creatine kinase aid in catalytic activity, interfacial interactions between dimers, and possibly in the formation of octameric structures (Gross et al., 1994). Thus, these 2 tryptophan residues might aid in the hydrophobic association of GA with other inner membrane or matrix enzymes.

To further establish that renal mitochondrial GA is a product of alternative processing of its presequence, we attempted to sequence the 68-kDa subunit. We also attempted to radiosequence the 68-kDa subunit generated from the in vitro processing of [^3H]proline- or [^3H]leucine-labeled GA precursor. However, neither the amount of peptide isolated nor the level of radioactivity incorporated was sufficient to obtain interpretable sequencing data. Therefore, we are currently developing procedures to overexpress GA and the various chimeric constructs of GA (Srinivasan et al., 1995) in bacteria. Treatment of the purified precursor with MPP should yield an amount of the 68-kDa subunit sufficient for sequencing.


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: MPP, matrix processing peptidase; GA, glutaminase; PAGE, polyacrylamide gel electrophoresis; IMS, intramembrane space.

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


ACKNOWLEDGEMENTS

We thank Laurie Minamide for her assistance with two-dimensional gels.


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