(Received for publication, October 20, 1994)
From the
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, pGA, was created which
lacks the nucleotides that encode the amino acid residues 32 through 72
of GA. When transcribed and translated in vitro, p
GA
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
GA precursors suggest that the
68-kDa subunit may function to retain the mature GA within the
mitochondrial matrix.
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) ()(Kleiber et al., 1990;
Paces et al., 1993) and the matrix
intermediate peptidase (Kalousek et al., 1992). MPP is
composed of two subunits,
and
(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 ()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 (GA) 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.
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.
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
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
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
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.
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 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).
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).
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.
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.
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.
Figure 5:
Kinetics of
import of GA into isolated rat liver mitochondria. Radiolabeled
GA 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.
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
GA precursor was
synthesized in vitro and subjected to the same treatment,
processed
GA was observed to be preferentially recovered from the
IMS fraction. Thus,
GA 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 GA. Radiolabeled GA and
GA 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
GA peptides are indicated in
kilodaltons.
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.
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 GA was recovered primarily from
the IMS with some present in the matrix (Fig. 6). This suggests
that the processed
GA 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-
GA is still targeted to the matrix and that
the deletion does not affect translocation of the preprotein. The
alternative interpretation that processed
GA 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
GA 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
GA 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
[H]proline- or
[
H]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.