(Received for publication, October 20, 1994)
From the
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, GA
CAT, 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,
GA
CAT associates with mitochondria but is not
proteolytically processed and GA
CAT
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.
Renal glutaminase (GA) ()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 (
)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
-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
-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.
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.
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
GA
CAT(M). The abbreviations used are: MCS, multicloning site of the vector pBluescriptII
(SK
); MTS, mitochondrial targeting signal or
presequence.
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 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
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).
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 -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. pGA
CAT
and pGA
CAT 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.
pGA
CAT 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 -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.
Figure 4:
Translocation of
GACAT into isolated mitochondria.
[
S]Methionine-labeled
GA
CAT 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
, mature form 1; m
, 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
and m
. Interestingly, m
is
synthesized at a faster rate than m
(Fig. 5, panel B).
Figure 5:
Kinetics of import of
GACAT into isolated mitochondria. A,
labeled GA
CAT 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
, mature form 1; m
, mature
form 2.
To investigate whether m and m
arise by differential proteolytic processing of the presequence
by MPP, radiolabeled precursor of GA
CAT was
incubated for various times with purified rat liver MPP (Fig. 6). The action of MPP yields form i and the mature
subunits m
and m
. MPP processing results in the
generation of form i within the initial 3 min followed by the
simultaneous appearance of m
and m
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
, mature form
2.
When the mature forms of GACAT
were subjected to two-dimensional gel electrophoresis and fluorography (Fig. 7), m
(pI 6.1) was observed to be more basic
than m
(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
GA
CAT 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 GA
CAT 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
, mature form 1; m
, mature form 2.
Figure 8:
Processing of GACAT
in isolated mitochondria or by purified MPP.
[
S]Methionine-labeled GA
CAT
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
GA
CAT 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
GA
CAT and CAT synthesized in vitro were
subjected to two-dimensional gel electrophoresis and fluorography, the
mature form of GA
CAT 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 GA
CAT. 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 GA
CAT and CAT
synthesized in vitro. The processed forms of
GA
CAT 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.
Figure 11:
Processing of GACAT
and GA
CAT precursor in isolated
mitochondria. The GA
CAT (lanes 1 and 2) and GA
CAT (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
GA
CAT 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
, mature
form 1; m
, mature form
2.
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
and m
(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 GA
CAT
precursor and the GA precursor are essentially the same. The kinetics
of GA
CAT processing (Fig. 5) essentially
mimicked those of the GA precursor (Srinivasan et al., 1994). However, the final ratio of the subunits m
(analogous to the 66-kDa form of GA) and m
(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 GA
CAT 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
GA
CAT were subjected to two-dimensional gel
electrophoresis, m
was observed to be more basic than
m
(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
GA
CAT. 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 GA
CAT by the purified
MPP occurred at a rate equal to that observed for GA or
GA
CAT. 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 GA
CAT 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
GA
CAT 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
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
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
subunit obtained from GA
CAT.
In contrast, GA
CAT 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 GA
CAT
is identical with that of the GA
CAT which
generates m
. Nevertheless, the non-identity of the apparent
m
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.