(Received for publication, March 27, 1995; and in revised form, August 29, 1995)
From the
Mitochondrial processing peptidase (MPP) cleaves the signal sequence from a variety of mitochondrial precursor proteins. A subset of mitochondrial proteins, including rhodanese and 3-oxoacyl-CoA thiolase, are imported into the matrix space, yet are not processed. Rhodanese signal peptide and translated protein were recognized by MPP, as both were inhibitors of processing. The signal peptide of precursor aldehyde dehydrogenase consists of a helix-linker-helix motif but when the RGP linker is removed, processing no longer occurs (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G.(1993) J. Biol. Chem. 268, 19906-19914). Disruption of the helical signal sequence of rhodanese by the addition of the RGP linker did not allow cleavage to occur. However, addition of a putative cleavage site allowed the protein to be processed. The same cleavage site was added to 3-oxoacyl-CoA thiolase, but this protein was still not processed. Thiolase and linker-deleted aldehyde dehydrogenase signal peptides were poor inhibitors of MPP. It can be concluded that both a processing site and the structure surrounding this site are important for MPP recognition.
The majority of mitochondrial proteins are synthesized on
cytosolic polyribosomes as precursor proteins with a signal sequence
that targets these proteins to the mitochondria. Import into the matrix
is usually accompanied by the removal of the signal sequence from the
precursor protein in one or two steps (Attardi and Schatz, 1988; Hartl et al., 1989). A general processing peptidase called
mitochondrial processing peptidase (MPP) ()catalyzes the
removal of most signal sequences (Ou et al., 1989). For
precursors that are cleaved in two steps, the initial part of the
signal sequence is removed by MPP, leaving an octapeptide that is
cleaved from the precursor by the mitochondrial intermediate peptidase
(Kalousek et al., 1992).
MPP, which has been purified from Neurospora crassa (Hawlitschek et al., 1988), yeast
(Yang et al., 1988), and rat liver (Ou et al., 1989),
is a metalloprotease consisting of two nonidentical subunits termed
-MPP and
-MPP (Kleiber et al., 1990; Paces et
al., 1993). This processing enzyme belongs to a new class of
endoproteases since it does not appear to fit into any of the known
classes of proteases. Recently, attention has been given to the
processing activity in plants. In contrast to mammals and fungi, MPP
processing activity is integrated into the cytochrome c reductase complex of the respiratory chain. This suggests that, in
plants, this complex functions both in respiration and protein
processing (Glaser et al., 1994).
Although the processing enzyme does not recognize a specific amino acid sequence, a positional bias of amino acids has been identified. Most notably, arginine tends to exist at position -2, -3, -10, or -11 relative to the cleavage site (Gavel and von Heijne, 1990). Based on this observation, four cleavage motifs were suggested and are listed in Table 1. A further examination of a set of mitochondrial signal sequences revealed a preference for a large non-hydrophobic residue at position -2; a slight preference for hydrophobic residues at positions +1, -1, and -5; and an additional slight preference for large residues at position -5 (Schneider et al., 1995). Despite the identification of different cleavage motifs and positional preferences for various residues, there are signal sequence cleavage sites that do not have these characteristics. Thus, since there is no obvious cleavage motif for all processed proteins, it is believed that MPP also recognizes some unidentified higher-order structure.
Until recently, the only structural
information about a mitochondrial signal peptide was its theoretical
ability to form an amphipathic -helical structure. With the use of
two-dimensional NMR, a more detailed structural evaluation of several
signal peptides has been undertaken. The structure of four signal
peptides from proteins in which the signal sequence is processed has
been determined. NMR data indicates that the rat mitochondrial aldehyde
dehydrogenase (ALDH) signal peptide forms a helix-linker-helix
(Karslake et al., 1990); the signal peptide of yeast
cytochrome oxidase subunit IV (COX IV) forms a helix followed by a
random coil (Endo et al., 1989); F
-ATPase
-subunit signal peptide forms a helix followed by a less stable
helix (Bruch and Hoyt, 1992), and the signal peptide from malate
dehydrogenase forms a flexible helix (MacLachlan et al.,
1994). It appears that each of these signal sequences has a helix with
some degree of flexibility before the MPP cleavage site.
A group of
mitochondrial matrix proteins that do not possess a cleavable signal
sequence exists. Each of these proteins has a signal necessary for
import into the mitochondria, yet lack an element(s) essential for
processing. Such proteins include rhodanese (Miller et al.,
1991), 3-oxoacyl-CoA thiolase (thiolase) (Arakawa et al.,
1987), isopropyl malate synthase (Beltzer et al., 1988),
chaperonin 10 (Ryan et al., 1994), and the -subunit of
the human electron-transfer flavoprotein (Finocchiaro et al.,
1993). Recently, the structure of four of the nonprocessed signal
peptides has been determined by NMR. It appears that rhodanese and
thiolase signal peptides are similar in that each forms a long,
continuous helix through the initial 14 or 21 residues, respectively
(Hammen et al., 1994). Additionally, if the linker from the
ALDH signal peptide is deleted, this protein is imported into
mitochondria, yet is no longer processed. The structure of this
linker-deleted signal peptide has also been solved and has been shown
to form a long, continuous helix (Thornton et al., 1993). The
signal peptide from the mitochondrial chaperonin 10 is different
because its signal peptide forms a helix-turn-helix (Jarvis et
al., 1995). Before the structure of the chaperonin 10 signal
peptide was solved, it was suggested that MPP recognizes a flexible
signal peptide since the nonprocessed signal peptides from rhodanese,
thiolase, and linker-deleted ALDH form a continuous helix (Hammen et al., 1994).
Although a flexible signal peptide may be an important recognition factor for MPP, we show here that the lack of processing of certain proteins may be more complex and other factors may be necessary for recognition by MPP. In this paper, we demonstrate that certain nonprocessed proteins lack a processing site, while other nonprocessed proteins weakly interact with MPP.
Figure 1:
The N-terminal residues of rhodanese
are sufficient for import. Residues 23-55 were deleted from
rhodanese with the resultant protein remaining competent for import. In vitro import was performed as described under
``Experimental Procedures.'' Lanes 1-5 represent rhodanese. Lanes 6-10 represent rhodanese
23-55. For each assay, translated protein (lanes 1 and 6) was incubated with rat liver mitochondria for 30
min at 30 °C. One reaction mixture was subsequently treated with
proteinase K to remove nonimported protein (lanes 4 and 9), while another reaction mixture was left untreated (lanes 3 and 8). The protected protein in lanes 4 and 9 represents imported protein. To determine
susceptibility to protease digestion, rabbit reticulocyte lysate was
treated with proteinase K (lanes 2 and 7).
Additionally, mitochondria were disrupted with 1% Triton X-100 and
subsequently treated with proteinase K to confirm the presence of
protein within the mitochondria (lanes 5 and 10).
After incubation of mitochondria with
rhodanese or rhodanese 23-55, proteinase K was added to
destroy protein not imported into the mitochondria. Presumably, the
protein remaining after proteinase K digestion resides within the
organelle. For proteins processed after import, one can observe a
smaller, cleaved protein on an SDS-polyacrylamide gel. Since rhodanese,
a known matrix-space enzyme, is not processed after import, it was
difficult to know if rhodanese was imported into the mitochondria or
remained outside the mitochondria and was resistant to proteinase K.
Newly translated rhodanese was digested by proteinase K, as shown in Fig. 1, lane 2. To confirm the presence of rhodanese
within the organelle after import, mitochondria were disrupted with
Triton X-100. Following disruption, protein released from the
mitochondrial interior was digested with proteinase K. Approximately
10% of native rhodanese remained protease-resistant after mitochondrial
disruption (Fig. 1, lane 5). However, this nondigested
protein represented a small fraction of the total imported protein. As
expected, the molecular weight of the protein imported into the
mitochondria did not change, indicating that both rhodanese and
rhodanese
23-55 were not processed.
Figure 2:
Sequence and structure of pALDH,
rhodanese, and thiolase signal peptides. A, structure of
native and linker-deleted pALDH signal peptides. The helical segments
were determined by NMR analysis in a hydrophobic environment and are underlined (Karslake et al., 1990; Hammen et
al., 1994). B, structure of native rhodanese and
rhodanese-RGP signal peptides. Underlined regions indicate
helical regions of the native rhodanese signal peptide as determined
using NMR (Hammen et al., 1994). Broken lines under
the rhodanese-RGP signal peptide indicate presumed disruption of the
helix in this signal peptide. C, the amino acid sequence of
rhodanese/R-3 and rhodanese-RGP/R-3 with an engineered R-3 (RXYS)
recognition site (Gavel and von Heijne, 1990). The processing site is underlined. D, the amino acid sequence of thiolase
with an engineered R-3 processing site. The native thiolase sequence,
GAYG, was changed to RAYS.
To confirm this lack of processing, rhodanese and rhodanese-RGP, as well as pALDH and linker-deleted ALDH, were incubated with purified MPP. In accordance with the results after import, rhodanese, rhodanese-RGP, and linker-deleted ALDH were not processed by the purified enzyme, while pALDH was processed. Hence, it appears that MPP must recognize more than a disrupted N-terminal signal sequence for processing.
Figure 3:
Rhodanese and pALDH signal peptides
inhibit pCOX IV processing. Various concentrations of pALDH ()
and rhodanese (
) signal peptides were used to inhibit the
processing of pCOX IV, which was translated in reticulocyte lysate and
incubated with purified MPP for 1 h, as described under
``Experimental Procedures.'' The percent processing was the
amount of product produced (mature protein), divided by the total
amount of protein in the reaction (mature + precursor (M +
P)). Processing in the absence of peptide was arbitrarily set at
100%.
Although the rhodanese signal peptide inhibited MPP, it remained unknown whether full-length rhodanese interacted with MPP. To address this question, fully translated rhodanese (from rabbit reticulocyte lysate) was used as a potential inhibitor of pCOX IV processing. Rhodanese, translated for 1 h in reticulocyte lysate, was capable of inhibiting pCOX IV processing, with half-maximal inhibition estimated to be approximately 10 pM. Rabbit reticulocyte lysate alone had no effect on processing, indicating that the inhibition of processing was due to rhodanese and not some other component present in the lysate. Additionally, bovine serum albumin at 10 µM, as a control for nonspecific protein interactions, had no effect on processing. Furthermore, purified commercial rhodanese, folded into its native conformation, had no effect on processing at a concentration of 10 µM. Therefore, it appears that rhodanese translated in rabbit reticulocyte lysate could interact with MPP, even though it lacked an element essential for processing.
Figure 4: Cleavage of rhodanese possessing an added MPP recognition site. In vitro import and processing assays were performed as described under ``Experimental Procedures.'' A, import of rhodanese/R-3 and rhodanese-RGP/R-3. Lanes 1-3 represent rhodanese/R-3. Lanes 4-6 represent rhodanese-RGP/R-3. Lanes 1 and 4, rhodanese/R-3 and rhodanese-RGP/R-3 translated in reticulocyte lysate, prior to import. Lanes 2 and 5, import in the absence of proteinase K treatment. Lanes 3 and 6, addition of proteinase K. B, processing after incubation with purified MPP. Lanes 1 and 2 represent rhodanese/R-3. Lanes 3 and 4 represent rhodanese-RGP/R-3. Lanes 1 and 3, translated rhodanese/R-3 and rhodanese-RGP/R-3. Lanes 2 and 4, incubation of translated protein with MPP for 1 h.
Thiolase/R-3 behaves like the nonprocessed protein, linker-deleted ALDH. Both proteins have a signal peptide that forms a helix which is followed by a processing site, yet neither is processed. To determine if either native thiolase or linker-deleted ALDH was recognized by MPP, the corresponding signal peptides were used as potential inhibitors of pCOX IV processing. Although both inhibited, inhibition was half-maximal at approximately 130 µM for thiolase peptide and approximately 210 µM for linker-deleted ALDH peptide. These concentrations were two to three times higher than the concentration necessary for rhodanese and pALDH peptides to inhibit processing. Additionally, both proteins were translated in reticulocyte lysate for use as potential inhibitors of pCOX IV processing. Unlike rhodanese, these proteins were poor inhibitors of MPP processing. Thus, not all nonprocessed proteins are capable of inhibiting MPP, even if they possess an R-3 site.
Figure 5:
Trypsin-resistance of nonprocessed
proteins. Each of the nonprocessed proteins, rhodanese (),
thiolase (&cjs2100;), thiolase/R-3 (&cjs2110;), and linker-deleted ALDH
(&cjs2113;), was translated for 30 or 60 min at 30 °C. After the
60-min translation, an aliquot was removed and incubated at 15 °C
for an additional 180 min, or a total of 240 min after the start of
translation. Protein remaining after a 1-min trypsin digestion was
quantitated and compared at the indicated time points. Experiments were
repeated in duplicate or triplicate and the results were
averaged.
The pmAspAT had been shown to become trypsin-resistant after an extended incubation at 15 °C. Therefore, after translating the nonprocessed proteins for 60 min at 30 °C, these proteins were incubated for an additional 180 min (a total of 240 min after the start of translation) at 15 °C so protein folding may occur. Each nonprocessed protein became more resistant to trypsin with time (Fig. 5), indicating that proteins were slowly folding or possibly aggregating.
Experiments with MPP reported above had been performed on proteins translated for 60 min. The nonprocessed proteins, although, were the most sensitive to trypsin after a 30-min translation. It is presumed that most of the protein remained unfolded at this time. Therefore, as a further verification that folding was not a factor for the lack of processing by MPP, processing assays were performed on these proteins which had been translated for 30 min. Processing by MPP still did not occur. Thus, it appears that these nonprocessed proteins, even in an unfolded conformation, are lacking an element essential for cleavage by MPP.
Many endopeptidases are known to exist. Some, such as trypsin or chymotrypsin, cleave nonspecifically, while others, such as thrombin, recognize a specific cleavage site. MPP is unique among endopeptidases in that it acts on hundreds of different signal sequences, yet removes the signal sequence in a single and specific cleavage reaction. MPP falls into a new class of metalloendoproteases since it lacks the zinc-binding site -His-Glu-Xaa-Xaa-His- found in the majority of metalloendoproteases, such as thermolysin (Rawlings and Barrett, 1991). The specificity and mechanism of MPP remain enigmatic in that MPP cleavage sites differ among precursor proteins and not all precursors have one of the cleavage motifs listed in Table 1. Since there is a significant amount of variability in the signal sequence among different precursors, it appears that higher structural features may also be important for MPP recognition and cleavage.
It is unknown in what state of folding a protein is processed after import into the mitochondria. It is believed that when proteins first reach the matrix space, they are maintained in a loosely folded conformation by heat shock protein 70 (hsp 70) until they are able to interact with mitochondrial heat shock protein 60 (hsp 60). Subsequent to this interaction, the protein is folded into its native conformation (Stuart et al., 1994). Does MPP recognize the loosely folded protein associated with a heat shock protein, or does MPP cleave a folded protein? Rhodanese, translated in rabbit reticulocyte lysate (which contains various cytosolic factors and heat shock proteins that maintain proteins in a loosely folded state), inhibits MPP processing, suggesting that MPP may recognize an unfolded or partially folded protein. Purified rhodanese, commercially available, is not an inhibitor of processing by MPP, even at concentrations a thousand times greater than rhodanese translated in rabbit reticulocyte lysate, showing that folded proteins may not interact with MPP.
One may speculate that the rate of folding or extent of folding of an imported protein may influence its ability to be processed. A protein which immediately folds after import may not have time to interact with MPP. Perhaps certain nonprocessed proteins are folded more quickly than are processed proteins. It may be argued that the proteins not processed in this study may fold too rapidly to allow presentation of a putative cleavage site to MPP. However, we do not feel that this is the case for two reasons. First, each of the altered proteins used in this study remained import-competent. It has been proposed that proteins are imported in a loosely-folded conformation and are only imported if they do not have an opportunity to refold or aggregate (Glick and Schatz, 1991). This suggests that each of our nonprocessed proteins were also maintained in a loosely-folded conformation. Second, we showed that the nonprocessed proteins used in this study were initially very susceptible to digestion by trypsin, but with time, underwent a slow conversion to a trypsin-resistant state. These presumably unfolded proteins were not recognized by MPP, suggesting that these nonprocessed proteins lack a feature necessary for cleavage.
Generally, signal
peptides are believed to form amphiphilic -helices, at least in a
hydrophobic milieu. The amino acid composition of mitochondrial signal
peptides is characterized by the presence of numerous basic (especially
arginine) and hydrophobic residues. Acidic residues are rarely found
(Roise and Schatz 1988; von Heijne et al., 1989). Although the
three-dimensional structure of the
- and
-subunits of MPP has
not been solved, secondary structural predictions indicate that both
subunits possess an amphiphilic, highly negative-charged helix (Paces et al., 1993). Perhaps this region of MPP interacts with the
positive-charged signal sequence. Previous studies have indicated the
importance of basic residues, not only near the site of cleavage, but
also in N-terminal regions of the signal peptide (Ou et al.,
1994). For instance, it has been shown that synthetic peptides with a
greater number of arginine residues are better inhibitors of MPP than
less highly charged peptides. Additionally, the precursor protein,
pre-adrenodoxin, which possesses a 58-amino acid signal sequence, was
examined to show the importance of arginine residues for processing.
Deletion of the positive-charged middle portion of this signal sequence
prevented processing, as did mutation of the arginine residues between
the 25th and 37th residue (Ou et al., 1994).
Although positive-charged residues in the signal sequence are important, it is not yet well understood what structural requirements are essential for MPP recognition and processing. By making a nonprocessed protein processable, and, conversely, by making a processed protein nonprocessable, we may be in a better position to elucidate characteristics necessary for the cleavage of a signal sequence from a precursor protein. ALDH was made nonprocessable by removal of the RGP linker (Thornton et al., 1993). The addition of the linker to rhodanese did not make it processable. Rhodanese, was made processable by the addition of an R-3 cleavage site (rhodanese/R-3). However, the presence of a cleavage site is not sufficient for the processing of all proteins. Both linker-deleted ALDH and thiolase/R-3 have a helix and cleavage site, yet, unlike rhodanese/R-3, these proteins were not processed. Additionally, studies using synthetic peptides as inhibitors of MPP indicate that, unlike pALDH and rhodanese peptides, linker-deleted ALDH and thiolase peptides were poor inhibitors of MPP.
The thiolase signal peptide has a low hydrophobic moment and does
not appear to be as amphiphilic as the rhodanese or pALDH signal
peptide. A helical wheel representation of the thiolase signal peptide
(the N-terminal 14 amino acids) shown in Fig. 6indicates that,
instead of being an amphiphilic helix, hydrophobic residues are found
around the entire wheel. The linker-deleted ALDH peptide, although
amphiphilic and having a relatively high hydrophobic moment, displays
one face of a helix with six hydrophobic residues, which differs from
native ALDH (Fig. 6). Perhaps MPP does not recognize a
hydrophobic signal peptide. Hydropathy plot analysis indicates that
three nonprocessed proteins, thiolase, isopropyl malate synthase, and
the -subunit of the electron-transfer flavoprotein, all have a
greater degree of hydrophobicity at their N-terminal relative to pALDH
and rhodanese (data not shown).
Figure 6: Helical wheel diagram. The helical wheel diagrams are indicated for residues 1-14 thiolase, 1-22 rhodanese, 1-16 linker-deleted ALDH, and 1-19 native pALDH signal peptides. These helical wheels correspond to helical regions determined by two-dimensional NMR. The native ALDH signal peptide consists of a helix-linker-helix. Since there are 3.6 residues per turn of a helix, the 3-amino acid linker (residues RGP) may maintain the N- and C-terminal helices in phase with each other. Therefore, the linker was included in the helical wheel representation of native ALDH. Hydrophobic residues are boxed.
Other possibilities may be
envisioned for the lack of processing of certain proteins. Rigidity of
the signal sequence or the presence of negative charges within the
signal sequence may affect processing by MPP. A greater number of
negative charges in the signal sequence may prevent an interaction with
the negative-charged regions within MPP. Three nonprocessed proteins
with negative charges in the signal sequence are isopropyl malate
synthase (Beltzer et al., 1988), chaperonin 10 (Ryan et
al., 1994), and the -subunit of the electron-transfer
flavoprotein (Finocchiaro et al., 1993). Each of these
proteins possess two negative charges within the first 20 N-terminal
residues. However, of the nonprocessed proteins used in this study,
only rhodanese has one negative charge.
Many mitochondrial signal sequences contain one or more glycine or proline residues which may confer flexibility. Perhaps the positive-charged signal sequence interacts with MPP and then must be properly positioned to allow cleavage. Without this flexibility, the cleavage site may not be accessible to catalytic residues within MPP. Structural analysis by two-dimensional NMR has demonstrated that signal peptides from cleaved precursors have a certain degree of flexibility, while signal peptides from noncleaved proteins tend to form longer, more stable helices in a hydrophobic environment (Thornton et al., 1993; Hammen et al., 1994). We previously proposed that this feature of nonprocessed proteins may be one reason for the lack of processing. However, the addition of a linker to rhodanese (rhodanese-RGP) did not allow processing, indicating that lack of flexibility may not be a major reason for native rhodanese being noncleavable.
We show here that the structure of the signal peptide and a processing site are factors essential for cleavage by MPP. However, since the known structures of signal sequences were determined in a micellar environment, which is known to induce helicity, it is difficult to make definite conclusions about structural features of the signal peptide which are necessary for MPP recognition. A signal sequence may display different structural features in different environments, such as the environment in a proteinaceous import receptor or in the matrix. Additionally, an interaction with MPP may induce a conformation in the signal sequence that is compatible with cleavage. Although the nonprocessed proteins used in this study did not appear to fold too rapidly to allow presentation of a processing site, further investigation is necessary to pinpoint structural features necessary for cleavage. As more is learned about the structure of MPP and the relationship between this structure and its interaction with a signal sequence, a greater understanding of signal peptide recognition will be obtained.