(Received for publication, September 8, 1994; and in revised form, October 27, 1994)
From the
Chaperonin 10 (Cpn10) is one of only a few mitochondrial matrix
proteins synthesized without a cleavable targeting signal. Using a
truncated form of Cpn10 and synthetic peptides in mitochondrial import
assays, we show that the N-terminal region is both necessary and
sufficient for organellar targeting in vitro. To elucidate the
structural features of this topogenic signal, peptides representing
residues 1-25 of rat Cpn10 were synthesized with and without the
naturally occurring N-terminal acetylation. H NMR
spectroscopy in 20% CF
CH
OH, H
O
showed that both peptides assume a stable helix-turn-helix motif and
are highly amphiphilic in nature. Chemical shift and coupling constant
data revealed that the N-terminal helix is stabilized by N-acetylation, whereas NOE and exchange studies were used to
derive a three dimensional structure for the acetylated peptide. These
findings are discussed with respect to a recent model predicting that
targeting sequences forming a continuous
-helix of more than 11
residues cannot adopt a conformation necessary for proteolysis by the
matrix located signal peptidases (Hammen, P. K., Gorenstein, D. G., and
Weiner, H.(1994) Biochemistry 33, 8610-8617).
Of the several hundred proteins found in mitochondria, only a small subset (13 in mammals) are encoded by mitochondrial DNA. The remaining nuclear encoded proteins are synthesized on cytosolic ribosomes and transported into their correct mitochondrial compartment because of specific topogenic signals (for review see Hartl et al., 1989). Signals for targeting to the matrix have been characterized in considerable detail. They are usually located at the N terminus of the protein and typically consist of 15-40 amino acids, which generally are proteolytically removed by matrix-located proteases. These transient topogenic signals (referred to as presequences) have been defined in a large number of mitochondrial precursors, and although no apparent sequence homologies have been established, they are rich in hydrophobic and basic residues. The absence of sequence homologies suggests that recognition of the topogenic signals by the mitochondrial import and processing apparatus is caused by a common structural feature, most likely an amphiphilic positively charged structure (von Heijne, 1986).
A small number of
mitochondrial matrix proteins, namely rhodanese (Miller et
al., 1991), 3-oxoacyl-CoA thiolase (Amaya et al., 1988),
the -subunit of the human electron transfer flavoprotein
(Finocchiaro et al., 1993), the mitochondrial ribosomal
protein YmL8 (Matsushita and Isono, 1993) and chaperonin 10 (Rospert et al., 1993; Ryan et al., 1994) are unique because
sequence data have established that the targeting signal remains in
these proteins following import. Little is known about these topogenic
signals, and although they are believed to reside within the N-terminal
region, this has only been demonstrated experimentally for the
14-16 N-terminal residues of 3-oxoacyl-CoA thiolase (Arakawa et al., 1990).
We previously demonstrated that rat Cpn10 ()is synthesized without a cleavable presequence and yet is
imported into mitochondria in a fashion characteristic of archetypical
mitochondrial precursors (Ryan et al., 1994). We noted that
the N-terminal sequence possesses a high amphiphilic potential between
residues 1 and 15 and considered this to act as a mitochondrial
targeting signal. It was further suggested that the in vivo acetylation of Ala
enhances the efficiency of this
signal through an increase of the helical and amphiphilic potential,
which in turn augments the ability of the protein to interact with and
traverse the mitochondrial membrane.
In this study we demonstrate
that the targeting information resides within the N-terminal region of
mature Cpn10. To further characterize this topogenic signal, peptides
representing the first 25 amino acids of Cpn10 were synthesized with
(Ac-Cpn10) and without
(Cpn10
) N-terminal acetylation. The peptides
inhibited mitochondrial import of pre-ornithine transcarbamylase
(p-OTC), and their structures in aqueous trifluoroethanol (20% v/v)
were determined using NMR spectroscopy. Both peptides adopted stable
helix-turn-helix motifs. An increased N-terminal helix stability in the
acetylated peptide (Ac-Cpn10
) suggests that this
post-translational modification assists mitochondrial import, although
to only a subtle extent.
The analogous peptide was also synthesized with N-acetylation (Ac-Cpn10). Both peptides
were purified to homogeneity by reverse-phase HPLC, and their identity
was confirmed by amino acid sequencing, amino acid analysis, and
electrospray mass spectrometry ((M
Cpn10
: 2923.4 ± -0.4; expected
2923.4) (M
Ac-Cpn10
2965.3
± 0.2; expected 2965.4)).
For import competition studies using
Ac-Cpn10 and Cpn10
, peptides
in 10 µl of 20% (v/v) isopropanol were mixed with freshly
synthesized [
S]p-OTC in 10 µl of rabbit
reticulocyte lysate (Promega) and added to rat liver mitochondria (50
µg of protein) in 40 µl of import buffer. Import of
[
S]p-OTC proceeded at 30 °C for 15 min
before mitochondria were pelleted and subjected to SDS-PAGE in 12%
Tris-glycine gels (Fling and Gregerson, 1986) followed by PhosphorImage
analysis (Molecular Dynamics).
Before import reactions, porcine I-Cpn10
and
I-Cpn10
were denatured by the
addition of saturating amounts of urea. Four µl (6000 cpm) were
withdrawn and mixed simultaneously with
H-p-OTC in 20
µl of rabbit reticulocyte lysate plus rat liver mitochondria (100
µg of protein) in 76 µl of import buffer. Import proceeded at
30 °C for 60 min before electrophoresis in Tris-Tricine gels
(Schägger and von Jagow, 1987) and quantitation by
PhosphorImage analysis.
H NMR
spectra were recorded on Bruker AMX 300, 500, and 600 spectrometers.
Two-dimensional NMR spectra were recorded in the phase-sensitive mode
using time-proportional phase incrementation for quadrature detection
in the f
dimension (Redfield and Kunz, 1975; Marion and
Wüthrich, 1983). The water proton signal was
suppressed by low power irradiation during the relaxation delay (1.8 s)
and during the mixing time of NOESY experiments. Spectra were
referenced to the residual proton signal of d
-TFE at 3.96
ppm, calibrated externally using 3-(trimethylsilyl)-1-propanesulfonic
acid (TSP).
TOCSY experiments were recorded using an MLEV-17 mixing
scheme (Bax and Davis, 1985) and with mixing times of 120 ms. Double
quantum-filtered correlation spectroscopy experiments (Rance et
al., 1983) were used to measure J
coupling constants and corrected for line width contributions of
the dispersive peaks using a peak deconvolution routine (Marion and
Wüthrich, 1983). Line widths were typically 5 Hz.
NOESY experiments were acquired at 500 and 600 MHz with mixing times of
250 ms.
Two-dimensional experiments were collected over 4096 complex
data points. Usually 512 increments of 48 scans were acquired over a
spectral width corresponding to 11 ppm for both dimensions. Up to 64
scans/increment were acquired for less sensitive NOESY experiments, and
only 8 scans/increment and 256 slices were required for diagnostic
TOCSY experiments. Up to 800 increments were collected for improved
resolution in the f dimension in the correlation
spectroscopy and NOESY experiments.
The data were processed on a
Silicon Graphics (SGI 4D/30) computer using the UXNMR software package
(version 1.1). The f dimension was zero-filled to 4096 real
data points, with both dimensions being multiplied by a QSINE function
prior to Fourier transformation. A GM window function with a
Gauss/Lorentz function coefficient of 0.3 and line broadening of
8
Hz was applied to some NOESY spectra to improve peak resolution for
assignment purposes. Polynomial base-line correction was used to
improve the appearance of the spectrum.
NOE cross-peak intensities were measured using the integration function within the Felix software package (Hare Research, Inc.). These were classified as strong, medium, and weak corresponding to interproton distance restraints of 1.8-2.7, 1.8-3.5, and 1.8-5.0 Å respectively (Clore et al., 1986). Methyl cross-peak intensities were divided by 3, and appropriate pseudoatom corrections were applied to nonstereo-specifically assigned methyl and methylene protons (Wüthrich et al., 1983).
Figure 1:
The N-terminal region of Cpn10 is
required for import into mitochondria. A, A protein mixture
consisting of porcine Cpn10 (Cpn) and amino-acids
31-101 of Cpn10 (Cpn
) was radiolabeled
with
I (lane 1). The mixture was denatured in 8 M urea, mixed with rabbit reticulocyte lysate, and then
incubated with mitochondria for 60 min at 30 °C (lane 2).
The imported products were inaccessible to proteinase K (lane
3). 2,4-Dinitrophenol (DNP) treatment prior to and
proteinase K digestion subsequent to the import reaction were also
performed (lane 4). [
H]p-OTC was used as
a control protein in each import reaction (data not shown). Import
reactions were analyzed by Tris-Tricine SDS-PAGE and PhosphorImaging
(Molecular Dynamics). B, The radiolabeled bands shown in panel A were quantitated, and the percentage of starting
material imported into mitochondria was
calculated.
In order to confirm
the targeting properties of the N-terminal region of Cpn10, a peptide
corresponding to Ala-Val
was synthesized
with (Ac-Cpn10
) and without N-terminal acetylation
(Cpn10
) and used in competitition studies with the
mitochondrial precursor protein p-OTC. Both peptides were shown to
inhibit the mitochondrial import of p-OTC and to a similar degree (Fig. 2). Whereas the concentrations of Cpn10 peptides required
for effective competition were relatively high although not uncommon
for mitochondrial targeting sequences (Hoyt et al., 1991; Pak
and Weiner, 1990), this inhibition appears specific since 100
µM concentrations of an unrelated peptide (myosin light
chain kinase substrate; KKRAARATSNVFA) had no effect (data not shown).
Taken together, the evidence shows that the N-terminal region of Cpn10
is not only necessary but also sufficient for targeting to
mitochondria.
Figure 2:
Both Ac-Cpn10 and
Cpn10
inhibit p-OTC import. A,
0-100 µM of Ac-Cpn10
or
Cpn10
as indicated were incubated with
mitochondria (100 µg of protein) in the presence of
S-labeled p-OTC in a total volume of 100 µl. Import
was performed for 15 min at 30 °C, and reactions were terminated by
centrifugation and addition of SDS-PAGE sample buffer to the
mitochondrial pellet. B, imported
[
S]p-OTC was quantitated by PhosphorImage
analysis following Tris-glycine SDS-PAGE. 100% import refers to the
level of import in the absence of competing
peptides.
Figure 3:
Region of a 120-ms TOCSY NMR spectrum of
Ac-Cpn10 (20% TFE, 120-ms mixing time, 25 °C,
500 MHz) showing the assignments of intraresidue side-chain
connectivities.
Figure 4:
NH-H (upper panel) and NH-NH (lower panel) regions of a 250-ms NOESY NMR spectrum of
Ac-Cpn10
(20% TFE, 250-ms mixing time, 25 °C,
500 MHz) showing intraresidue and sequential connectivities for each
residue in the amino acid sequence.
Figure 5:
Deviation of Cpn10 (a) and Ac-Cpn10
(b)
H
shifts from their ``random coil'' values
(Wüthrich, 1986) in 20% TFE at 25 °C
(
-
). The upfield
trend between residues 3 and 7 and residues 10 and 22 is indicative of
an
-helical conformation within these regions. Panel c shows the difference between the
H chemical shifts upon
acetylation of Cpn10
(
-
).
Figure 6:
Summary of NOE connectivities measured
for Cpn10 (a) and
Ac-Cpn10
(b) in 20% TFE, 25
°C. The intensities of the NOE cross-peaks are indicated by the thickness of the line, grouped into strong, medium,
and weak. Overlapping and, therefore, ambiguous cross-peaks are
indicated by an asterisk. Connectivities not expected to be
present because of the lack of the specified protons (i.e. Pro
NH) are shaded.
J
scalar coupling constants measured as greater than 8.0 Hz
(
) or less than 6.0 Hz (
) are indicated, as are amide
protons identified as exchanging slowly with solvent
(
).
In
Ac-Cpn10, d
(i, i) NOEs are also generally stronger than d
(i, i + 1) NOEs in
the stretch between residues 2 and 8, helping to confirm that a helix
also exists in this region of the peptide. In the equivalent region of
Cpn10
, however, the d
(i, i + 1) NOE
connectivities are, on average, stronger, suggesting a significant
population of random conformations is present (Dyson and Wright, 1988).
It is therefore likely that a helix in Cpn10
between residues 2 and 8 is less stable than its counterpart in
Ac-Cpn10
. Medium strength connectivities between
sequential NHs were observed along most of the length of both peptides.
This is consistent with the peptides showing helical conformations.
The observation of medium range NOE connectivities, particularly the
series of d(i, i + 3)
and d
(i, i + 3)
NOEs, show conclusively that both peptides have a high propensity to
form an
-helix between residues 11 and 22 (helix II) and another
between residues 3 and 8 (helix I). d
(i, i + 2), d
(i, i + 4) and d
(i, i + 2) NOE
connectivities are also diagnostic of the presence of helical
structures (Wüthrich, 1986). NOE signals from
residues 22-24 fell in particularly overlapped regions of the
spectra, and NOEs that may have been present were not observed. The
extent of helix II is therefore not precisely determined. Also of note
is the NOE data in light of the coupling information obtained for the
two peptides. The large
J
coupling constant, measured for residue 8 in both peptides,
coincides with the apparent end of helix I. It is likely that a turn
interrupts the structure at this point. The NOE information is
consistent with this, although there is insufficient data to define the
type of turn that may be present.
A family of
30 structures was calculated using the NOE-based interproton distance
restraints in a simulated annealing/energy minimization protocol. 107
inter- and 96 intraresidue constraints, as well as three hydrogen bond
distance constraints were applied in these calculations to NHs of
residues 15, 16, and 17. Torsion angle constraints were not applied
since coupling constant measurements, while displaying a low trend at
the N terminus, were neither below 5 Hz nor well above 8 Hz (Wagner et al., 1987). The derived structures were consistent with the
experimental constraints, to within 0.1 Å for 93% of the NOE
constraints in all 30 structures. No NOE constraint in any structure
was violated by greater than 0.5 Å. Structural convergence was
also assessed from the calculation of angular order parameters (AOPs)
for each amino acid residue in the sequence. These are a measure of the
correlation of the and
angles as a score out of 1
(Detlefsen, 1991). Fig. 7shows these values calculated for the
10 derived Ac-Cpn10
structures with lowest NOE
energies. The AOP is close to 1 between residues 11 and 20, indicating
the good definition of the structure in the region of helix II. High
AOPs are also observed for the region comprising residues 3-7,
indicating a good convergence in the region of helix I. As expected for
a proline residue, a high AOP is also observed for the
angle of
residue 10. AOPs are low toward the C terminus of the peptide,
indicating a lack of structural constraint in this region. For residues
22-24, this may not be caused by a lack of structure but by a
lack of NMR data for this region. The variation in structural
definition may be seen by noting that the average pairwise root mean
square deviation over backbone atoms for the helical regions are 1.38
and 0.89 Å, whereas it is 4.89 Å over the molecule as a
whole.
Figure 7:
Angular order parameters for backbone
torsion angles of the 10 structures calculated for
Ac-Cpm10 best satisfying the NOE distance
constraints. For each residue, Ø and
are shown as filled and empty bars, respectively. The convergence
to a common backbone configuration is seen for the region between
residues 3 and 8 and, particularly, between 10 and
21.
Fig. 8shows two superpositions of the 10 lowest NOE energy structures, one in which residues 2-7 are superimposed and the other in which residues 10-20 are superimposed. The two helices cannot be simultaneously overlaid because of the flexible linker region between residues 7 and 10. The set of structures confirms that the NOE distance constraints are consistent with the helix-turn-helix motif qualitatively observed and is a visual aid for better understanding the properties of this region of Cpn10 containing the mitochondrial targeting signal.
Figure 8:
Superposition of backbone atoms of the 10
structures calculated for Ac-Cpn10 best satisfying
the NOE distance constraints. The structures are superimposed over
residues 2-7 (a) and 10-20 (b),
corresponding to helices I and II. No structural restraint is observed
for other regions of the molecule.
A small proportion of nuclear encoded mitochondrial proteins appear to be synthesized without a cleavable presequence. In this report we investigated one of these proteins, namely chaperonin 10 from rat liver, and showed that the mitochondrial targeting information resides within the first 30 amino acids of the protein (Fig. 1). Thus, like 3-oxoacyl-CoA thiolase (Arakawa et al., 1990) and most likely rhodanese (Zardeneta and Horwitz, 1992), the noncleavable mitochondrial targeting signal of Cpn10 is located at the N terminus.
To further characterize the topogenic signal of Cpn10, two peptides
(Ac-Cpn10 and Cpn10
) were
synthesized and structurally analyzed by NMR in the hope that essential
structural features required for mitochondrial targeting eventually can
be identified from a data base of targeting sequence structures.
Additionally comparison with cleavable targeting sequences may help
delineate structural features, if any, required by processing
peptidases.
The NMR studies showed that Cpn10 contains a helix-turn-helix motif in 20% TFE at 25 °C and
that N-terminal acetylation increases the helical propensity of the
first helix. The evidence, derived from
H chemical shifts,
coupling constants, and NOE (
J
)
measurements as well as the observation of slow exchange NHs suggests
that the structure exists in a significant proportion of the peptide
population. The structural stability of these peptides is particularly
high as compared with other mitochondrial signal sequences, which have
required much higher percentages of membrane mimetic agents, such as
TFE and micelles, and low temperatures to promote helix formation (Endo et al., 1989; Karslake et al., 1990; Bruch and Hoyt,
1992; Hammen et al., 1994). This warranted the calculation of
three-dimensional structures for the visualization of the peptide.
It can be seen from Fig. 9that helix I, spanning residues
2-7, is highly amphiphilic in nature (hydrophobic moment =
0.77). Thus our structural studies are consistent with and strengthen
the current model for mitochondrial import in which the signal sequence
is required to present a hydrophobic face to the import machinery
(Roise et al., 1986). The evidence suggests that a classical
-helix is adopted and consists of approximately two turns. The
turn that exists between residues 7 and 10, while defining the
termination of helix I, is not sufficiently constrained for
classification into a particular type. However, the lack of NOE or slow
exchange data suggests that this region is fairly flexible.
Figure 9:
View of the N-terminal region of a
calculated structure of Ac-Cpn10, showing the
amphiphilic nature of helix I. Side-chains are color-coded according to
hydrophobicity with charged species in red or blue,
uncharged hydrocarbons in green, and aromatics in brown as follows: Ala
, green (acetyl group
highlighted pink), Gln
red;
Ala
, green; Phe
, orange;
Arg
, dark blue; Lys
, dark
blue; Phe
, orange; Leu
, green; and pro
, light
blue.
Helix
II, spanning residues 10-22, is of particularly high stability
and may extend further, but because of the overlap of residue
22-24 resonances, NMR data was scant for this region of the
peptide. The amphiphilic nature of helix II is not so strong
(hydrophobic moment = 0.12), and it may not constitute a crucial
part of the targeting signal, although presentation of a hydrophobic
face is clearly possible at several regions along the signal sequence (Fig. 10). The relatively high stability of helix II may be
caused by its length or by the presence of salt bridges. Whereas there
is no direct NMR evidence for salt bridges in
Ac-Cpn10, it could be seen that the two Arg-Glu
pairs (14 and 18, 19 and 23) in helix II are held in close proximity (Fig. 10). In the absence of competing ionic interactions, it is
highly likely that these bridges are contributing to its structural
stability and serve to accommodate negative charges in mitochondrial
targeting signals as predicted by Lemire et al.,(1989).
Figure 10:
View of a calculated structure of
Ac-Cpm10 showing the positions of the charged
residues. Salt bridges between residues 14 and 18 and residues 19 and
23 may help to stabilize helix II. The color coding is as
described in the legend to Fig. 9.
A
particularly interesting feature of the Cpn10 signal sequence is the
post-translational acetylation of the N terminus. In eukaryotic cells
up to 80% of all proteins are N-acetylated (Brown and Roberts,
1976), but the biological significance of this modification is in most
cases not clear. In the current study the structural properties of
Cpn10 were compared with those of the N-acetylated analogue, and it was found that this modification
clearly enhanced the helical propensity of helix I. It has been
demonstrated that a stable N-terminal amphiphilic helix is a likely
requirement for successful import (Roise et al., 1986; Wang
and Weiner, 1993). It is therefore possible that N-acetylation
assists import of Cpn10, but this cound not be concluded from the
indirect analysis presented in Fig. 2in which the effect of
Cpn10
and Ac-Cpn
on the
targeting of the mitochondrial precursor protein p-OTC was
assessed.
The mechanism by which acetylation mediates helical
stabilization is interesting to contemplate. It is well established
that the -helix, because of the orientation of the amide repeating
unit, possesses a dipole, effecting a positive potential at the N
terminus and a negative potential at the C terminus of the helix (Hol,
1985; DeGrado et al., 1989). This has been used to rationalize
the observation that compensating charged groups at either end of the
helix can help to stabilize the structures (Shoemaker et al.,
1987). In particular, specific studies of N-group blockage by N-acetylation have been shown to increase the helicity of a
peptide in solution (Venkatachalapathi et al., 1993). Our
results showing that removal of the N-terminal positive charge results
in an increased helix I stability further support these findings.
Additionally, the removal of the charge on Ala
may assist
membrane transport through the extension of the amphiphilic face of
helix I. As can be seen in Fig. 9, acetylated Ala
is
positioned on the hydrophobic side of the amphiphilic helix, where an
N-terminal positive charge would otherwise reduce the hydrophobic
moment. The N-acetylated helix may therefore have better
membrane binding properties than the charged species.
The
helix-turn-helix motif observed for Cpn10 is
comparable with several other mitochondrial signal sequences also
structurally analyzed (Fig. 11). Cytochrome c oxidase
subunit IV (Endo et al., 1989); the
-subunit of the
F
-ATPase complex (Bruch and Hoyt, 1992), and rat liver
aldehyde dehydrogenase (Karslake et al., 1990) all assume
N-terminal amphiphilic helices of approximately two to three turns,
followed by a flexible region and, in the case of the later two
proteins, a second stretch of helix (Fig. 11). Of interest is
the fact that these three signal sequences all undergo processing upon
import, unlike the signal sequence of Cpn10. Other mitochondrial
proteins possessing nonprocessed signal sequences include 3-oxoacyl-CoA
thiolase and rhodanese, which have each been shown to possess long,
continuous helices (Hammen et al., 1994). Cpn10 therefore is
the first example of an imported protein containing a nonprocessed
signal sequence with a helix-turn-helix motif. Our data therefore show
that whereas a flexible region within a helical signal sequence may be
required for productive interaction with the processing protease (Wang
and Weiner, 1993; Hammen et al., 1994), it is not a sufficient
criterion for proteolysis. The structural requirement for proteolysis
therefore remains elusive although, as pointed out by Hammen et
al.(1994), signal sequences with long helical structures appear
less likely to undergo processing than signal sequences adopting only
short regions of continuous helical structure. The presence of the long
helix II in Ac-Cpn10
is consistent with this view,
but mutational and further structural studies of the topogenic signals
listed in Fig. 11are required to resolve these questions.
Figure 11:
Schematic diagram showing structural
details of mitochondrial signal sequences as determined by
two-dimensional NMR spectroscopy. Helical and nonhelical regions are
indicated as well as whether mitochondrial processing of the signal
sequence occurs. COXIV, cytochrome c oxidase subunit
IV (Endo et al., 1989); F-ATPase
,
-subunit of the F
-ATPase complex (Bruch and Hoyt,
1992); ALDH, aldehyde dehydrogenase (Karslake et al.,
1990); Cpn10, Chaperonin 10 (this study); ALDH
(11-13), ALDH mutant lacking a flexible linker
comprising residues 11-13 (Thornton et al., 1993); RHODANESE and THIOLASE, 3-oxoacyl-CoA thiolase (Hammen et
al., 1994).