(Received for publication, January 11, 1996; and in revised form, February 20, 1996)
From the
PIM1 protease in mitochondria belongs to a conserved family of
ATP-dependent proteases, which includes the Escherichia coli Lon protease. Yeast cells lacking PIM1 are largely
defective in degrading misfolded proteins in the mitochondrial matrix,
are respiratory deficient, and lose integrity of mitochondrial DNA. In
order to analyze whether E. coli Lon protease is functionally
equivalent to mitochondrial PIM1 protease, yeast cells lacking the PIM1 gene were transformed with a construct consisting of a
mitochondrial targeting sequence fused onto the Lon protease. In these
cells, the fusion protein was expressed and imported into mitochondria,
and the targeting sequence was removed. In the absence of PIM1
protease, the E. coli Lon protease mediated the degradation of
misfolded proteins in the matrix space in cooperation with the
mitochondrial hsp70 system. These cells maintained the integrity of the
mitochondrial genome and the respiratory function at 30 °C but not
at 37 °C. Stabilization of mitochondrial DNA in pim1 cells depended on protein degradation by the E. coli Lon
protease, as a proteolytically inactive Lon variant was not capable of
substituting for a loss of PIM1 protease. These results demonstrate
functional conservation of Lon-like proteases from prokaryotes to
eukaryotes and shed new light on the role of Lon-like proteases in
mitochondrial biogenesis.
Mitochondrial homeostasis depends on the coordinated synthesis
and degradation of nuclear and mitochondrially encoded proteins.
Energy-dependent proteases exist in various subcompartments of
mitochondria that mediate the removal of abnormal proteins and ensure
the proper stochiometry of multienzyme complexes. In the mitochondrial
matrix space of yeast and mammalian cells, an ATP-dependent protease
has been identified that is very similar to the Escherichia coli Lon protease (Desautels and Goldberg, 1982; Watabe and Kimura,
1985; Wang et al., 1993; Kutejova et al., 1993; Van
Dyck et al., 1994; Suzuki et al., 1994). The yeast
homologue PIM1 protease, like the Lon protease, is a homo-oligomeric
enzyme. It performs essential functions in mitochondrial biogenesis
(Van Dyck et al., 1994; Suzuki et al., 1994). Cells
lacking the PIM1 gene are respiratory deficient and accumulate
mitochondrial DNA with extensive deletions (rho phenotype). PIM1 protease controls selective mitochondrial
protein turnover and the proteolytic breakdown of misfolded proteins in
the matrix space (Suzuki et al., 1994; Wagner et al.,
1994). Efficient proteolysis by the PIM1 protease depends on the
mitochondrial hsp70 machinery that stabilizes abnormal polypeptides in
a soluble state, susceptible to degradation (Wagner et al.,
1994).
The Lon protease of E. coli shares 33% sequence
identity with PIM1 protease (Van Dyck et al., 1994; Suzuki et al., 1994). It is required for the degradation of misfolded
proteins and of specific proteins with regulatory functions in E.
coli during cell division and cell wall synthesis (Gottesman and
Maurizi, 1992; Goldberg, 1992). The single ATP-binding sequence is
conserved between prokaryotic and eukaryotic Lon-like proteases.
Studies on the E. coli Lon protease have demonstrated the
requirement of ATP for proteolytic activity (Chung and Goldberg, 1981;
Charette et al., 1981; Goldberg and Waxman, 1985; Waxman and
Goldberg, 1986; Fischer and Glockshuber, 1994). ATP binding promotes
the oligomerization of the Lon protease, ()whereas ATP
hydrolysis is required for the proteolytic breakdown of polypeptides.
The carboxyl-terminal domain contains a conserved serine residue that
most likely represents the nucleophile attacking the peptide bond of
the substrate, as a mutation in Ser
of E. coli Lon protease abolishes the proteolytic activity (Amerik et
al., 1991; Fischer and Glockshuber, 1993).
Mature PIM1 protease and the E. coli Lon protease differ considerably in the molecular mass of their subunits (122 and 87 kDa, respectively). Differences in the sequences are apparent mainly in the amino-terminal region. The first 18 amino acid residues of PIM1 protease exhibit characteristics of mitochondrial targeting sequences and thus are believed to target the protein to mitochondria. However, the function of amino-terminal regions of the mature PIM1 protease, not present in E. coli Lon protease, is obscure. Interestingly, the sequences of this region in yeast and human PIM1 protease are rather similar (Wang et al., 1993, 1994; Amerik et al., 1994). Another characteristic of PIM1 protease, not found in other Lon-like proteases, is an extended spacer region between the ATPase and the carboxyl-terminal proteolytic domains.
In order to investigate the possible functional equivalence of the yeast and E. coli Lon-like proteases, we carried out a complementation analysis in Saccharomyces cerevisiae. A chimeric protein consisting of a mitochondrial targeting sequence and the E. coli Lon protease was constructed and expressed in a yeast strain lacking the PIM1 gene. This hybrid protein was imported into yeast mitochondria in vivo. Lon protease exhibited proteolytic activity within the mitochondrial matrix and was capable of replacing PIM1 protease in its function of maintaining mitochondrial DNA. Analysis of a Lon protease variant with an impaired proteolytic activity revealed that the peptidase activity is required for the stabilization of the mitochondrial genome. These results demonstrate the functional conservation of yeast PIM1 protease and E. coli Lon protease and thus provide further experimental support for the endosymbiotic origin of mitochondria. On the other hand, E. coli Lon protease did not support maintenance of respiratory function at high temperatures in the absence of PIM1 protease, pointing to functional differences between prokaryotic and mitochondrial Lon-like proteases.
In order to increase expression of the
chimeric protein, Su9 (1-69)-Lon was cloned into the multicopy
vector pVT100-U (Vernet et al., 1987). In parallel, hybrid
proteins were generated carrying point mutations in serine 679 (S679A)
or in lysine 362 (K362A). DNA fragments encoding parts of the E.
coli LON gene were isolated by restriction digest of pLON,
containing the wild type or mutant genes (Fischer and Glockshuber,
1993, 1994), with NdeI and NcoI, which was filled in
with Klenow. The DNA fragments were cloned into M13-Su9
(1-69)-Lon that had been digested with XbaI, filled in
with Klenow, and NdeI. By this procedure, hybrid genes were
generated encoding the amino-terminal portion of the precursor of
subunit 9 of N. crassa F-ATPase (residues
1-69) and wild type or mutant forms of E. coli LON. The
hybrid genes were isolated as a SacI-EcoRV fragment
and cloned into the SacI-PvuII sites of pVT100-U. The
resulting constructs pVT100-U-Su9 (1-69)-Lon
were used for yeast transformation.
Figure 1:
Expression and
subcellular localization of Su9 (1-69)-Lon in S.
cerevisiae. pim1 mutant cells expressing Su9
(1-69)-Lon from a CEN-based or a multicopy plasmid were grown on
YP medium containing 3% glycerol at 30 °C. Cell extracts were
prepared and split into a mitochondrial (Mitochondria) and a
postmitochondrial fraction (Cytosol) by differential
centrifugation as described previously (Caplan and Douglas, 1991). In
addition, cells (0.25 optical density units) were lysed by alkaline
extraction (Total). Cytosol (90 µg) and mitochondria (90
µg for expression of Lon protease from CEN-based plasmid (LON
) or 10 µg for expression of
Lon protease from multicopy plasmid (LON
), respectively) were subjected to
SDS-PAGE and analyzed by Western blotting with polyclonal antibodies
directed against E. coli Lon protease (
-Lon),
Bmh2p (
-Bmh2p, for brain modulosignalin homologue; Van
Heusden, et al.(1992)) and Mge1p (
-Mge1p, for
mitochondrial GrpE). Bmh2p and Mge1p are markers for the cytosol and
mitochondrial matrix fraction, respectively. As a reference, Su9
(1-69)-Lon synthesized in reticulocyte lysate in vitro and purified E. coli Lon protease (15 ng) were subjected
to SDS-PAGE in parallel. p, precursor form of Su9
(1-69)-Lon; m, mature form of Su9
(1-69)-Lon.
Figure 2:
Suppression of the respiratory deficiency
of pim1 cells by E. coli Lon protease. 2-Fold
serial dilutions of saturated cultures were spotted onto YP medium
containing 3% glycerol and incubated at 30 or 37 °C. Abbreviations
are as defined in the Fig. 1legend.
The growth rate of pim1 cells complemented with Su9 (1-69)-Lon, however, was lower
as compared with wild type cells. Moreover, expression of E. coli Lon protease did not promote growth of
pim1 mutant
cells on nonfermentable carbon sources at 37 °C (Fig. 2).
This might indicate specific functions of PIM1 protease within
mitochondria that cannot be performed by the prokaryotic homologue.
Alternatively, the level of Lon protease might be limiting for growth
under these conditions. To distinguish between these possibilities, Su9
(1-69)-Lon was cloned into a multicopy yeast expression vector
under the control of the ADH1 promotor and expressed in a
pim1 mutant strain. E. coli Lon protease was
recovered exclusively in the mitochondrial fraction upon cell
fractionation (Fig. 1, lanes 4-6). When compared
with the expression from a CEN-based plasmid, E. coli Lon
protease accumulated at an approximately 50-fold higher level within
yeast mitochondria under these conditions. The growth rate of the cells
on nonfermentable carbon sources at 30 °C increased significantly (Fig. 2). Thus, when expressed from a CEN-based plasmid, the
level of E. coli Lon protease within mitochondria is limiting
for growth. Overexpression of Lon-like proteases, either of PIM1
protease in yeast or of Lon protease in E. coli, on the other
hand, was observed to inhibit cell growth (Goff and Goldberg, 1987). (
)A precise regulation of the cellular activity of Lon-like
proteases must therefore exist. It remains to be determined whether
this is achieved by regulation of protein synthesis or by
posttranslational processes.
The presence of E. coli Lon
protease even at high levels did not maintain mitochondrial respiration
at higher temperature (Fig. 2). pim1 cells
expressing Su9 (1-69)-Lon accumulated aberrations in the
mitochondrial genome at 37 °C as demonstrated by mating with a
mit
tester strain (data not shown). The
temperature-dependent growth phenotype could be the consequence of a
low specific activity or a low thermostability of Lon protease in the
mitochondrial environment. Alternatively, functional differences
between the prokaryotic and eukaryotic homologue have to be envisioned.
In order
to investigate whether E. coli Lon protease can also degrade
polypeptides presented by mitochondrial hsp70, we employed two model
proteins that fail to attain their native conformation after import
into the mitochondrial matrix and thus are subject to proteolysis.
Mouse DHFR, when fused to the amino acids 1-167 of cytochrome b, is targeted to mitochondria and sorted to the
intermembrane space. Exchange of specific amino acids in the sorting
signal of cytochrome b
results in targeting of the
hybrid protein to the matrix space
(b
(1-167)
-DHFR; Schwarz et
al.(1993)). The matrix targeting sequence is removed upon import
by the matrix-localized processing peptidase, yielding the so-called
i-form, and a further, so far unidentified protease generates the
slightly smaller i*-form. The unfolded cytochrome b
moiety becomes complexed by the mitochondrial hsp70 machinery and
degraded by PIM1 protease. This results in the formation of a fragment f that corresponds to the folded DHFR domain (Wagner et
al., 1994; Fig. 3). To investigate whether Lon protease can
replace its mitochondrial homologue in this reaction, the stability of
b
(1-167)
-DHFR was analyzed in
pim1 mutant mitochondria containing E. coli Lon
protease. b
(1-167)
-DHFR was found to be
degraded in
pim1 mutant mitochondria in the presence of
Lon protease (Fig. 3). The efficiency of fragment formation was
relatively low. However, fragment formation was strictly dependent on
Lon protease, as b
(1-167)
-DHFR was
stable in mitochondria lacking PIM1 protease (Fig. 3). The
product of proteolysis by Lon protease was indistinguishable in size
from the fragment generated by PIM1 protease, suggesting a similar
substrate specificity of both proteases.
Figure 3:
Degradation of
b(1-167)
-DHFR by E. coli Lon
protease in the mitochondrial matrix.
b
(1-167)
-DHFR was imported into
mitochondria isolated from wild type,
pim1, and
pim1 cells expressing Su9 (1-69)-Lon from a
CEN-based plasmid (
pim1LON
). After
completion of import, proteolysis was assessed as described under
``Materials and Methods.'' i and i*,
intermediate forms; f, fragment.
The proteolytic breakdown
of newly imported Su9 (1-69)-DHFR in the
mitochondrial matrix space was studied in similar experiments. The
exchange of amino acid residues in the DHFR domain
(Cys
-Ser
,
Ser
-Cys
, and
Asn
-Cys
) by site-directed mutagenesis
results in the destabilization of the DHFR domain, preventing the
binding of substrate molecules (Vestweber and Schatz, 1988). The
mutated DHFR domain was fused to the 69 amino-terminal amino acid
residues of subunit 9 of the F
-ATPase of N.
crassa, which comprise the matrix targeting signal (Su9
(1-69)-DHFR
). Wild type Su9 (1-69)-DHFR
was imported in parallel as a control. The imported, loosely folded
DHFR domain was degraded with a half-time of about 15 min, whereas wild
type DHFR was stable under these conditions (Fig. 4A).
In contrast to wild type DHFR, the loosely folded DHFR was found in
association with mitochondrial hsp70 upon coimmunoprecipitation with
antibodies against hsp70, reflecting the involvement of the
mitochondrial hsp70 machinery in the degradation of this misfolded
protein (data not shown). Proteolysis is mediated by PIM1 protease as
demonstrated by the stability of the mutated DHFR domain within
mitochondria lacking PIM1 protease (Fig. 4B). In
pim1 mutant mitochondria containing E. coli Lon
protease, degradation of misfolded DHFR was again observed (Fig. 4B). This provides further evidence for the
proteolytic activity of E. coli Lon protease with misfolded
polypeptide chains as substrates.
Figure 4:
Degradation of the misfolded
DHFR by Lon-like proteases in the mitochondrial
matrix. A, Su9 (1-69)-DHFR
and Su9
(1-69)-DHFR
were imported into wild type
mitochondria. After completion of import, samples were further
incubated at 30 °C to allow proteolysis to occur. B, Su9
(1-69)-DHFR
was imported into mitochondria
isolated from wild type,
pim1, and
pim1 cells expressing Su9 (1-69)-Lon from a CEN-based plasmid (
pim1LON
), and proteolysis was analyzed
upon further incubation of the samples at 30 °C. C, after
import as in B, mitochondria were lysed at a concentration of
1 mg/ml by incubation in 0.1% Triton X-100, 10 mM MOPS/KOH,
pH7.2, 150 mM NaCl, 5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride for 10 min at 4 °C under
vigorous mixing. Aggregation of the DHFR
domain was
determined by centrifugation for 15 min at 25,000
g.
DHFR
in the supernatant was precipitated with 12.5%
trichloroacetic acid. The pellet fraction was dissolved in SDS-PAGE and
sample buffer. Both fractions were then subjected to SDS-PAGE. In A, B, and C, total material after import was
set to 100%.
Degradation of the mutant DHFR
moiety occurred at a reduced rate in pim1 mitochondria
containing Lon protease when compared with wild type, similar to what
was observed for b
(1-167)
-DHFR (see
above). To further characterize the fate of the nondegraded fraction,
the solubility of mutated DHFR was analyzed in wild type mitochondria,
in
pim1 mitochondria and in
pim1 mitochondria containing Lon protease. Aggregates of mutant DHFR
accumulated in the pellet fraction in the absence of PIM1 protease (Fig. 4C). Release of misfolded polypeptide chains from
mitochondrial hsp70 followed by aggregation in the absence of PIM1
protease has been reported earlier (Wagner et al., 1994). When
compared with
pim1 mitochondria, aggregation of mutant
DHFR was diminished in the presence of E. coli Lon protease,
as a fraction of the misfolded polypeptide chain was degraded (Fig. 4C). The nondegraded form of mutant DHFR,
however, was apparently released from molecular chaperone proteins,
leading to its aggregation.
These observations demonstrate that the E. coli Lon protease is active within yeast mitochondria and indicate that misfolded polypeptide chains associated with the mitochondrial hsp70 machinery are substrates of the E. coli Lon protease. Lon protease was also found to mediate the proteolysis of polypeptides stabilized by the hsp70 system at 37 °C (data not shown). Therefore, the inability of the prokaryotic enzyme to maintain respiration at 37 °C in the absence of PIM1 protease is apparently not caused by an impaired cooperation with molecular chaperones under these conditions.
The expression of mutant Su9
(1-69)-Lon did not confer on
pim1 mutant cells the ability to grow on nonfermentable carbon sources
at 30 °C, in contrast to the overexpression of the wild type form
of Su9 (1-69)-Lon (Fig. 5A). Thus, the
proteolytic activity of E. coli Lon protease is required for
the maintenance of respiratory competence.
Figure 5:
Complementation depends on the proteolytic
activity of E. coli Lon protease. A, pim1 cells complemented with Su9 (1-69)-Lon, Su9
(1-69)-Lon
, or Su9 (1-69)-Lon
were grown on synthetic complete media containing 2% glucose at
30 °C. 2-Fold serial dilutions of saturated cultures were then
spotted onto YP medium containing 3% glycerol and incubated at 30
°C. B, isolated colonies were selected on synthetic
complete medium containing 3% glucose at 30 °C and then crossed
with a mit
tester strain that carries a mutation in
the OXI2 gene encoding subunit III of cytochrome c oxidase (MAT
ade1 mit
).
Diploids were replica-plated on YP medium containing 3% glycerol and
grown at 30 °C.
Cells expressing Su9
(1-69)-Lon were able to grow on nonfermentable
carbon sources although at a strongly reduced rate (Fig. 5A). Most likely, this is due to a residual
activity of the mutant Lon protease in ATP binding and hydrolysis. The
K362A mutant of Lon protease was observed to exhibit a very weak
proteolytic activity in vitro (about 2% of wild type activity;
Fischer and Glockshuber(1994)).
The integrity of the mitochondrial
genome was analyzed by crossing the pim1 strain
complemented with mutant Su9 (1-69)-Lon with a mit
tester strain carrying a mutation in the mitochondrial OXI2 gene (Fig. 5B). The respiratory defect of oxi2 cells was not complemented by mating with a
pim1 strain expressing Su9 (1-69)-Lon
, pointing
to aberrations in the mitochondrial DNA (Fig. 5B).
Furthermore, replacement of serine 1015 by alanine in PIM1 protease was
found to cause aberrations in mitochondrial DNA. (
)In
contrast, complementation was observed with Su9
(1-69)-Lon
(Fig. 5B). Apparently,
when overexpressed, a rather low specific proteolytic activity of Lon
protease is sufficient to stabilize the mitochondrial genome.
We demonstrate in the present study conservation of function of Lon-like proteases in prokaryotes and mitochondria. E. coli Lon protease can substitute for mitochondrial PIM1 protease in several respects; the prokaryotic homologue preserves the integrity of the mitochondrial genome and maintains respiration competence of the cells at 30 °C in the absence of PIM1 protease. E. coli Lon protease exerts proteolytic activity within yeast mitochondria. Similar to PIM1 protease, it mediates the proteolytic breakdown of misfolded proteins in the mitochondrial matrix that are stabilized against aggregation by the hsp70 machinery. The interplay between folding and proteolytic reactions in mitochondria is not well understood. In particular, the question remains to be answered whether the transfer of polypeptides from molecular chaperone proteins to PIM1 protease occurs by direct interaction between the PIM1 protease and the chaperone proteins. The ability of E. coli Lon protease to degrade polypeptides presented by hsp70 is remarkable, as the cooperation of chaperone proteins with PIM1 protease appears to be rather specific. hsp78, a ClpB homologue in the mitochondrial matrix space has recently been demonstrated to stabilize misfolded proteins against aggregation. However, in contrast to polypeptides associated with hsp70, hsp78-bound proteins are not substrates of PIM1 protease (Schmitt et al., 1995).
PIM1 protease belongs to a group of nuclear encoded genes that are essential for mitochondrial genome stability including components of the replication system, the protein synthesis apparatus, and nucleotide biosynthesis. E. coli Lon protease can substitute for a loss of PIM1 protease and preserves the integrity of the mitochondrial genome at 30 °C in the absence of PIM1 protease. Mutational analysis revealed that a proteolytically active Lon-like protease in the matrix is required for the maintenance of mitochondrial DNA. Proteolytic activation of a mitochondrial protein through specific processing by PIM1 protease might be a process involved in preserving the integrity of the mitochondrial genome. Alternatively, PIM1 protease might regulate the turnover rate of a regulatory protein that negatively affects DNA stability. In any case, the requirement of the proteolytic activity for mitochondrial genome integrity is illustrated by two observations: low amounts of active protease as well as large amounts of Lon protease with a mutation in the ATPase domain are sufficient to stabilize mitochondrial DNA.
Interestingly, E. coli Lon protease does not entirely
substitute for PIM1 protease. At 37 °C, the respiratory competence
of pim1 mutant cells and integrity of mitochondrial DNA
were not restored upon expression of E. coli Lon protease.
This might be the consequence of a low specific activity or a low
thermostability of the prokaryotic homologue in the mitochondrial
environment. Although the Lon protease was proteolytically active in
the matrix space, it was rather inefficient in degrading misfolded
proteins. An increase in the growth rate of
pim1 mutant
cells on nonfermentable carbon sources at 30 °C was observed when
high levels of Lon protease were accumulated within mitochondria.
Respiratory competence of
pim1 mutant cells at 37 °C,
on the other hand, was still not restored. As E. coli Lon
protease was capable of degrading misfolded polypeptides in the
mitochondrial matrix space under these conditions, it seems unlikely
that this is the consequence of a low specific activity or a low
thermostability of E. coli Lon protease within mitochondria.
Rather, additional activities of PIM1 protease have to be envisioned.
This might also include variations in the substrate specificity. It
will be interesting to link these nonoverlapping activities to protein
domains that are not conserved between the mitochondrial and the
prokaryotic homologue.