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(Received for publication, July 11, 1995; and in revised form, August 23,
1995) From the
Most mitochondrial precursor proteins are processed to the
mature form in one step by mitochondrial processing peptidase (MPP),
while a subset of precursors destined for the matrix or the inner
membrane are cleaved sequentially by MPP and mitochondrial intermediate
peptidase (MIP). We showed previously that yeast MIP (YMIP) is required
for mitochondrial function in Saccharomyces cerevisiae. To
further define the role played by two-step processing in mitochondrial
biogenesis, we have now characterized the natural substrates of YMIP. A
total of 133 known yeast mitochondrial precursors were collected from
the literature and analyzed for the presence of the motif
RX(
Mitochondrial intermediate peptidase (MIP) ( Since the
molecular characterization of rat MIP(8) , a family of
structurally related but primarily cytosolic enzymes, thimet
oligopeptidases, has been defined(9, 10) . The
prototype of this family is the rat testes thimet oligopeptidase EC
3.4.24.15, which has homologues in bacteria, Saccharomyces
cerevisiae, and mammals(9) . Although there is evidence
that EC 3.4.24.15 may play a role in the processing or catabolism of
pharmacologically active peptides(9) , the natural substrates
and biological roles of thimet oligopeptidases are not known in most
cases. Likewise, the biological role of the proteolytic cleavage
carried out by MIP is not yet understood, in part because only a
handful of natural substrates of this peptidase are known. To date,
cleavage by MIP has been demonstrated, in vitro or in
vivo, for only seven precursors from S.
cerevisiae(11, 12) , Neurospora
crassa(13, 14) , rat(15, 16) ,
and man(1) . On the other hand, several observations indicate
that MIP is important for mitochondrial function. Chromosomal
disruption of the MIP1 gene causes loss of respiratory
competence in S. cerevisiae(3) . Moreover, this locus
is conserved in eukaryotes(8, 10) , and MIP1 homologues from Schizophyllum commune and rat liver can
rescue the phenotype of mip1 Unlike MIP1, the S. cerevisiae
MAS1/MIF1 and MAS2/MIF2 genes, which encode the two
structurally related subunits of yeast MPP, are essential for yeast
viability(17, 18) ; as such, MPP is believed to be
required for global mitochondrial protein processing(19) . In
contrast, yeast MIP (YMIP) must be required for the biogenesis of a
specific subset of mitochondrial proteins, as MIP1 inactivation leads to loss of respiratory function without
affecting the viability of the facultative anaerobe S.
cerevisiae(3) . We have previously shown that MIP1 disruption results in failure to cleave at least two
nuclear-encoded respiratory chain components, the cytochrome c oxidase subunit IV (CoxIV) and the iron-sulfur protein of the bc
The coding
sequences of the previously cloned yeast genes COXIV(26) , DHSA1(27) , DLDH1(28) , CYP3(29) , MRPS28(30) , tufM(31) , and RIM1(32) were polymerase chain reaction-amplified from total
genomic DNA of wild-type yeast, using primers complementary to the
published sequence of these genes. All genes were cloned in pGEM-3Z
downstream from the T7 polymerase promoter, and their 5`-coding regions
were analyzed by DNA sequencing. Each gene was transcribed in
vitro, and the mRNA was translated in the presence of
[ Processing
reactions were analyzed directly by SDS-PAGE and fluorography. The
following separating gels (total length, 12.5 cm) were used: T =
12.5% for CoxIV, CYPC, and RIM1, T = 10.4% for MRPS28, T
= 8.3% for F
The identification of a larger number of octapeptide-containing
proteins from a single organism may help elucidate the nature of the
substrates cleaved by MIP. To characterize the octapeptide-containing
proteins of S. cerevisiae, yeast mitochondrial precursor
sequences were collected from Swiss Protein (May 1995). We found 133
mitochondrial matrix and inner membrane protein precursors, including
56 precursors for which the leader peptide cleavage site has been
defined by amino acid sequencing of the mature N terminus (Table 2, A-D), and 77 precursors for which the mature N
terminus is not known (Table 2E). (
Precursors of
the first group were aligned according to the mature N terminus, and
the leader peptide cleavage sites were analyzed for the presence of
three motifs, XRX(
Yeast mitochondrial
precursors for which the mature N terminus is not known (77 precursors; Table 2E)
The yeast pF
Figure 1:
YMIP activity is required for
maturation of yeast R-10 precursors. In vitro translated,
Different processing patterns were observed with pDLDH and pCYPC. In
addition to a typical R-10 motif, these precursors contain an arginine
residue at -3 or -2, respectively; thus, they may be
cleaved in two steps or one step, depending on the preferred MPP
cleavage site. Incubation of pDLDH with wild-type versus
mip1
To carry
out import assays using mitochondria isolated from mip1(G578L) Isolated mitochondria were initially tested for
their ability to import pF
Figure 2:
mip1(G578L)
Similar to pF These results are consistent with the pattern of processing shown by
pCoxIV, pDHSA, and pMRPS28 upon incubation with wild-type and mip1 Import of pDLDH by mip1(G578L)
The in vitro translated ptufM and pRIM1
were incubated with wild-type and mip1(G578L) Similar to pCoxIV, pRIM1 was processed
to predominantly mature form upon import into wild-type yeast
mitochondria (Fig. 2, lane 32), and mature RIM1 was
protected from externally added trypsin (lane 34). An
intermediate-size polypeptide was accumulated in mip1(G578L) These data
indicate that at least three components of the yeast mitochondrial
genetic machinery, MRPS28, RIM1, and tufM, require YMIP activity for
normal biogenesis.
Two different isogenic sets of strains were used in genetic
crosses: mip1
Because the number of yeast mitochondrial proteins identified to
date is still relatively small, these observations do not exclude the
possibility that proteins involved in other metabolic functions may be
cleaved by YMIP. However, the YMIP substrates identified to date are
totally consistent with the observed phenotype of mip1
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 27366-27373
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)(F/L/I)XX(T/S/G)XXXX(
),
typical of precursors cleaved by MPP and MIP. We found characteristic
MIP cleavage sites in two distinct sets of proteins: respiratory
components, including subunits of the electron transport chain and
tricarboxylic acid cycle enzymes, and components of the mitochondrial
genetic machinery, including ribosomal proteins, translation factors,
and proteins required for mitochondrial DNA metabolism. Representative
precursors from both sets were cleaved to predominantly mature form by
mitochondrial matrix or intact mitochondria from wild-type yeast. In
contrast, intermediate-size forms were accumulated upon incubation of
the precursors with matrix from mip1
yeast or intact
mitochondria from mip1 yeast,
indicating that YMIP is necessary for maturation of these proteins.
Consistent with the fact that some of these substrates are essential
for the maintenance of mitochondrial protein synthesis and
mitochondrial DNA replication, mip1
yeast undergoes loss
of functional mitochondrial genomes.
)is a
component of the mitochondrial protein import machinery required for
the maturation of a subset of nuclear-encoded precursor proteins
targeted to the matrix or inner membrane (1, 2, 3) (for a review, see (4) ).
These precursors are characterized by a three amino acid motif,
RX()(F/L/I)XX(T/S/G)XXXX(
), at
the carboxyl terminus of their leader peptide(5, 6) .
In the matrix, the mitochondrial processing peptidase (MPP) initially
cleaves the motif two peptide bonds from the arginine residue, leaving
a characteristic octapeptide sequence at the protein amino terminus;
the octapeptide is then cleaved by MIP to yield mature
protein(1, 2) . Active MIP is a soluble monomer of 75
kDa and presents the unusual characteristic of being a thiol-dependent
metallopeptidase (7) . Positioning of the octapeptide at the
substrate amino terminus and a large hydrophobic residue at P-8 are
essential features for cleavage by MIP(2) , a substrate
specificity which is not shared by other known peptidases.
yeast(10) ,
indicating that crucial substrates in this pathway must have been
conserved as well. complex (Fe/S)(3) . In this study, we
analyze the leader peptide cleavage sites of known yeast mitochondrial
precursors and show that a significant fraction of these proteins
contain typical MIP cleavage sites. We demonstrate that the natural
substrates of YMIP include not only proteins required for respiration,
but also components of the mitochondrial genetic apparatus essential
for mitochondrial protein synthesis and mtDNA replication. As the
latter are required for mtDNA maintenance(20) , loss of YMIP
activity indirectly leads to mitochondrial genome instability.
Yeast Strains, Growth Media, and Plasmids
The
yeast strains used in this study are listed in Table 1. The YPD,
YPEG, SD, SEG, and semisynthetic media were as described
previously(3) . Plasmid pGEM-3Z (Promega) was used for in
vitro protein expression. Two centromeric plasmids, YCp50 (21) and YCplac22(22) , which carry URA3 and TRP1, respectively, were used for construction of the mip1(G578L) mutant.
Genetic Crosses
Haploid cells of opposite mating
types and different growth requirements were mixed in patches on YPD
plates and left at 30 °C for 8-10 h. Diploid cells were
selected based on their ability to grow on SD plates lacking the growth
requirements of the two parental haploid strains; they were
subsequently replica-plated onto YPEG and SEG plates, and growth was
scored after 5 days at 30 °C, as described in Myers et
al.(20) .Construction of (G578L)
A MIP1(G578L)-c-myc allele, coding for a
YMIP-c-myc fusion protein with a glycine to leucine
substitution at position 578 of the predicted YMIP sequence, was
synthesized by site-directed mutagenesis, using two primers, sense
primer 1, 5`-GCAAATATATGGTATTAGATTAGA-3` (bp 1,337-1,360 of MIP1), upstream from a unique KpnI site in the MIP1 gene, and antisense primer 4,
5`-GTAAATGCTTTTTAAATTTATCAC-3` (bp 2,323-2,300 of MIP1),
downstream from a unique EcoNI site. These two sites flank the
region encoding the putative YMIP catalytic domain(10) . The
G578L mutation was introduced using two overlapping primers, antisense
primer 2, 5`-TAGCACATCTTGTTAAACTTATGTTTTGCA-3` (bp 1,870-1,841)
and sense primer 3, 5`-TGCAAAACATAAGTTTAACAAGATGTGCTA-3` (bp
1,841-1,870), containing the desired codon change (underlined).
Two DNA fragments of 534 and 483 bp were initially amplified from
wild-type MIP1 using primers 1 and 2, and primers 3 and 4,
respectively. A 987-bp fragment was then synthesized in a reaction
containing the two overlapping polymerase chain reaction products plus
sense primer 1 and antisense primer 4. This mutated fragment was
digested with KpnI and EcoNI and substituted for the
corresponding wild-type fragment in a MIP1-c-myc fusion
gene(3) . The resulting MIP1(G578L)-c-myc gene was
cloned in YCplac22 and introduced by transformation into a mip1Mutant Yeast
Strain
mutant complemented by a wild-type allele of MIP1 on YCp50. The MIP1(G578L)-c-myc allele was
exchanged with wild-type MIP1 using a URA3/5-fluoroorotic acid counterselection
procedure(23) . The growth phenotype of mip1(G578L) yeast was characterized on solid YPEG
medium at 25 and 37 °C. The YMIP(G578L)-c-myc protein was
detected by immune blotting using a monoclonal anti-c-myc (9E10) antibody, as described previously(3) .
Mitochondrial Import and Processing of Yeast
Mitochondrial Precursor Proteins
Mitochondria were isolated
according to the procedure of Daum et al.(24) . To
obtain coupled mip1(G578L) mitochondria, it was
necessary to use nonpermissive conditions that cause incomplete
inactivation of YMIP(G578L), such that the isolated mitochondria are
defective in MIP activity but maintain some degree of respiratory
function and import competence. The mip1(G578L)
yeast were grown in semisynthetic medium at room temperature to
2-4 OD
. Spheroplasts were subsequently prepared by
treatment with Zymolyase 20T for 30 min at the nonpermissive
temperature (37 °C). Mitochondria were then isolated at 4 °C by
homogenization of the spheroplasts and differential centrifugation.
Either freshly isolated or previously frozen mitochondria were used in
import assays, as described in Yaffe(25) . Preparation of
matrix fractions and determination of protein concentration were
carried out as described previously(3) .
S]methionine using a coupled
transcription/translation system (Promega Biotech Inc.) (3) .
The precursor for the S. cerevisiae F
ATPase
subunit 
(pF) (33) was similarly
synthesized and used in control reactions. In vitro processing
with crude matrix fractions, mitochondrial import assays, trypsin
treatment of import reactions with or without addition of 1% Triton
X-100, and reisolation of mitochondrial pellets by centrifugation were
carried out as described previously(3) ., T = 7.3% for DLDH and
tufM, using a stock solution of acrylamide:bisacrylamide =
40:1.7, and T = 7.7% for DHSA, using a stock solution of
acrylamide:bisacrylamide = 30:0.8 (T denotes the total
concentration of acrylamide and bisacrylamide). Separating gels were
overlaid with T = 4% stacking gels. Electrophoresis at room
temperature began at 180 V, was shifted to 240 V after the samples had
completely entered the separating gel, and was continued for an
additional 20 min (CoxIV; CYPC; RIM1), 40 min (F), 75
min (tufM), 90 min (DHSA; MRPS28) or 120 min (DLDH) after the samples
had reached the bottom of the separating gel.
Cleavage Site Motifs in Yeast Mitochondrial Protein
Precursors
In two previous studies(5, 6) ,
amino acid sequence analysis in the region surrounding the mature N
terminus of about 50 mitochondrial precursor proteins from lower
eukaryotes and mammals revealed that precursors cleaved in two steps by
MPP and MIP share a highly conserved three-amino acid motif at the C
terminus of their leader peptide. The motif,
RX()(F/L/I)XX(T/S/G)XXXX(
),
includes an arginine at -10, a large hydrophobic amino acid
(phenylalanine, leucine, or isoleucine) at -8, and a small
hydroxylated residue (serine or threonine) or glycine at -5 from
the amino terminus of the mature protein (i.e. the MIP
cleavage site). Because the -10 arginine is at -2 from the
first cleavage site (i.e. the MPP cleavage site), eight amino
acids within the motif (i.e. the octapeptide) separate the two
cleavages. Not counting homologous sequences, about 13 precursors
potentially cleaved in two steps by MPP and MIP were identified on the
basis of this motif (5) . Since then, two-step processing via
formation of an octapeptide-containing intermediate has been
demonstrated for seven proteins: the S. cerevisiae CoxIV (11) and Fe/S (12) precursors, the N. crassa Fe/S (13) and cyclosporin A-binding protein (14) precursors, the rat malate dehydrogenase
precursor(16) , and the rat (15) and human (1) ornithine transcarbamoylase precursors. Taken together, the
octapeptide-containing precursors identified to date represent a rather
heterogeneous group of proteins, as they include proteins targeted to
the matrix or the inner membrane and involved in a variety of metabolic
functions. The lack of any obvious functional correlation among these
proteins represents a hindrance in understanding the role of MIP.
)
)X(S/X)
(R-2), XRX(Y/X)(
)(S/A/X)
(R-3), and XXX(
)X(S/X) (R-none), which
are found in precursors that are cleaved in one step by
MPP(5, 6) , and the motif
RX(F/L/I)XX(T/S/G)XXXX(
) (R-10), which
is typical of precursors that are cleaved in two sequential steps by
MPP and MIP(5, 6) . Eleven of the 56 precursors
conformed to the R-2 motif (Table 2A); one of these precursors
(CYPC) also contained an R-10 motif. Twelve precursors conformed to the
R-3 motif (Table 2B); one of them (DLDH) also contained a R-10
motif, while another precursor (SODM) contained arginine at both the
-2 and -3 positions. Together, the R-2 and R-3 precursors
represent about 39% of the 56 sequences analyzed. Twelve precursors
conformed to the R-10 motif (Table 2D); two of these precursors
also contained a R-2 (CYPC) or R-3 (DLDH) motif. The CYPC precursor was
subsequently shown to be cleaved in one step, while two-step processing
was observed for the DLDH precursor (see below). Therefore, CYPC and
DLDH were identified as R-2 and R-10 precursors, respectively. Another
precursor (RM20) contained arginine at -10 and phenylalanine at
-8 but alanine instead of serine, threonine, or glycine at
-5; this precursor was also included in the R-10 group. These 11
R-10 precursors represent about 20% of the sequences analyzed.
Twenty-two precursors (R-none) did not contain arginine at any of the
three critical positions, -2, -3, or -10 (Table 2C). One precursor (DHA1) contained arginine at -10
but not phenylalanine, leucine, or isoleucine at -8, nor serine,
threonine, or glycine at -5. Because a closely related precursor
sequence (DHA2) contains asparagine at the -10 position, the DHA1
precursor was included in the R-none precursors. Together, R-none
precursors represent about 41% of the sequences analyzed.
Analysis of Yeast MIP Cleavage Sites
On the basis
of previous observations, the 11 R-2, 11 R-3, and 23 R-none (Table 2, A-C) precursors are predicted to be cleaved in
one step by yeast MPP(5, 6) . When the leader peptides
of these precursors were compared to those of the R-10 precursors, no
obvious differences were observed except for the presence of the
RX(F/L/I)XX(T/S/G)XXXX motif in the latter.
A phenylalanine was present at the -8 position in 8 of the 11
R-10 precursors, while isoleucine and leucine were found at this
position in one and two precursors, respectively. Although glycine is
frequently found at the -5 position in mammalian octapeptides (5) , (
)serine or threonine was found at the
-5 position in 10 of the 11 R-10 yeast precursors, while alanine
was found at -5 in one case (RM20). While serine appeared to be
preferred at the -7 position, no particular amino acid(s) were
preferred at any of the remaining 5 positions within the octapeptide.
On the other hand, the 11 octapeptide sequences did seem to have a
similar amino acid composition, with each containing serine or
threonine at two or more positions, often clustered together.
Furthermore, when the overall amino acid composition of the
octapeptides was analyzed, one of the six smallest amino acids, serine,
threonine, alanine, valine, proline, or glycine, was found at 47
positions (53% of the total number of amino acids). While a positively
charged residue, arginine or lysine, was present at 7 positions, no
acidic residues were found in any of the 11 octapeptides. No particular
amino acid residue(s) appeared to be preferred at the +1 or
+2 positions, nor at any other positions within the first 10 amino
acids of the mature protein, consistent with the fact that no consensus
sequence has been identified on the C-terminal side of known MIP
cleavage sites(5, 6) . Because six of the yeast R-10
precursors have now been shown to be processed by YMIP (3, 11, 12) (this study), we conclude that
the octapeptides of these 11 proteins reflect the cleavage specificity
of the yeast peptidase. In addition to the motif
RX(F/L/I)XX(T/S/G)XXXX, the general features
of these octapeptides can be used to predict new MIP substrates in
yeast and other species.Prediction of YMIP Substrates
The 56 precursors
with a known mature N terminus were also analyzed for the presence of
the motif RX(F/L/I)XX(T/S/G)XXXX over the
entire length of their leader peptide. In addition to the 11 R-10
precursors described above, the motif was found in the leader peptide
of seven precursors, but the motif was not associated with the mature N
terminus of these proteins. Furthermore, while the CYPC precursor
contained the motif precisely upstream from the mature N terminus, this
precursor did not show two-step processing (see below). Thus, the motif
was found in 19 precursors and was associated with the leader peptide
cleavage site in 11 of them (58%). As processing of seven of these
precursors has now been analyzed (3, 11, 12) (this study) and only CYPC is not
cleaved in two steps, we suggest that, in yeast sequences which contain
the motif RX(F/L/I)XX(T/S/G)XXXX upstream
from the mature N terminus, YMIP cleavage can be predicted with a high
level of confidence. However, until a larger protein sample is
analyzed, the processing pattern of putative YMIP-cleaved precursors
may require confirmation by biochemical studies. were then analyzed for the presence of the
motif RX(F/L/I)XX(T/S/G)XXXX within amino
acids 1-50. This region should be sufficient to include the
leader peptide cleavage sites of most precursors, as suggested by the
fact that, of the 56 yeast precursors with known mature N terminus
analyzed in this study, only three (CCPR, RM32, and RM41) contain
leader peptides longer than 50 amino acids. We found a typical
RX(F/L/I)XX(T/S/G)XXXX motif in 18 of the 77
precursors (Table 2E). With the exception of the EFGM octapeptide
which contained a glutamate residue, the amino acid composition of the
18 putative octapeptides was characterized in most cases by a
predominance of small hydroxylated amino acids, similar to the amino
acid composition of authentic octapeptides. Thus, it is likely that
many of these 18 octapeptides represent actual YMIP cleavage sites.
Known and predicted R-10 precursors comprise about 22% of the 133
sequences in our compilation, which suggests that YMIP is involved in
the biogenesis of a significant fraction of mitochondrial proteins.YMIP Activity Is Required for Processing of R-10
Precursors in Vitro
Two of the yeast precursors in our
compilation, pCoxIV and pFe/S, have been previously shown to be
processed in two sequential steps via formation of an
octapeptide-containing intermediate(11, 12) .
Additionally, we have shown previously that YMIP is required for
maturation of pCoxIV and pFe/S in vivo(3) . To our
knowledge, two-step processing has not been previously reported for any
of the remaining yeast R-10 precursors identified in this study;
therefore, we have analyzed the proteolytic processing of in vitro translated pCoxIV and other yeast precursors predicted to be
cleaved by YMIP. The previously cloned genes encoding precursors for
CoxIV(26) , DHSA(27) , MRPS28(30) ,
DLDH(28) , and CYPC (29) were used in in vitro transcription/translation reactions to produce radiolabeled
polypeptides, which were then processed in vitro using
mitochondrial matrix fractions prepared from either wild-type or mip1 yeast. The latter strain is totally deficient in MIP
activity but contains levels of MPP activity which are comparable to
wild-type(3) . is processed to
the mature form by MPP in vivo(33) and in
vitro(1) , and was therefore used as a model for one-step
processing. Upon incubation with a matrix fraction from wild-type
yeast, pF was processed to the mature form (Fig. 1, lanes 2 and 4); mature
F was similarly produced when the precursor was
incubated with a matrix fraction from mip1 yeast (lanes 3 and 5). A very different pattern of
processing was observed with pCoxIV, which was processed to
predominantly mature form with small amounts of intermediate form
(iCoxIV) by wild-type matrix (lanes 7 and 9), whereas
only iCoxIV was produced upon incubation of pCoxIV with mip1 matrix (lanes 8 and 10). We have
shown previously that this processing pattern is characteristic of
precursors cleaved in two steps by MPP and MIP(1, 7) .
The processing patterns of pDHSA (lanes 11-15) and
pMRPS28 (lanes 16-20) were very similar to that of
pCoxIV: mature forms were produced upon incubation of these precursors
with matrix from wild-type yeast (pDHSA, lanes 12 and 14; pMRPS28, lanes 17 and 19), while
intermediate-size species accumulated upon incubation with mip1 matrix (pDHSA, lanes 13 and 15;
pMRPS28, lanes 18 and 20), indicating that, similar
to pCoxIV, these precursors require YMIP activity for maturation.
S-labeled precursor proteins were incubated with 3 µg (lanes 2, 3, 7, 8, 12, 13, 17, 18, 22, 23, 27, and 28) or 5 µg (lanes 4, 5, 9, 10, 14, 15, 19, 20, 24, 25, 29, and 30) total protein of mitochondrial matrix from wild-type or mip1
yeast, as indicated at the bottom of the
figure, for 20 min at 27 °C (3) and analyzed directly by
SDS-PAGE and fluorography. Lanes 1, 6, 11, 16, 21, and 26 contain the translation only.
The electrophoretic positions of precursor (p), intermediate (i), and mature (m) forms are
indicated.
matrix yielded two protein species of roughly similar
electrophoretic mobility, although the polypeptide formed in the
presence of mip1 matrix (lanes 23 and 25) was slightly larger than mature DLDH (lanes 22 and 24). This pattern was clearly different from the
one-step processing of pF (lanes 1-5).
Further, the same pattern was reproduced in several different sets of
processing reactions, and was also observed with mitochondrial import
assays (see below). Thus, although a single protein band was detected
upon incubation of pDLDH with wild-type matrix (lanes 22 and 24), an intermediate-size protein was formed by mip1 matrix (lanes 23 and 25), strongly
suggesting that pDLDH requires YMIP activity for maturation and that
the presence of an arginine residue at -3 from the mature amino
terminus of this precursor is not sufficient to direct cleavage by MPP.
On the other hand, pCYPC was processed identically by wild-type (lanes 27 and 29) and mip1 matrix (lanes 28 and 30). This pattern was very similar to
the one-step processing of pF1 (lanes 1-5) and
clearly distinct from the processing patterns of all other R-10
precursors analyzed. This indicates that pCYPC does not require YMIP
activity for maturation, presumably because the R-2 motif can direct
cleavage of this precursor by MPP alone, an interesting result given
that CYPC is cleaved in two steps in N. crassa(14) .
Similarly, pFe/S is cleaved in two steps in yeast (12) and N. crassa(13) , but in one step in
mammals(35) . Therefore, while the overall function of MIP is
conserved in eukaryotes(10) , its requirement for maturation of
particular precursors may be different depending on the organism.YMIP Activity Is Required for Maturation of R-10
Precursors in Intact Mitochondria
Import reactions were carried
out to assess whether the R-10 precursors require YMIP for normal
biogenesis in intact mitochondria. Mitochondria isolated from mip1 yeast could not be used in this experiment, however,
because these organelles were unable to import radiolabeled precursors
efficiently (not shown). As YMIP inactivation causes multiple defects
of respiratory chain complexes (3) and loss of functional mtDNA
(see below), mip1 mitochondria must present a severe
energy impairment which affects their ability to import precursor
proteins in vitro. This is consistent with previous reports
that import into respiration-deficient mitochondria can be performed in vitro only under specialized conditions(25) . To
overcome this problem, we constructed a mip1 temperature
sensitive (ts) mutant which contains a chromosomally disrupted
copy of wild-type MIP1 and is complemented by a MIP1-c-myc allele on a centromeric vector. The encoded YMIP-c-myc protein contains a single amino acid substitution, glycine for
leucine, at residue 578 of the YMIP sequence. G578 is found 16 residues
C-terminal from the second histidine in the motif
F-H-E-X-G-H-(X)-H-(X)-G-(X)
-D-(X)-E-X-P-S-(X)-E,
which is shared by all known MIP and thimet oligopeptidase sequences
and probably represents the active site of these enzymes (10) .
Conservative replacements of single amino acids in this motif caused
complete loss of YMIP function in most cases. (
)On the other
hand, the mip1(G578L) mutant expressed wild-type
levels of YMIP(G578L) and was able to grow slowly on YPEG at 25 °C
but not at 37 °C (not shown), indicating that partial YMIP function
is retained by this mutant at the permissive temperature.
yeast, we established nonpermissive
conditions which lead to only incomplete inactivation of YMIP(G578L),
such that the respiratory function and thereby the import competence of
these organelles is maintained. For this purpose, mip1(G578L)
yeast were grown at 25 °C and
shifted to the nonpermissive temperature for only 30 min during the
preparation of spheroplasts; mitochondria were similarly prepared from
wild-type yeast.
(Fig. 2, lanes
1-5). Similar to wild-type mitochondria (lane 2), mip1(G578L) mitochondria were able to process
pF
to the mature form (lane 3). When
mitochondria were treated with trypsin upon import and reisolated by
centrifugation, significant amounts of mature protein were associated
with the mitochondrial pellets (wild-type, lane 4; mip1, lane 5), protected from the
externally added trypsin, while only residual amounts of precursor were
found in the corresponding supernatants (not shown). Precursor and
mature F
were degraded when trypsin treatment of
wild-type and mip1 mitochondria was carried out
in the presence of the membrane detergent Triton X-100 (not shown).
These results demonstrate that mip1(G578L)
mitochondria are similar to wild-type mitochondria in their
ability to import pF
and to process this precursor to
the mature form.
mitochondria accumulate intermediate-size polypeptides. In
vitro translated,
S-labeled precursor proteins were
incubated with intact isolated yeast mitochondria, prepared from
wild-type or mip1(G578L)
yeast; addition
of wild-type or mip1(G578L)
mitochondria
to the import reactions is indicated by the plus sign (+) at the bottom of the figure. Aliquots corresponding to 10% of a total
import reaction were analyzed directly by SDS-PAGE and fluorography (lanes 2, 3, 7, 8, 12, 13, 17, 18, 22, 23, 27, 28, 32, and 33). Lanes
1, 6, 11, 16, 21, 26,
and 31 contain translation only. Upon import, some reactions
were treated with trypsin (0.4 mg/ml, final concentration) for 5 min on
ice, followed by soybean trypsin inhibitor (1 mg/ml, final
concentration). Mitochondria were then reisolated by centrifugation,
and the pellets (lanes 4, 5, 9, 10, 14, 15, 19, 20, 24, 25, 29, 30, 34, and 35)
and supernatants (not shown) were analyzed separately by SDS-PAGE.
Aliquots corresponding to 50% of each mitochondrial pellet were
analyzed; trypsin addition is indicated by the plus sign (+) at
the bottom of the figure.
, pCoxIV was
efficiently processed by wild-type mitochondria to the mature form (lane 7), which was protected from externally added trypsin (lane 9). In contrast, iCoxIV was predominantly accumulated by mip1(G578L) mitochondria (lane 8).
However, upon trypsin treatment and reisolation of the mitochondria by
centrifugation, only small amounts of mature CoxIV and traces of iCoxIV
were detected (lane 10). Similar to pCoxIV, both pDHSA and
pMRPS28 were processed to predominantly mature form upon import into
wild-type yeast mitochondria (pDHSA, lane 12; pMRPS28, lane 17), while intermediate-size polypeptides were
accumulated in mip1(G578L)
mitochondria (pDHSA, lane 13; pMRPS28, lane 18). Small quantities of iDHSA (lane 15) and larger of iMRPS28 (lane 20) were
trypsin-protected, while all proteins were fully degraded when trypsin
treatment was carried out in the presence of Triton X-100 (not shown).
mitochondrial matrix (Fig. 1) and further
indicate that YMIP activity is required by these precursors for normal
biogenesis. Two-step processing of pDHSA is consistent with our
previous observation that, in addition to complete defects of succinate
cytochrome c reductase and cytochrome c oxidase, mip1 mitochondria also present a 90% reduction of
succinate dehydrogenase activity(3) . The fact that only very
small amounts of iCoxIV and iDHSA could be recovered in trypsin-treated
mitochondria may indicate that these intermediates have been only
partially translocated by mip1 mitochondria;
alternatively, iCoxIV and iDHSA may be rapidly degraded inside the
mitochondrion. The latter possibility is supported by the observation
that iCoxIV, but not iFe/S, is rapidly degraded in mip1
mitochondria in vivo(3) , suggesting that a defect in
YMIP activity may have different effects on different substrates. mitochondria
yielded an intermediate-size protein (lane 23) which was
protected from externally added trypsin (lane 25), while a
smaller band, mature DLDH, was detected upon import of this precursor
by wild-type mitochondria (lanes 22 and 24). This
processing pattern was similar to the one obtained upon incubation of
pDLDH with matrix fractions from mip1
and wild-type
yeast, respectively, further supporting the conclusion that this
precursor requires YMIP for normal biogenesis. To optimize the
separation of intermediate- from mature-size proteins, we used
different electrophoretic conditions for each of the precursors
analyzed in this study; however, the processing pattern of pDLDH
indicates that a difference of only eight amino acids between a
particular intermediate and the corresponding mature protein may not
always be sufficient to separate these two species by standard
SDS-PAGE. In such cases, matrix fractions and/or isolated mitochondria
that are totally or partially deficient in MIP activity are required
for accumulation and detection of intermediate-size species. These
problems may explain why two-step processing has seldom been reported
despite the fact that a number of R-10 precursors are known.YMIP Cleaves Components of the Yeast Mitochondrial
Genetic Machinery
One of the R-10 precursors analyzed above,
pMRPS28, is an essential component of the mitochondrial
ribosome(30) . To further investigate the role of YMIP in the
biogenesis of mitochondrial genetic components, the single-stranded
DNA-binding protein RIM1 (32) and elongation factor tufM (20) were analyzed. The RIM1 precursor presents a typical
octapeptide motif upstream from the mature N terminus (Table 2D).
Although the mature N terminus of tufM has not been determined, by
comparison to the highly homologous Escherichia coli elongation factor TuA, yeast tufM is predicted to contain an
amino-terminal leader peptide of 37 amino acids(31) , with a
potential R-10 cleavage site displaced one residue from the predicted
mature N terminus. mitochondria, as described above. The tufM precursor was
processed to an intermediate-size form by mip1
mitochondria (Fig. 2, lane 28), and this protein
was protected from externally added trypsin (lane 30); a
slightly smaller protein, presumably mature tufM, was detected upon
incubation of ptufM with wild-type mitochondria (lane 27) and
was also protected from trypsin (lane 29). This processing
pattern is different from the one-step processing of
pF
(lanes 1-5) and very similar to the
pattern observed for pDLDH. mitochondria (lane 33) and
was protected from externally added trypsin (lane 35),
indicating that pRIM1 is processed in two steps. Discrete amounts of a
mature-size protein were detected in lane 33; however, only
traces of this protein were detected in the mitochondrial pellet after
trypsin treatment (lane 35). Because a mature-size band was
detected in the total translation reaction in the absence of
mitochondria (lane 31), we conclude that most of the
mature-size protein band detected in lane 33 does not
represent a bona fide mature RIM1 species, but rather, a
nonspecific translation product which is not inside the mitochondria
and is thus degraded by externally added trypsin.
Loss of YMIP Function Has a Secondary Effect on mtDNA
Stability
Chromosomal disruption of the genes encoding MRPS28 (30) , RIM1(32) , and tufM (20) has been
previously shown to result in respiration-deficient phenotypes with
loss of functional mtDNA. If octapeptide cleavage is required for
normal function of these and, perhaps, other components of the
mitochondrial genetic system, loss of YMIP function should indirectly
cause mitochondrial genome instability. We showed previously that the mip1 phenotype can be rescued if an heterozygous
disruption strain is transformed with a centromeric vector carrying MIP1 prior to sporulation(3) . In contrast, haploid mip1 cells could not be rescued by transformation with MIP1 (not shown), suggesting that YMIP inactivation had
resulted in some irreversible change in the mitochondria of these
cells, presumably loss of intact mitochondrial genomes. The integrity
of mtDNA in mip1 cells was then analyzed by genetic
methods. Y34, wild-type Y193, and ycl57w Y191; and mip1 Y6040 and Y6043, and
wild-type Y6041 and Y6042 (Table 1). The mip1
mutants Y34, Y6040, and Y6043 failed to complement a 
tester, which is devoid of mtDNA, as well as three mit testers, which contain point mutations
in the mitochondrial genes COXI (i.e. OXI3), COXIII (i.e. OXI2), and COB1, respectively (Table 3). On the other hand, zygotes from a cross of a
tester, carrying normal mtDNA, with Y34 gave
rise to 93%
diploid strains (not shown). These
results are consistent with important deletions, if not complete loss,
of mtDNA in mip1
cells. In contrast, the and mit strains were complemented by
the ycl57w
mutant and the wild-type strains (Table 3), indicating that the loss of functional mtDNA in mip1 mutants is independent of the genetic background of
the parental strains.
The Known YMIP Substrates Are Functionally
Related
Yeast mitochondrial proteins which mediate global
protein import are essential for cell viability(19) . Although
the putative YMIP substrates seem to represent a significant fraction
of imported mitochondrial proteins, inactivation of YMIP causes loss of
mitochondrial oxidative phosphorylation without affecting viability (3) . Thus, the actual fraction of mitochondrial proteins
cleaved by YMIP may be smaller than we have predicted. Alternatively,
proteins that are normally cleaved by YMIP may be at least partially
functional in mip1 yeast. A third possibility is that
only a specific subset of precursors contain octapeptides cleaved by
YMIP, such that inactivation of the peptidase results in loss of
respiratory function without affecting other metabolic pathways. The
available evidence supports the last idea. The 11 R-10 precursors in Table 2D, include two distinct but functionally related sets of
proteins. The first are components required for respiratory function:
respiratory chain subunits, CoxIV(26) , Fe/S(36) ,
DHSA(27) , tricarboxylic acid cycle enzyme components,
DLDH(28) , IDHP(37) , MDHM(38) , and
ferrochelatase, HEMZ(39) , the enzyme which catalyzes the final
step in the heme biosynthetic pathway. Genetic inactivation was
previously carried out for six of these components and resulted in
respiration-deficient phenotypes with intact
mtDNA(26, 28, 36, 37, 39, 56) .
The second set includes essential components of the mitochondrial
protein translational machinery, MRPS28(30) ,
PET122(40) , and RM20 (41) , and a protein essential
for mtDNA replication, RIM1(32) . Genetic inactivation of these
components results in respiration-deficient phenotypes with loss of
mtDNA. If the same type of analysis is extended to the remaining 18
potential R-10 precursors (Table 2E) similar results are obtained
in 16 cases, including components required for respiratory function,
ABC1(42) , ACON(43) , ATP0(44) ,
COQ2(45) , SDH3 (46) , mitochondrial protein
translation, tufM(31) , EFGM(47) , MRS2 (48) ,
MSS1(49) , PET111(50) , RF1 M(51) ,
RMO6 (52) , PET123(53) , MST1(54) , and mtDNA
recombination and repair, PIF1 (55) , and PHR(34) .
yeast. Thus, further analysis of these substrates in mip1 mutants should help clarify the role of two-step processing in
respiratory and mtDNA function.
),
FATPase subunit ; pF, precursor
F; PAGE, polyacrylamide gel electrophoresis; bp, base
pair(s).)))
We thank R. A. Rollins for excellent technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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