(Received for publication, November 17, 1995; and in revised form, January 9, 1996)
From the
Apocytochrome c is synthesized in the cytoplasm,
transported to the mitochondrial intermembrane space, and subsequently
covalently attached to heme in a reaction catalyzed by the enzyme
cytochrome c heme lyase. We have investigated the amino acid
sequences in cytochrome c which are required for mitochondrial
import, using a systematic series of site-directed alterations of the CYC7-H3 gene which encodes iso-2-cytochrome c in the
yeast Saccharomyces cerevisiae. Import of the altered
apocytochromes c was assayed in yeast strains that
overexpressed cytochrome c heme lyase. Under these conditions,
there was efficient mitochondrial accumulation of forms of
apocytochrome c which are incapable of having heme covalently
attached. In fact, all apocytochromes c containing deletions
located to the carboxyl-terminal side of His efficiently
accumulated in the mitochondria of strains overexpressing heme lyase,
even though all but one of these deletion-containing proteins were
incapable of heme attachment. A minimum length of polypeptide chain at
the extreme amino terminus of cytochrome c, rather than any
specific sequence element in this region, appears to be required for
efficient mitochondrial import. Certain amino acid substitutions in the
region extending from Gly
to Leu
, at residue
Phe
and at residue His
, lead to reduced
mitochondrial import of apocytochrome c, resulting from
stalling of the altered apocytochrome c in partially imported
states.
Most mitochondrial proteins, including cytochrome c, are encoded in the nucleus, synthesized in the cytoplasm, and then transported across one or more mitochondrial membranes. For several of these imported mitochondrial proteins, the sequences that direct them to mitochondria, and to the correct subcompartment of mitochondria, have been identified. Such sequences are generally located near the amino terminus of the imported protein, often as part of a presequence that is cleaved during or after import.
Cytochrome c is
translocated into the mitochondrial intermembrane space from its site
of synthesis in the cytoplasm along a pathway that differs in several
respects from pathways followed by other, more extensively studied,
mitochondrial proteins that are targeted to the same mitochondrial
subcompartment. Cytochrome c is initially synthesized as
apocytochrome c, lacking heme. During or after import into
mitochondria, heme is covalently attached via stereospecific thioether
linkages to two cysteine residues in the protein. This reaction is
catalyzed by the enzyme cytochrome c heme lyase (CCHL), ()which is located in the mitochondrial intermembrane space,
predominantly associated with the inner membrane. Heme attachment is
accompanied by a transition from a partially extended conformation to
the native compact structure. Cytochrome c does not contain
any cleaved targeting sequence. No protease-sensitive receptor on the
external surface of mitochondria mediates the initial binding and
import of apocytochrome c. Cytochrome c import does
not depend on the presence of an electrochemical potential across the
inner mitochondrial membrane or on the presence of ATP (for review see
Hartl et al., 1989; Stuart and Neupert, 1990; Kiebler et
al., 1993; Hannavy et al., 1993; Dumont, 1995).
The signals in the cytochrome c sequence which are responsible for the mitochondrial import have not been well characterized previously. Since cytochrome c does not contain a cleavable signal presequence at the amino terminus of the protein, the import signal must be a part of the mature sequence. Furthermore, sequences important for import are unlikely to reside at the extreme amino terminus of cytochrome c, since deletions and replacements of up to 11 residues at the amino terminus of iso-1-cytochrome c of yeast (Sherman and Stewart, 1973; Baim et al., 1985; Hampsey et al., 1988) do not prevent normal targeting and maturation.
Sequences responsible for subcellular targeting of many proteins have been identified through mutational alteration of the relevant structural genes followed by analysis of the subcellular distribution of the altered protein. Application of this approach to cytochrome c has been problematic because of the difficulty of separating effects on heme attachment from effects on import. Failure to attach heme to cytochrome c leads to accumulation of the precursor, apocytochrome c, in the cytoplasm. Heme attachment can be blocked by competition with a heme analog that cannot be incorporated into holocytochrome c, by mutational alteration of cytochrome c, or by a lack of CCHL, the enzyme catalyzing the attachment (Hennig and Neupert, 1981; Dumont et al., 1988, 1991; Nargang et al., 1988). Such coupling between heme attachment and import is consistent with a mechanism in which apocytochrome c is reversibly transported across the mitochondrial outer membrane and subsequently trapped in the intermembrane space by the protein folding reaction accompanying CCHL-mediated heme attachment. However, direct participation of CCHL in import has not been completely ruled out (see Dumont, 1995; Mayer et al., 1995).
Two previous mutational studies of sequences responsible for targeting cytochrome c to mitochondria were conducted in systems where mitochondrial localization could be assayed independent of heme attachment. The first of these was carried out in vitro, assaying association of Drosophila melanogaster apocytochrome c with isolated mouse liver mitochondria in the presence of a cytoplasmic factor from wheat germ (Hakvoort et al., 1990; Sprinkle et al., 1990). For unknown reasons, mitochondrial association was not accompanied by a significant level of heme attachment in this system. The second of the mutational studies was carried out in vivo, using gene fusions between yeast iso-1-cytochrome c sequences and the chloramphenicol acetyltransferase sequence (Nye and Scarpulla, 1990a, 1990b). In this system, the presence of a large folded protein fused to cytochrome c prevented correct localization to the intermembrane space, which, in turn, prevented efficient heme attachment. Both mutational studies concluded that cytochrome c contains two targeting sequences that are at least partially redundant, one in the first 60 residues of the protein, and the other in a more carboxyl-terminal region.
We have investigated the sequences involved in targeting cytochrome c to mitochondria using an in vivo system that is closely related to the authentic pathway of import in the yeast Saccharomyces cerevisiae. To separate the effects on heme attachment from effects on import, we have capitalized on a previous observation that high level expression of CCHL allows apocytochrome c to be efficiently imported into mitochondria in the absence of heme attachment (Dumont et al., 1991). In this system, import of apocytochrome c appears to be driven by direct binding to the heme lyase rather than by conversion to holocytochrome c (Dumont et al., 1991; Mayer et al., 1995), thus allowing evaluation of the import competence of altered cytochromes c that are not capable of heme attachment.
We report the effects of a systematic set of deletions, as well as selected point mutations, on the import of yeast iso-2-cytochrome c into mitochondria. Levels of mitochondrial apocytochrome c were assayed by immunoblotting subcellular fractions of strains expressing high levels of CCHL. In addition, examination of heterozygous diploid strains revealed that expression of certain altered apocytochromes c prevented the maturation and targeting of coexpressed normal cytochrome c. Some of the altered cytochromes c appeared to be stalled at specific stages of translocation into mitochondria. Taken together, these analyses have led to the identification of amino acid residues required for cytochrome c import into mitochondria and to the detection of partially translocated forms of cytochrome c which accumulate when certain sequence elements are missing.
For construction of the cyc7-H3-87 allele (see Fig. 1) with the 12CA5 epitope replacing the first 14 amino acids of the protein, oligonucleotides OL91.072 and OL91.038 were used with plasmid pAB1386 in the polymerase chain reaction (PCR) to amplify the epitope-containing CYC7-H3 gene as described by Scharf (1990). The sample was cycled 30 times with denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min and 72 °C for 4 min. The PCR product was treated with restriction endonuclease SalI and self-ligated. Reverse PCR using this same protocol was then performed on the circular product with oligonucleotides OL91.300 and OL91.298 as primers, thereby creating the deletion. The PCR fragment was treated with restriction endonuclease BamHI and self-ligated. The circularized product was subjected to an additional round of PCR using oligonucleotides OL91.335 and OL91.257 as primers. Plasmid YCp50 was digested with restriction endonuclease NruI, and a single T was added to each end of the DNA molecule with deoxy-TTP through the action of terminal deoxynucleotidyl transferase. Finally, the PCR fragment containing the CYC7-H3 allele with the epitope and the deletion was ligated to the modified YCp50 vector to construct a plasmid pAB1326.
Figure 1:
Scheme for making the deletion mutation cyc7-H3-87 with PCR. Two arrows inside the
plasmid pAB1386 denote two oligonucleotides that are used to PCR
amplify the Ept + iso-2 gene containing the 12CA5 epitope
inserted upstream of the CYC7 gene. The directions of the arrows indicate the 5` 3` direction of the
oligonucleotides. The arrow that is parallel to the circle represents the oligonucleotide that is fully complementary to the
plasmid sequence. The tail at the beginning of the arrow represents the part of the oligonucleotide sequence which is not
complementary to the plasmid sequence. The Ept + iso-2
gene is represented by a solid box. The restriction
endonuclease SalI (S) sites are marked by short
lines. The PCR product was digested with SalI and
self-ligated at 16 °C for 12 h. Reverse PCR was performed on the
ligated plasmid with two oligonucleotides that incorporated a BamHI (B) site at the 5` ends of their sequences. The
PCR product was digested with BamHI and self-ligated. A third
round of PCR was then used to amplify the mutated gene. Plasmid YCp50
was digested with restriction endonuclease NruI (N).
The blunt ended DNA fragment was incubated with dideoxy-TTP (ddTTP) and terminal deoxynucleotidyl transferase (TDTase) at 37 °C for 1 h. A single T was added to two 3`
ends of the molecule. The modified plasmid was then ligated with the
mutated gene, resulting in plasmid pAB1326, which contained cyc7-H3-87.
Figure 2:
Summary of the mutations in the
iso-2-cytochrome c and their effects. The various alterations
of iso-2-cytochrome c are listed on the left. Normal denotes the normal iso-2-cytochrome c. No
iso-2-cyt. c denotes the total deficiency of iso-2-cytochrome c. Deletions are denoted by , followed by the first and
last residue, for example,
(Ala1-Lys14) denotes the
mutant in which the sequence from Ala
to Lys
is deleted. Amino acid replacements are indicated using arrows, for example, Cys23
Ala23 denotes the
mutant in which the cysteine residue at position 23 is replaced by the
alanine residue. Ept +
(Ala1-Lys14) denotes
the mutant in which the amino-terminal peptide from Ala
to
Lys
is replaced by the 12CA5 epitope. The iso-2-cytochrome c constructs are schematically shown in the center column of the figure. For panels A and B, the solid
box is the intact amino acid sequence of iso-2-cytochrome c, and the open box is the part of iso-2-cytochrome c sequence which has been deleted. The hatched box is
the 12CA5 epitope. Amino acid residues Cys
and
Cys
, the sites of the heme attachment, are shown above the
protein constructs. For panel C, only part of the
amino-terminal region is shown. Deletions are again indicated by the open boxes, and amino acid replacements are shown above the
protein constructs. The results are listed on the right. Holo-Cyt. c refers to the total amount of holocytochrome c in intact cells. Internal Cyt. c refers to the amount of
cytochrome c inside the mitochondria which is protected from
digestion by externally added proteinase K (see the Mito.+Pro.K lane of Fig. 4). Asso. Cyt. c refers to the amount of cytochrome c which is associated
with the mitochondria after the mitochondria are isolated and washed
(see the Mito. lane of Fig. 4). Holo. in 2N refers to the amount of holocytochrome c produced in the
heterozygous diploid strains and reflects the extent of displacement of
holocytochrome c by the altered apo-iso-2-cytochromes c. The cytochrome c level that is associated with the
mitochondria and the level inside the mitochondria were calculated from
the comparisons of the intensities of the cytochrome c bands
of the cytoplasm and the relevant mitochondria fractions when various
amounts of total protein of each fraction were loaded in the Western
blot analyses. For purposes of this calculation, mitochondria were
assumed to constitute 2% of total cellular
protein.
Figure 5:
Low temperature (-196 °C)
spectrophotometric recordings of a series of diploid strains. The
following representative strains contained the indicated levels of
normal cytochrome c. A, B-9609, 100% (cyc1- CYC7-H3
CYC1 cyc7-
); B, B-9610, 50% (cyc1-
cyc7-
CYC1 cyc7-
); C, B-9592, 40% (cyc1-
cyc7-H3-81
CYC1 cyc7-
); D, B-9588, 30% (cyc1-
cyc7-H3-77
CYC1 cyc7-
); E,
B-9591, 20% (cyc1-
cyc7-H3-80
CYC1
cyc7-
); F, B-9608, 15% (cyc1-
cyc7-H3-99
CYC1 cyc7-
); G,
B-8482, 0% (cyc1-
cyc7-
cyc1-
cyc7-
).
Figure 4: Subcellular distributions of cytochrome c in some representative yeast strains. The subcellular fractionation and Western blot procedures are described under ``Materials and Methods.'' The allele number of the yeast strain is listed on the left. Descriptions of the alleles are listed on the right and are also presented in Table 2. The cytochrome c bands of each fraction are shown in the middle of the figure. The Crude extract lane refers to the cytochrome c in the mixture of cytoplasm and mitochondria after the yeast cells were disrupted, and unbroken cells and nuclei have been removed by low speed centrifugation. The Cytoplasm lane refers to the cytochrome c in the supernatant after the mitochondria have been removed by centrifugation. The Mitochondria lane refers to the cytochrome c that remains with the mitochondria after they have been pelleted and washed. Mito.+Pro.K refers to the cytochrome c in the mitochondria after they have been treated with proteinase K. Mito.+Trit.+Pro.K refers to the cytochrome c after the mitochondria have been broken by 0.5% Triton X-100 and then treated with proteinase K. A total of 100 µg of protein was loaded on the Crude extract and Cytoplasm lanes, and 10 µg was loaded on the Mitochondria, Mito.+Pro.K, and Mito.+Trit.+Pro.K lanes.
Because iso-2-cytochrome c normally constitutes only about 5% of the total complement of cytochrome c, making it difficult to detect, we have used the CYC7-H3 allele, which constitutively overproduces iso-2-cytochrome c to a level that is approximately the same as the normal level of iso-1-cytochrome c (McKnight et al., 1981). This increased expression is due to a deletion that removes the normal promoter and fuses the translated region of iso-2-cytochrome c to an upstream promoter (McKnight et al., 1981; Melnick and Sherman, 1993).
Each strain of haploid series 1 has the following relevant
genetic properties: a single copy of the cyc7-H3-xx allele
integrated into the chromosome; a cyc1- deletion causing
a total lack of iso-1-cytochrome c; a normal copy of the CYC3 gene encoding CCHL; and a multicopy plasmid
p[CYC3]
leading to overproduction of
CCHL driven by the actin promoter. As described previously (Dumont et al., 1991) and discussed above, overproduction of CCHL
leads to efficient accumulation of apocytochrome c inside
mitochondria when heme attachment does not occur. Such accumulation
appears to be mediated by a direct interaction between apocytochrome c and CCHL in a nonproductive enzyme-substrate complex located
in the mitochondrial intermembrane space (Dumont et al., 1991;
Mayer et al., 1995; Dumont, 1995). The combination of genetic
elements in haploid series 1 allows examination of the effects of cyc7-H3-xx alleles on mitochondrial accumulation of
apocytochrome c under conditions where heme is not attached to
the protein. Mitochondrial accumulation of mutant cytochromes c was assayed by immunoblotting of subcellular fractions derived
from the relevant yeast strains. The antibodies used recognize both
holocytochrome c and apocytochrome c.
Initially, the studies described below were performed with the cyc7-H3-xx alleles to which a 12CA5 epitope tag from influenza hemagglutinin had been added (Kolodziej and Young, 1991). However, in some cases, the presence of this tag appeared to alter the intracellular distribution of cytochrome c (see below). Comparison of the immunologic reactivity of anti-epitope-tag antibodies with that of anti-cytochrome c antibodies demonstrated that the affinity-purified polyclonal anti-cytochrome c antibodies efficiently recognized all of the mutant cytochromes c that we tested. Thus, anti-cytochrome c antibodies rather than anti-epitope-tag antibodies were used in the assays of intracellular cytochrome c distributions presented below.
The level of holo-iso-2-cytochrome c in each member of
this series was estimated by low temperature (-196 °C)
spectroscopic examination of intact yeast cells. The ability of each
altered iso-2-cytochrome c to function in electron transport
was estimated from the ability of each strain to grow on media
containing nonfermentable carbon sources, such as glycerol or ethanol.
The levels and functions were assigned values in comparison with the
control strain cyc1- CYC7-H3 CYC3
.
Thus, each of the diploid strains contains the normal level of
CCHL, a single copy of the cyc7-H3-xx encoding one of the
altered iso-2-cytochromes c, and a single copy of CYC1 encoding normal iso-1-cytochrome c.
Using low temperature spectroscopy of intact cells, the level
of holocytochrome c in each of the strains of this series was
compared with the level in two control strains. One control strain
contains two normal cytochrome c alleles. The other contains
one normal cytochrome c allele accompanied by a deletion of
the other allele. The level of holocytochrome c in the double
hemizygous CYC7-H3/cyc7- CYC1
/cyc1-
control strain was
defined as 100%. The level of holocytochrome c in the
heterozygous cyc7-
/cyc7-
CYC1
/cyc1-
control strain
corresponded to 50% of the previous one. We used diploid series in
which the normal cytochrome c allele was iso-1-cytochrome c (cyc1-
cyc7-H3-xx CYC3
CYC1
cyc7-
CYC3
) rather than diploid series in which the
normal cytochrome c allele was iso-2-cytochrome c (cyc1-
cyc7-H3-xx CYC3
cyc1-
CYC7-H3 CYC3
) because
apo-iso-1-cytochrome c is much more labile than
apo-iso-2-cytochrome c; therefore, the efficiency of
production of holocytochrome c should be more sensitive to the
inhibition caused by the presence of an altered apo-iso-2-cytochrome c.
The results presented below reveal that all diploid strains, except for the control strain, contained less than 50% of holocytochrome c, indicating that all of the altered apo-iso-2-cytochromes c partially displaced the normal forms. Furthermore, several of the diploid strains contained as little as 15% of the normal level, suggesting that these altered forms had a higher affinity than the normal protein for some components involved in import or maturation of cytochrome c.
The mutations causing less than 50% of holocytochrome c in the heterozygous strains are formally designated as dominant-negative or semidominant-negative mutations. Such dominant-negative mutations are generally encountered in the single copy heterozygous state when the mutant form of the protein has an abnormally high affinity for some other cellular component, thus displacing the normal form.
Figure 3: Amino acid sequence of iso-2-cytochrome c in the yeast S. cerevisiae.
The levels of holocytochromes c in haploid series 2 are presented in Fig. 2. Representative results of low temperature spectrophotometric recordings are shown in Fig. 5. Analysis of the subcellular distributions included: (i) determination of the amount of cytochrome c that was associated with mitochondria but not necessarily protected from proteinase K digestion; (ii) determination of the amount of cytochrome c inside mitochondria which was protected from digestion with externally added proteinase K; and (iii) determination of the susceptibility of each cytochrome c to proteinase K digestion in the presence of Triton X-100, which disrupts the mitochondrial membrane. Based on these measurements, each of the 25 mutationally altered iso-2-cytochromes c could be assigned to one of five classes (Class I-Class V, see Table 4). All but Class II and Class V altered cytochromes c were deficient in heme attachment.
Six of the 10 deletions spanning the CYC7 gene gave rise to phenotypes typical of Class I mutations. The
deletions in this class included
(Val
-Arg
) and the five deletions
encompassing the region from Thr
to Lys
,
comprising most of the carboxyl-terminal half of the molecule. Each of
these mutant apo-iso-2-cytochromes c lacked heme attachment.
Furthermore, each was imported into mitochondria and interfered with
the formation of holo-iso-1-cytochrome c in diploid strains
with approximately the same efficiency as the mutations involving
substitutions for the cysteine residues involved in heme attachment.
Thus it appeared that these deletions did not reside in sequences
required for mitochondrial import or binding to CCHL.
The
(Ile
-Lys
) deletion has been
included in this class of mutants although the percentage of this form
of apocytochrome c localized within mitochondria was slightly
lower than that seen in the other members of this class. This less
efficient accumulation suggests that sequences at the extreme carboxyl
terminus of cytochrome c may play a minor role in some stages
of targeting or binding to CCHL. In a diploid strain, expression of the
(Ile
-Lys
) deletion inhibited
formation of normal holocytochrome c to the same extent as
other Class I mutations.
The
(Gly
-Leu
) and
(Phe
-Arg
) deletions each exhibited
only partial defects in the accumulation of mitochondrial apocytochrome c compared with the entire
(Gly
-Arg
) deletion. This indicated
that sequences from each of these two smaller regions might be
important for this process. The phenotypes of the
(Phe
-Arg
) deletion and the single
substitution Phe
Ala
were
indistinguishable, indicating that Phe
might be the
critical residue in the Phe
-Arg
region.
Thus, Phe
and one or more residues in the
Gly
-Leu
region appeared to be required
for efficient mitochondrial accumulation of apocytochrome c.
Even though the (Gly
-Gln
)
apocytochrome c did not accumulate inside mitochondria to any
appreciable extent, expression of this altered apocytochrome c in the heterozygous diploid strain displaced the formation of the
normal holocytochrome c to the same extent as the Class I
apocytochromes c, which were accumulated efficiently inside
mitochondria (see Fig. 2).
Since even normal apocytochrome c did not associate with mitochondria that lacked CCHL (Dumont et al., 1991), it seemed likely that binding the
(Gln
-Lys
) apocytochrome c on the mitochondrial surface would depend on CCHL. This was tested
by measuring mitochondrial association of this mutant in a strain
expressing a single copy of CYC3 (encoding CCHL), instead of
the overexpressing strains used in the other subcellular localization
study. The lower level of CCHL reduced the amount of mitochondrially
associated cytochrome c from 40 to 3% of the total cellular
level. Thus, interaction with CCHL was required to maintain even
surface-exposed precursor in association with mitochondria.
His appears to be the key residue in this region
responsible for the accumulation of apocytochrome c in a
protease-inaccessible compartment of mitochondria. Apocytochrome c containing a
(Thr
-Lys
)
deletion accumulated in a protease-protected state with the same
efficiency seen for Class I mutants. Furthermore, the single
substitution His
Ala
exhibited a
phenotype indistinguishable from deletion of the entire region from
Gln
to Lys
.
Not all of
the apocytochrome c containing the
(His
- Tyr
) deletion was converted
into holocytochrome c. Approximately 30% of the
immunologically detectable protein was found in the cytoplasm. Since
strains expressing this mutant contained a normal overall cellular
level of immunologically detectable cytochrome c, 70% of which
was mitochondrial, there appeared to be additional apocytochrome c inside mitochondria. The existence of this population of
mitochondrial apocytochrome c, which was presumably bound to
CCHL, was consistent with the observed partial displacement of normal
holo-iso-1-cytochrome c by the
(His
-Tyr
) mutant in diploid
strains.
The sequences in cytochrome c which are responsible for directing the protein from its site of synthesis in the cytoplasm to its functional site on the outer surface of the mitochondrial inner membrane have not been well characterized previously. A major difficulty in identifying such sequences has been the requirement for heme to be covalently attached to apocytochrome c to observe efficient import. This makes it difficult to distinguish sequence determinants that are important for heme attachment and concomitant folding of the protein from sequence determinants that are actually involved in subcellular targeting.
To circumvent these difficulties, we have made use of a previous observation that altered forms of apocytochrome c which are incapable of having heme covalently attached can be imported efficiently into mitochondria that have an abnormally high level of CCHL (Dumont et al., 1991). Determination of the subcellular distribution of apocytochrome c in such CCHL-overproducing strains has the following advantages for the identification of sequences involved in targeting. (i) The assay is performed in vivo with a system that is known to be capable of efficiently importing and maturing normal cytochrome c. (ii) Comparisons can be made among different forms of cytochrome c which are incapable of undergoing covalent heme attachment. (iii) The assay does not involve the creation of fusion proteins that could alter targeting. (iv) Apo-iso-2-cytochrome c is stable in the cytoplasm and mitochondria, allowing immunologic assay of the subcellular distribution of the protein.
In a
previous analysis of single amino acid replacements at 16 different
sites throughout iso-1-cytochrome c which caused deficiency of
function, only replacements at Cys, Cys
, and
His
(iso-2-cytochrome c numbering system)
completely prevented the formation of holocytochrome c (Hampsey et al., 1988). Thus, except for these three
amino acid residues that comprise and adjoin the actual site of
covalent heme attachment, it seems likely that no short motifs are
required for the covalent attachment of heme. On the other hand, the
failure of the numerous deletion-containing cytochromes c to
be converted into holocytochrome c indicates that successful
catalysis of heme attachment by CCHL requires the existence of an
overall structure. Since most of the deletion-containing apocytochromes c accumulate efficiently inside mitochondria, structural
distortions that prevent heme attachment do not necessarily prevent
binding to CCHL, and furthermore, the actual site of heme attachment in
cytochrome c may not be the same as the sequence determinants
involved in the binding to heme attaching enzyme.
The failure of most of the deletion-containing alleles of iso-2- cytochrome c to undergo covalent heme attachment is in sharp contrast to the report of Veloso et al.(1984) that partially purified CCHL is capable of attaching heme to a peptide corresponding to residues 1-25 of horse apocytochrome c. Either the efficiency of the reaction in vitro is very low, or the sequence requirement for CCHL action is much more stringent in vivo.
Wild type eukaryotic cytochromes c from
numerous species vary considerably in their amino-terminal regions.
Many of these proteins are nine amino acid residues shorter than
iso-2-cytochrome c, and some are two amino acid residues
longer than iso-2-cytochrome c (Hampsey et al., 1988;
Moore and Pettigrew, 1990). In spite of this variation, the import and
heme attachment processes of cytochrome c appear to be
fundamentally the same in all eukaryotes. For example, horse cytochrome c, expressed under control of the normal yeast
iso-1-cytochrome c promoter, was produced in yeast at a level
corresponding to 66% of the normal abundance of iso-1-cytochrome c, even though the amino terminus of horse cytochrome c corresponds to Gly of iso-2-cytochrome c (Hickey et al., 1991).
A minimal requirement for the length of the amino-terminal region of a cleavable mitochondrial matrix targeting signal fused to dihydrofolate reductase has been observed by Ungermann et al.(1994). In that case, the minimal length appeared to be required for the ATP-dependent interaction of the precursor with mitochondrial Hsp70. In the case of cytochrome c, the length could be required for stable interaction with the mitochondrial outer membrane, with CCHL, or with some yet-to-be identified factors. However, it is surprising that the length requirement for cytochrome c resides at the extreme amino terminus of the protein, since cytochrome c contains no cleavable targeting signal, and the other targeting signals that we have uncovered are located at least 15 residues from this end. This suggests either that there is nonspecific anchoring of the end of the chain in order to expose some more specific signals farther from the end or that the extreme amino terminus is involved in stabilizing a translocation-competent conformation. The amino-terminal region of apocytochrome c could also enhance membrane-active properties that would facilitate penetration across the mitochondrial outer membrane (Jordi et al., 1989b).
There is one previous report of a cytoplasmic factor involved in cytochrome c import in an in vitro system (Hakvoort et al., 1990). Furthermore, a cytoplasmic factor called mitochondrial import stimulation factor from rat liver has been found to promote mitochondrial import of proteins other than cytochrome c. The ATPase activity of mitochondrial import stimulation factor was stimulated by the presence of unfolded mitochondrial precursors, including apocytochrome c but not holocytochrome c. This stimulation of ATPase was reversed by the presence of mitochondrial outer membranes, apparently because of binding of the mitochondrial import stimulation factor-precursor complex to sites on the membranes (Hachiya et al., 1994). Import of cytochrome c into mitochondria does not appear to depend on cytoplasmic heat shock-related proteins to the same extent as import of other mitochondrial proteins, since, in contrast to other mitochondrial precursors, apocytochrome c can be imported efficiently into isolated mitochondria from wheat germ translation systems, which are at least partially deficient in such heat shock-related proteins (Dumont et al., 1988; Murakami et al., 1988).
There appear
to be at least two sequence determinants in the region
Gly-Arg
, which affect mitochondrial
accumulation, including Phe
and at least one residue in
the region Gly
-Leu
. The equivalent of
Phe
is found in the sequence of every known eukaryotic
cytochrome c (see Hampsey et al., 1988), in agreement
with the possible importance of this residue. The residues found at the
equivalent of positions Ala
, Thr
, and
Leu
vary considerably among eukaryotic species and have
been mutated with little effect on cytochrome c function.
Gly
is invariant among eukaryotic cytochromes c,
suggesting that it could play an important role. However, replacements
of Gly
by alanine, serine, cysteine, or aspartic acid
residues allowed the formation of substantial levels of holocytochromes c which were at least partially functional (Hampsey et
al., 1988; Auld and Pielak, 1991). Thus, deletion of the region
Gly
-Leu
might lead to the loss of
functionally redundant sequence elements or disrupt a local
conformation that was not affected by replacement of any single amino
acid residue.
Sprinkle et al.(1990) found that substitutions of glutamine, glutamic acid, and asparagine for lysine residues at the equivalent of positions 14, 16, and 17 (iso-2-cytochrome c numbering system) in D. melanogaster cytochrome c led to loss of import. The lysine residues at positions 16 and 17 are not conserved in iso-2-cytochrome c. The third of these lysine residues, at position 14, is not indispensable based on the results of amino-terminal deletions described above. Nonetheless, our identification of a range of mutations over the amino-terminal 25% of the sequence of cytochrome c which lead to defects in mitochondrial accumulation targeting agrees with the identification by Sprinkle et al.(1990) of lesions in this region which are capable of preventing targeting. In contrast to these results, Nye and Scarpulla (1990b), assaying for targeting of an iso-1-cytochrome c-chloramphenicol acetyltransferase fusion protein, found that they could delete as much as the first 67 amino acids of the cytochrome c sequence without affecting targeting to mitochondria. The results reported in the present manuscript do not support the proposal by Nye and Scarpulla (1990b) that there is functional redundancy between amino and carboxyl regions of the protein.
Because single localized deletions were examined in our study, we have not ruled out the possibility that, in addition to the sequences we uncovered, additional alterations at two or more nonadjacent sites might lead to impaired mitochondrial accumulation.
The observation that
apocytochrome c lacking His fails to associate
with mitochondria unless they contain high levels of CCHL confirms the
role of CCHL in maintaining the stalled form of the precursor. The
simplest explanation of this result is that the altered apocytochrome c adopts a membrane-spanning topology, rendering it accessible
to external proteases at the same time as it is bound to CCHL inside
the mitochondrial outer membrane. However, CCHL has been reported to be
associated predominantly with the mitochondrial inner membrane, not the
outer membrane (Dumont et al., 1991). There are a number of
ways of reconciling these findings. (i) CCHL may not interact directly
with the stalled precursor but, rather, act in an indirect way to
maintain the association of the altered apocytochrome c with
mitochondria. (ii) CCHL may transiently become associated with the
outer membrane in vivo, or it may be localized to sites where
the two membranes are in close contact. (iii) The apparent localization
of CCHL to the inner membrane in isolated mitochondria may result from
redistribution during fractionation.
We have not resolved the question of whether the same regions of cytochrome c which we have determined to be necessary for mitochondrial accumulation are also sufficient for accumulation. Expression of amino-terminal regions of apocytochrome c as short fragments in vivo does not result in accumulation of sufficient quantities of protein for detection using an epitope tag, presumably because of rapid degradation (data not presented). On the other hand, fusion of apocytochrome c fragments to a larger carrier protein can lead to mistargeting (see Nye and Scarpulla, 1990a).
Figure 6:
Model for mitochondrial import of
cytochrome c. Cytochrome c is synthesized in the
cytoplasm as apocytochrome c (Apo), lacking heme.
Apocytochrome c binds to a putative factor in the cytoplasm (Cytoplasmic factor). Class III mutants (Table 4) appear
to interact with the hypothetical cytoplasmic factor but are unable to
translocate across the mitochondrial outer membrane. Apocytochrome c diffuses reversibly across the mitochondrial outer membrane
and binds to cytochrome c heme lyase (CCHL), which is located
on the mitochondrial inner membrane facing the intermembrane space.
Class IV mutants involving His (Table 4) appear to
bind to CCHL while remaining exposed to the outside surface of the
mitochondria. Apocytochrome c can bind to CCHL even in the
absence of heme attachment, as indicated by the Class I (Table 4)
mutants. After heme attachment, catalyzed by CCHL, holocytochrome c (Holo) folds into its native globular state, is released
from CCHL, and remains trapped in the intermembrane
space.
1. After synthesis in the cytoplasm, apocytochrome c may associate with a cytoplasmic factor. The existence of such a factor is suggested by our observation that expression of altered forms of apocytochrome c which do not bind or enter mitochondria inhibits the formation of the coexpressed normal cytochrome c.
2. Apocytochrome c, either alone or in conjunction with a cytoplasmic factor, associates with mitochondria and adopts a membrane-spanning topology. Upon becoming exposed to the intermembrane space, sequences near the amino terminus of apocytochrome c bind either directly to CCHL or to a CCHL-associated factor. Such a factor would normally be present in excess relative to CCHL, or its level would increase upon overexpression of CCHL. Both the amino and carboxyl termini exhibit membrane-active properties, although only amino-terminal fragments are able to translocate across the lipid bilayer (Zhou et al., 1988; Jordi et al., 1989a, 1989b). However, it is not currently known whether the pathway of import of cytochrome c involves a direct interaction with the lipid phase.
3. His of apo-iso-2-cytochrome c interacts with CCHL or some other CCHL-associated component of the
translocation machinery in order to allow the carboxyl-terminal portion
of the protein to be translocated into the mitochondrial intermembrane
space.
4. If CCHL has remained on the inner membrane, it associates with the precursor following diffusion across the intermembrane space.
5. Altered apocytochromes c that are unable to have heme covalently attached remain trapped in the intermembrane space in a dead end complex with CCHL. Normal precursors are covalently linked to heme in a reaction catalyzed by CCHL. Folding to the native globular structure occurs concomitant with this reaction.
6. Finally, matured normal precursors are released into the intermembrane space. In their folded form, they cannot translocate back across the outer membrane (see Fig. 6).