In an attempt to clarify this point, we examined the process in Saccharomyces cerevisiae cells synthesizing cytochrome P450scc as its native precursor pCYP11A1 or versions in which its N
Trang 1Effects of various N-terminal addressing signals on sorting
and folding of mammalian CYP11A1 in yeast mitochondria
Irina E Kovaleva, Lyudmila A Novikova, Pavel A Nazarov, Sergei I Grivennikov
and Valentin N Luzikov
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
Topogenesis of cytochrome P450scc, a resident protein of the
inner membrane of adrenocortical mitochondria, is still
obscure In particular, little is known about the cause of its
tissue specificity In an attempt to clarify this point, we
examined the process in Saccharomyces cerevisiae cells
synthesizing cytochrome P450scc as its native precursor
(pCYP11A1) or versions in which its N-terminal addressing
presequence had been replaced with those of yeast
mitoch-ondrial proteins: CoxIV(1–25) and Su9(1–112) We found
the pCYP11A1 and CoxIV(1–25)-mCYP11A1 versions to
be effectively imported into yeast mitochondria and
subjec-ted to proteolytic processing However, only minor portions
of the imported proteins were incorporated into
mito-chondrial membranes, whereas their bulk accumulated as
aggregates insoluble in 1% Triton X-100 Along with
pre-viously published data, this suggests that a distinguishing
feature of the import of the CYP11A1 precursors into yeast
mitochondria is their easy translocation into the matrix
where the foreign proteins mainly undergo proteolysis or aggregation The fraction of CYP11A1 that happens to be inserted into the inner mitochondrial membrane is effectively converted into the catalytically active holoenzyme Experi-ments with the Su9(1–112)-mCYP11A1 construct bearing a re-export signal revealed that, after translocation of the fused protein into the matrix and its processing, the Su9(67–112) segment ensures association of the mCYP11A1 body with the inner membrane, but proper folding of the latter does not take place Thus it can be said that the most specific stage of CYP11A1 topogenesis in adrenocortical chondria is its confinement and folding in the inner mito-chondrial membrane In yeast mitochondria, only an insignificant portion of the imported CYP11A1 follows this mechanism
Keywords: yeast mitochondria; import; sorting; folding; aggregation
Cytochrome P450sccis a resident protein of adrenocortical
mitochondria In co-operation with adrenodoxin and
adrenodoxin reductase it carries out conversion of
choles-terol into pregnenolone Cytochrome P450sccis synthesized
in the cytoplasm as a precursor [1] that is imported into
mitochondria, where it becomes an integral protein of the
inner membrane [2] At least two peculiarities of its
topogenesis are still unclear First, this protein cannot be
extracted from the membrane by carbonate treatment,
although it does not contain any distinct transmembrane
domains Second, the import of the cytochrome P450scc
precursor into mitochondria is tissue-specific In fact,
pCYP11A1 is imported into adrenocortical and liver
mito-chondria but not into heart mitomito-chondria [3,4] On the other
hand, the import of various versions of pCYP11A1 has been
demonstrated with mitochondria of COS-1 [5] and yeast [6] cells Moreover, even plant mitochondria can import pCYP11A1 [7] Mitochondria of transformed yeast cells exhibit side-chain cleavage activity in reconstituted systems, the activity being detected in the inner membrane fraction [6,8] These data seem to suggest similar topogenesis of CYP11A1 in various organisms However, no details of its import into heterologous mitochondria have been studied in the above publications Efforts in this direction were undertaken in the experiments with isolated yeast mitochon-dria and in vitro synthesized bovine pCYP11A1 [9] It turned out that upon import into mitochondria the protein accu-mulates mainly in the matrix in precursor and mature forms Only a trace amount of the protein was found in the inner membrane fraction This was in discord with the earlier data [10] according to which pCYP11A1 does not leave the inner membrane upon its import into isolated adrenocortical mitochondria and subsequent maturation Thus, the crucial distinction of the import of pCYP11A1 into yeast mito-chondria is that, for unknown reasons, the protein is rapidly translocated into the mitochondrial matrix The imported protein largely undergoes proteolysis by matrix protease Pim1p, which additionally testifies to such translocation [9] Proteolysis competes with aggregation dominating in mito-chondria with mutant forms of Pim1p or mtHsp70 In contrast to pCYP11A1, the Su9(67–112)-CYP11A1(75–481) version was detected in a considerable amount in the inner membrane fraction where it was directed by the re-export
Correspondence to I E Kovaleva, Belozersky Institute of Physical
and Chemical Biology, Moscow State University,
119899 Moscow, Russia Fax: + 7 095 939 3181,
E-mail: kovaleva@genebee.msu.su
Abbreviations: pCYP11A1, precursor form of CYP11A1;
mCYP11A1, mature form of CYP11A1; SMP,
submito-chondrial particles.
Enzymes: CYP11A1 (EC 1.14.15.6); adrenodoxin reductase
(EC 1.14.15.4).
(Received 26 March 2002, revised 6 November 2002,
accepted 20 November 2002)
Trang 2mechanism [11] using the N-terminal transmembrane
domain of the Su9 protein The Su9(1–112) segment is
known to ensure the initial stage of the import of Su9 into the
matrix followed by two-stage processing and reinsertion of
its N-terminus into the inner membrane in such a way that
the transmembrane domain spans the membrane while the
14-mer extension becomes exposed into the intermembrane
space The Su9(1–112) sequence fused to N-termini of some
foreign proteins is capable of carrying out the same transport
function [12] The membrane-bound
Su9(67–112)-CYP11A1(75–493) form was degraded by the membrane
Yta10p-12p proteolytic complex [9], which testifies to its
improper folding Unfortunately, the in vitro experiments
could not provide information about conversion of
CYP11A1 into a catalytically active holoenzyme Using
Saccharomyces cerevisiae yeast expressing CYP11A1
ver-sions (CYP11A1s) with different topogenic signals, in this
work we planned to answer the following questions: (a) how
do various topogenic signals influence the sorting of
CYP11A1s in yeast mitochondria; (b) what is the relation
between the contents of membrane-bound and aggregated
forms of the imported CYP11A1 and (c) how effective is the
conversion of membrane-bound CYP11A1 into the
cataly-tically active form? Such information revealing specific
features of CYP11A1 import into foreign mitochondria
might be helpful both for understanding the topogenesis of
this protein in nature and for assessing its possibilities in
transgenic organisms
Materials and methods
Media for cultivation of yeast and bacteria were from Difco
Laboratories (USA) 22(R)-hydroxycholesterol and
d-ami-nolevulinic acid were from Sigma (USA) Restriction
endo-nucleases, Klenow fragment, and T4 DNA ligase were from
Fermentas (Lithuania) Plasmids were maintained and
am-plified in Escherichia coli JM-109 (Promega, USA) cDNAs
encoding recombinant proteins were used for transformation
of Saccharomyces cerevisiae 2805 (aMATpep4::HIS3 prb1-d
can 1 GAL2 His3 ura3–5) deficient in vacuolar proteases
Rabbit polyclonal anticytochrome P450sccIgG,
adrenocor-tical mitochondria, purified bovine cytochrome P450scc,
adrenodoxin, and adrenodoxin reductase were generous gifts
from V M Shkumatov (State University of Belarus, Minsk)
DNAs and plasmids
Yeast expressing the shuttle vector pYeDP1-8/2 (pYeDP)
[13] was used for expression of cDNAs encoding
recombin-ant proteins This vector includes a galactose-inducible
chimeric promoter GAL10-CYC1, a terminator of yeast
phosphoglycerate kinase, and an auxotrophic marker
URA3 The pYeDP/Cox(1–25)-mCYP11A1 with the
pre-sequence of subunit IV of yeast cytochrome oxidase has
been prepared earlier in this laboratory [6] The pYeDP/
pCYP11A1 encoding CYP11A1 with its own N-terminal
addressing presequence was constructed by inserting cDNA
for human CYP11A1, a gift from W L Miller (University
of California, San Francisco, USA), into pYeDP at the
EcoRI and KpnI sites cDNA for a fusion protein composed
of mCYP11A1 preceded by the N-terminal fragment of
subunit 9 (Su9) of yeast F-ATPase, including its addressing
presequence and the N-proximal transmembrane segment, was constructed on the basis of pGEM4/Su9(1–112)-CYP11A1(75–493) [9] Full-size cDNA for mCYP11A1 was obtained by substituting the NcoI-BglII fragment of pTrc99A/mCYP11A1 [14] for the BamHI-BglI fragment in pGEM4/Su9(1–112)-CYP11A1(75–493) Prior to ligation, the BamHI end of the plasmid and the NcoI end of the excised cDNA were treated with Klenow fragment Hybrid cDNA encoding the Su9(1–112)-mCYP11A1 fusion protein was excised from pGEM4/Su9(1–112)-mCYP11A1 at EcoRI and KpnI sites and inserted into pYeDP pretreated with BamHI and KpnI The BamHI and EcoRI ends were treated with Klenow fragment prior to ligation The resulting pYeDP/Su9(1–112)-mCYP11A1 plasmid was used
to transform yeast cells To achieve low-copy expression of CoxIV(1–25)-mCYP11A1, the corresponding cDNA was inserted into pINT2 (an integrative vector with phospho-glycerate kinase promoter and URA3 marker; kindly pro-vided by D G Kozlov, Institute of Genetics and Selection
of Industrial Microorganisms, Moscow, Russia) by excising this cDNA from pYeDP/CoxIV(1–25)-mCYP11A1 with EcoRI and XbaI and subcloning it sequentially in pUC19 and pUC18 to obtain appropriate ends for insertion into pINT2 at BamHI and EcoRI sites Final ligation gave the pINT2/CoxIV(1–25)-mCYP11A1 construct
Transformation of yeast cells with pYeDP/pCYP11A1, pYeDP/Cox(1–25)-mCYP11A1, and pYeDP/Su9(1–112)-mCYP11A1 was carried out as described earlier [15] The yeast cells were grown in SD medium (0.67% yeast nitrogen bases, 1% ammonium sulfate and 2% glucose) for 24 h, and then for 14 h in a selective medium with 2% galactose added for promoter induction After this, the growth medium was also supplemented with 200 lMd-aminolevulinic acid
Isolation and fractionation of yeast mitochondria Yeast mitochondria were isolated as described in [16] and fractionated according to [4] with minor modifications Mito-chondria were suspended in 5 mMTris/HCl, pH 7.4, incu-bated for 20 min at 4°C to disrupt the outer membranes, and subjected to sonic disintegration The mitochondrial suspen-sion was then centrifuged for 15 min at 12 000 g The resulting suspension was centrifuged for 1 h at 106 000 g to obtain the submitochondrial particles (SMP) fraction
Assessment of aggregation of recombinant proteins
in yeast mitochondria Mitochondria (0.5 mg protein mL)1) were incubated in
20 mM Hepes/KOH, pH 7.4, with 150 mM NaCl, 1 mM
phenylmethyl sulphonyl fluoride, and 1% Triton X-100 for
30 min on ice The suspension was then centrifuged for
15 min at 25 000 g The supernatant proteins were precipi-tated with 10% (v/v) trichloroacetic acid, and the pellets were dissolved in SDS/PAGE buffer The content of CYP11A1 was estimated in Triton X-100 soluble and insoluble fractions by Western blot analysis
Western blot analysis
To estimate the contents of recombinant CYP11A1 ver-sions in mitochondria and 12 000 g supernatants from
Trang 3mitochondrial homogenates, we used cytochrome P450scc
-calibrated immunoblotting For this purpose, SDS/PAGE
was carried out with fixed amounts of isolated cytochrome
P450sccin 2.5–20 or 25–200 ng ranges, with the contents of
the protein in adjacent lanes differing by a factor of two
Then routine procedures were used for transferring the
proteins to nitrocellulose membranes [17] and their
detec-tion with rabbit anticytochrome P450sccIgG and conjugate
of anti-rabbit IgG with peroxidase This approach allowed
one to estimate the content of various CYP11A1 versions
with an accuracy of ± 50%
Assays of cholesterol side-chain cleaving activity
of various CYP11A1 versions
Mitochondria isolated from yeast cells expressing CYP11A1
versions were subjected to hypotonic shock and sonication
as indicated above The assays were carried out with
supernatants after centrifugation of mitochondrial
homo-genates at 12 000 g, which removed large mitochondrial
fragments and protein aggregates The reaction mixture
contained 200–500 lg of mitochondrial protein, purified
bovine adrenodoxin (0.8 nmole), adrenodoxin reductase
(0.2 nmole), and 25 nmoles of 22(R)-hydroxycholesterol in
0.5 mL of 30 mMphosphate buffer, pH 7.5, supplemented
with 0.05% (v/v) Tween 20 The samples were preincubated
for 20 min at 25°C The reaction was started by addition of
an NADPH-generating system including 0.1 mMNADPH,
5 mMglucose-6-phosphate, and glucose-6-phosphate
dehy-drogenase (1 UÆmL)1) (Serva, Germany) The reaction was
stopped after 30 min by plunging the samples into boiling
water for 15–20 s Then the samples were mixed with 3 mL
of 100 mMsodium phosphate buffer, pH 7.2, with 0.05%
(v/v) Tween 20, and cholesterol oxidase (0.1 U per sample)
(Serva, Germany) was added to convert pregnenolone
formed from 22(R)-hydroxycholesterol into progesterone
After a 45-min incubation at 37°C, the samples were
treated with ethyl acetate to extract the steroids The
extracts were evaporated to dryness and the content of
progesterone was determined with an ELISA test system
based on antiprogesterone antibodies The test kits were
kindly provided by Dr A G Pryadko (Institute of
Bioorganic Chemistry, Belarus)
Results
Import of recombinant forms of CYP11A1 into yeast
mitochondriain vivo
cDNAs encoding pCYP11A1, CoxIV(1-25)-mCYP11A1,
and Su9(1–112)-mCYP11A1 have been inserted into the
yeast expressing shuttle vector pYeDP CoxIV(1–25) is the
N-terminal addressing presequence of subunit IV of yeast
mitochondrial cytochrome oxidase, and Su9(1–112) is the
N-terminal part of the precursor of subunit 9 of yeast
mitochondrial Fo-ATPase Yeast cells expressing the above
recombinant proteins were used to isolate mitochondria in
which various forms of CYP11A1 were detected by
immunoblotting with anti-P450sccIgG Figure 1 shows that
all the CYP11A1 forms were imported into mitochondria
As we failed to register CO difference spectra of yeast
mitochondria containing the recombinant proteins, we have
estimated their intramitochondrial contents by cytochrome P450scc-calibrated immunoblotting The values obtained for CoxIV(1–25)-mCYP11A1, Su9(1–112)-mCYP11A1, and pCYP11A1 were 0.3%, 0.5% and 0.1% of total mito-chondrial protein, respectively
Appreciable distinctions were found at the stage of processing of the imported proteins (Fig 1) All of Su9(1)112)-mCYP11A1 is processed to a single product somewhat larger than mCYP11A1 because of the 45-mer addition at the N-terminus CoxIV(1–25)-mCYP11A1 is also effectively processed, although immunoblotting reveals some precursor in yeast mitochondria Similar results have been obtained upon expression of CYP11A1 with the presequence of subunit VI of yeast cytochrome oxidase [8]
A more complicated pattern was observed for pCY-P11A1 with its own N-terminal presequence In this case one could see three forms of the protein, i.e precursor, mature protein and semiprocessed precursor The lower content of mCYP11A1 might have resulted from the poor match of the yeast mitochondrial processing peptidase to the indigenous processing site in pCYP11A1
Fig 1 Western blots of mitochondria prepared from recombinant yeast strains producing pCYP11A1, CoxIV(1– –25)-mCYP11A1, and Su9(1– –112)-mCYP11A1 The samples of mitochondria isolated from recombinant yeast producing CoxIV-mCYP11A1, pCYP11A1, and Su9-mCYP11A1 were probed with anti-(cytochrome P450 scc ) IgG Mitochondria from these strains were loaded at 25, 25, and 10 lg protein per lane, respectively Purified bovine cytochrome P450 scc was
a marker at 20 and 500 ng protein per lane for the left and right panels, respectively In the scheme, p, i, and m correspond to precursor, intermediate, and mature forms of CYP11A1 Light and dark shaded boxes are targeting signal (ts) and mature protein sequence, respect-ively; empty box is the transmembrane domain (tm) of the Su9 precursor.
Trang 4In as much as in the three above cases the mitochondrial
fraction mainly contained mature or semiprocessed forms of
CYP11A1, one may assume that this fraction was not
significantly contaminated with the recombinant protein in
a cytoplasmic aggregated form
Relationship between aggregated and
membrane-bound forms of CYP11A1 in yeast mitochondria
Obviously, early post-translocational changes of a protein
imported into mitochondria can be revealed only in the
short-term in vitro experiments It has been shown [9] that
upon import of the in vitro synthesized pCYP11A1 into
isolated yeast mitochondria, only a minor portion of the
protein remains in the inner membrane, whereas its bulk is
transferred into the matrix where it undergoes proteolysis or
aggregation Therefore, expressing CYP11A1 with various
addressing presequences in yeast cells, one would like to
know the relationship between aggregated and
membrane-bound forms of the protein imported into mitochondria, in
particular, to estimate the efficiency of conversion of the
imported protein into a catalytically active form in foreign
surroundings
In our experiments, mitochondria isolated from the
transformed yeast cells after 14-h induction of pCYP11A1,
CoxIV(1–25)-mCYP11A1, and Su9(1–112)-mCYP11A1
were subjected to hypotonic shock followed by sonication
and centrifugation at 12 000 g Figure 2 shows the contents
of CoxIV(1–25)-mCYP11A1 in whole mitochondria and
the corresponding 12 000 g supernatant determined by
SDS/PAGE and Western blotting of the samples containing
equal amounts of total protein According to these data, the
content of the imported protein in the supernatant was
0.025% of total protein of the fraction, whereas that in
mitochondria was approximately 0.3% of total protein
Obviously, the supernatant containing soluble
mitochond-rial proteins and membrane vesicles had 10 times less
mCYP11A1 In contrast to mCYP11A1, the specific
content of phosphate carrier, a marker protein of the inner
mitochondrial membrane, was almost the same in whole
mitochondria and in the 12 000 g supernatant (Fig 2) This suggests that CoxIV(1–25)-mCYP11A1 imported into yeast mitochondria does not accumulate in the inner membrane after its processing
Figure 2 shows also the data for the pCYP11A1 version expressed in yeast In this case the difference in the combined contents of pCYP11A1 and iCYP11A1 in mitochondria and the 12 000 g supernatant was not so great as for the CoxIV(1–25)-CYP11A1 version (0.1% in mitochondria vs 0.05% in the supernatant) It is remark-able that the 12 000 g supernatant does not contain a detectable amount of mCYP11A1 Perhaps, its content in whole mitochondria is too low to be detected in the supernatant
Unlike the above experiments with CoxIV(1–25)-mCY-P11A1 and pCYCoxIV(1–25)-mCY-P11A1 constructs, fractionation of mito-chondria containing Su9(1–112)-mCYP11A1 yielded a
12 000 g supernatant with a considerable amount of the recombinant protein (Fig 2) similar to that in whole mitochondria (0.5% of total protein in both cases) This suggests that the Su9(1–112) fragment ensures effective insertion or anchoring of mCYP11A1 in the inner mito-chondrial membrane Recall that this amino acid sequence governs the import of fused proteins into the mitochondrial matrix and subsequent reinsertion into the inner membrane The treatment of yeast mitochondria imported
COX-IV-mCYP11A1 with 1% (v/v) Triton X-100 failed to wash out an appreciable amount of mCYP11A1 (Fig 3), which was indicative of its predominant aggregation Similarly, the pCYP11A1 and iCYP11A1 forms were insignificantly solubilized with 1% (v/v) Triton X-100 from yeast mito-chondria that had imported pCYP11A1 (Fig 3), which testifies to their prevalent aggregation Only the iCYP11A1 form was easily identified in the supernatant under these particular conditions; probably, pCYP11A1 and iCY-P11A1, which are the main forms of the imported foreign protein, differently associate with mitochondrial mem-branes Figure 3 shows that Su9(67–112)-mCYP11A1 is also poorly solubilized from mitochondria with 1% (v/v) Triton X-100 Upon similar treatment of adrenocortical mitochondria, cytochrome P450scc was essentially solubi-lized (Fig 3) However, even in this case solubilization was incomplete; therefore, we could not use the above data to quantitatively estimate protein aggregation
Figure 4 demonstrates that when the 12 000 g super-natant of mitochondria that had imported CoxIV(1–25)-mCYP11A1 was centrifuged at 106 000 g, CoxIV(1–25)-mCYP11A1 and
a minor amount of the precursor were found in the pellet mainly containing the inner mitochondrial membrane fragments (sonic SMP) In the case of mitochondria from yeast cells expressing pCYP11A1, the analogous SMP fraction contained pCYP11A1 and iCYP11A1 In both experiments, we could not see any CYP11A1 form in the high-speed supernatant Specific contents of mCYP11A1, pCYP11A1, and iCYP11A1 were much lower in the SMP fraction than in whole mitochondria, though the SMP fraction was enriched in phosphate carrier, the inner membrane marker (Fig 4) In contrast, fractionation of adrenocortical mitochondria yielded SMP with a cyto-chrome P450scccontent higher than that in whole organelles [6] One may conclude that in yeast mitochondria only minor amounts of the above CYP11A1 forms are associated
Fig 2 Comparative Western blots of mitochondria and their 12 000 g
supernatant fractions from recombinant yeast producing CoxIV(1–
–25)-mCYP11A1, pCYP11A1 and Su9(1– –112)-mCYP11A1 The samples of
mitochondria (M) from yeast producing recombinant proteins and
corresponding 12 000 g supernatant fraction (S) were probed with
anti-(cytochrome P450 scc ) IgG In all cases 20 lg of protein were
loaded per lane Markers: phosphate carrier (PC), an integral protein
of the inner membrane; Mge1p, a soluble matrix protein.
Trang 5with the mitochondrial membrane(s) while the bulk thereof
aggregates in the matrix
Clearly different results were obtained for the Su9(1–
112)-mCYP11A1 construct As follows from Fig 4, the
specific contents of its processed form are close in whole
yeast mitochondria and the SMP fraction, which is similar
to the data for phosphate carrier Thus, in contrast to
pCYP11A1, iCYP11A1, and mCYP11A1,
Su9(67–112)-mCYP11A1 is predominantly harboured by mitochondrial
membranes The impossibility of solubilizing this
recom-binant protein by the Triton X-100 treatment of
mitochon-dria (see above) suggests that it forms membrane-associated
aggregates However, these aggregates, unlike those of
other CYP11A1 forms, are not sedimented at 12 000 g
This indirectly indicates that aggregates of pCYP11A1,
iCYP11A1, and mCYP11A1 are not associated with
mitochondrial membranes and accumulate in the matrix
It is known that in some cases the aggregation of proteins
imported into mitochondria can be attenuated by lowering
their concentration [18] With this in mind, we have constructed a plasmid for expression of cDNA for CoxIV(1–25)-mCYP11A1 in an integrative vector under the control of the constitutive phosphoglycerate kinase promoter In this case the content of recombinant protein in mitochondria was reduced by an order of magnitude However, the bulk of CYP11A1 was again in the form of aggregates insoluble in 1% (v/v) Triton X-100 (data not shown)
Assays of cholesterol side-chain cleavage activity
of recombinant forms of CYP11A1 in yeast mitochondria
Knowing that recombinant forms of CYP11A1 imported into yeast mitochondria can exert catalytic activity only in a nonaggregated state, we used for the assays the 12 000 g mitochondrial supernatants As the latter obviously contain CYP11A1s as components of SMP, the results below should be taken as activities of the membrane-bound proteins It follows from the Table 1 that the rates of conversion of 22(R)-hydroxycholesterol into pregnenolone related to the total protein content of the 12 000 g supernatant are similar for the cells expressing pCYP11A1 and CoxIV(1–25)-mCYP11A1 The activity of the 12 000 g supernatant containing Su9(67–112)-mCYP11A1) proved
to be much lower This became even more evident with the specific activities of CYP11A1s Table 1 shows that for pCYP11A1 and CoxIV(1–25)-mCYP11A1 both the con-tents of the proteins and their specific activities are quite close Within the experimental error, they match the values obtained for a system reconstituted of purified bovine cytochrome P450scc, adrenodoxin, and adrenodoxin reduc-tase (Table 1) or for the solubilized membrane fraction of
E coli cells expressing mCYP11A1 supplemented with appropriate components [14] Thus, once cotranslocation-ally inserted into the inner membrane of yeast mitochon-dria, CYP11A1 is efficiently converted into holocytochrome P450scc Especially remarkable is that in the experiments with pCYP11A1 the 12 000 g supernatant contains mainly pCYP11A1 and iCYP11A1, not mCYP11A1 As in this case the specific activity related to the total content of these
Fig 3 Treatment of mitochondria from recombinant yeast producing CoxIV(1– –25)-mCYP11A1, pCYP11A1, or Su9-mCYP11A1, and adrenocortical mitochondria with Triton X-100 Mitochondria were treated with 1% (v/v) Triton X-100 and centrifuged at 25 000 g The resulting pellet (P) and supernatant (S) were probed with anti-(cytochrome P450 scc ) IgG or anti-(cytochrome P450 scc ) and anti-Hsp78 IgGs The presented P and S samples are from 50, 50, 60 and 20 lg of mitochondria including CoxIV-mCYP11A1, pCYP11A1, Su9-mCYP11A1, and cytochrome P450 scc , respectively Probing with anti-Hsp78 IgG was made to demonstrate complete separation of soluble and aggregated/complexed proteins.
Fig 4 Comparative Western blots of mitochondria and SMP fractions
from recombinant yeast strains producing CoxIV(1– –25)-mCYP11A1,
pCYP11A1, and Su9(1– –112)-mCYP11A1 The samples of
mitochon-dria (M) and corresponding SMP fractions (SMP) from yeast
produ-cing recombinant proteins were probed with anti-(cytochrome P450 scc )
IgG The M and SMP samples contained equal amounts of total
protein, which were 18, 25 and 20 lg per lane in the experiments with
mitochondria including CoxIV-mCYP11A1, pCYP11A1, and
Su9-mCYP11A1, respectively Markers: phosphate carrier (PC), an integral
protein of the inner membrane; Mge1p, a soluble matrix protein.
Trang 6forms was close to the activity of cytochrome P450sccin the
reconstituted system, we suggest that pCYP11A1 and/or
iCYP11A1 are catalytically active
The content of Su9(67–112)-mCYP11A1 was 10–20 times
higher while its specific activity was several hundred times
lower than the above values Hence, the Su9(1–112)
targeting signal governs effective binding of
Su9(67–112)-mCYP11A1 to the inner mitochondrial membrane
How-ever, such binding does not result in proper folding of the
polypeptide chain and formation of the active holoenzyme
Of course, cytochrome P450scc-calibrated immunoblotting
is only semiquantitative, but the observed effects exceed all
possible experimental errors
Discussion
In adrenocortical mitochondria, cytochrome P450scc is
known to be an integral protein of the inner membrane
[2,19] Two ways of achieving such localization have thus far
been considered [reviewed in 20]: the stop-transfer
mech-anism, i.e cotranslocational insertion of a polypeptide chain
into the inner membrane using stretches of hydrophobic and
nonpolar residues; and the re-export mechanism, i.e total
or partial translocation of a polypeptide chain into the
matrix followed by its processing and reinsertion into the
membrane by means of a specific N-terminal hydrophobic
stretch Analysis of the amino acid sequence of mCYP11A1
[21] shows that this protein has no regions capable of
realizing either of the above mechanisms Thus, the mode of
membrane insertion of CYP11A1 still remains puzzling It
has earlier been reported [10] that CYP11A1 perhaps
remains membrane-bound during the entire period of its
translocation into adrenocortical mitochondria and
matur-ation This might be accounted for by the presence of a
specific mitochondrial partner of pCYP11A1 that retards its
transmembrane translocation Besides, the addressing
pre-sequence of pCYP11A1 can be somehow involved in
confining this protein in the inner mitochondrial membrane
This specific presequence has an N-terminal region enriched
in hydrophobic amino acid residues, and negatively charged
residues in its C-terminal region [21], which is not typical of
the canonical presequences of cytoplasmically made
mito-chondrial precursor proteins
In contrast to adrenocortical mitochondria, the in vitro
synthesized pCYP11A1 is easily translocated into the matrix
of isolated yeast mitochondria, as follows from the results of
precise digitonin fractionation of mitochondria and the high
sensitivity of either form of imported pCYP11A1 to the
matrix protease Pim1p [9] In this work we show that upon expression of pCYP11A1 and CoxIV(1–25)-mCYP11A1 in yeast the precursor, semiprocessed, and mature proteins (CYP11A1s) accumulate in mitochondria mainly as aggre-gates Nevertheless, some amounts of CYP11A1s were found in the mitochondrial membranes We suggest that in yeast mitochondria the CYP11A1 precursors meet no serious hindrances to their translocation and thus largely
slip into the matrix, where the processed proteins cannot be properly folded and undergo proteolysis or aggregation To
a lesser degree this concerns pCYP11A1, which, owing to its noncanonical presequence, is better confined in the mem-brane of yeast mitochondria (Fig 5) This can explain why the SMP fraction contains mainly the pCYP11A1 and iCYP11A1 forms, and why their insertion into the mito-chondrial membranes is more effective than that of Cox (1–25)-mCYP11A1
Table 1 The content and specific cholesterol side-chain cleavage activity of various forms of recombinant CYP11A1 imported into yeast mitochondria The measurements were carried out with 12 000 g supernatants of mitochondria from yeast cells producing recombinant forms of CYP11A1 The activity is defined as the rate of conversion of 22(R)-hydroxycholesterol into pregnenolone at 37 °C The latter was quantitatively oxidized to progesterone with cholesterol oxidase.
Initial forms of
recombinant proteins
Content of CYP11A1 in assay sample, nmole (% total protein)
Activity (10 4 nmole progesteroneÆmin)1Æmg)1 total protein)
Activity (10 3 nmole progesterone min)1Ænmol)1 CYP11A1)
Fig 5 Putative pathways of import of various CYP11A1 precursor versions into yeast mitochondria and their intramitochondrial sorting (1) Su9(1–112)-mCYP11A1 is translocated into the matrix, processed, and reinserted into the membrane (yeast mitochondria); (2) CoxIV(1–25)-mCYP11A1 is rapidly processed and translocated into the matrix, with
a smaller portion being inserted into the membrane (yeast mitochondria); (3) pCYP11A1 is rather slowly processed and trans-located into the matrix, with the more essential portion being cotranslocationally inserted into the membrane (yeast mitochondria); (4) pCYP11A1 is cotranslocationally processed and inserted into the inner membrane (adrenocortical mitochondria).
Trang 7It has earlier been found [8] that, upon expression of
the pCoxVI-mCYP11A1 construct in S cerevisiae,
mCYP11A1 resides in the inner mitochondrial membrane
and the intermembrane contact sites However, these
gradient centrifugation experiments with mitochondrial
homogenates could not have quantitatively assessed the
balance of the import As follows from the present work, a
considerable amount of the imported CYP11A1 could have
been missed
The cytochrome P450scc-calibrated immunoblotting
allowed one to analyse the balance between the processes
of membrane insertion of CYP11A1s and their aggregation,
which takes place in the matrix, as follows from the in vitro
experiments [9] As the total protein contents in an aliquot
of yeast mitochondria and the corresponding 12 000 g
supernatant are close, one can compare the contents of
CYP11A1s Such a comparison clearly shows that for
CoxIV(1–25)-mCYP11A1 the content of the imported and
nonproteolysed protein is almost 10-fold higher in
mito-chondria than in the 12 000 g supernatant, which suggests
that only one-tenth of mCYP11A1 is inserted into the
membrane, thus escaping aggregation in the matrix It is
evident that for pCYP11A1 the combined contents of
precursor and intermediate forms of the recombinant
protein in mitochondria and 12 000 g supernatant differ
at least twofold
All the above membrane-bound CYP11A1s are
effi-ciently converted into the catalytically active form This
follows from their high specific side-chain cleavage activities,
which are of the same order of magnitude as that estimated
for mCYP11A1 expressed in E coli cells [14]
As to the Su9(67–112)-mCYP11A1 imported into
mito-chondria, it almost completely accumulates in the
mem-brane fraction: the mitochondria and the 12 000 g
supernatant contain equal relative amounts of the
recom-binant protein The Su9(1–112) segment is known to ensure
the initial stage of the import of Su9 into the matrix
followed by two-stage processing and reinsertion of its
N-terminus into the inner membrane [11] Most plausibly,
the Su9(67–112)-mCYP11A1 sequence imported into yeast
mitochondria misfolds in the matrix and does not refold
upon reinsertion into the inner membrane through the
Su9(67–112) fragment In fact, Su9(67–112)-mCYP11A1
detected mainly in the SMP fraction exhibits very low
side-chain cleavage activity and is poorly solubilized with
1% (v/v) Triton X-100
Thus, different topogenic signals in the CYP11A1
versions predetermine their sorting and conversions in
yeast mitochondria (Fig 5) Catalytically active
cyto-chrome P450scccan be accumulated in yeast mitochondria
in as much as the protein escapes aggregation in the
matrix However, some basic features of the import of its
precursor into mitochondria inevitably result in a low
yield of the active protein Analysis of the above data
definitely shows that the crucial moment in the
topo-genesis of cytochrome P450scc is not reception,
trans-membrane translocation, or proteolytic processing of its
precursor, but rather the confinement of the protein in the
inner membrane upon its import into adrenocortical
mitochondria This still unknown mechanism cannot be
adequately implemented in some foreign (e.g yeast)
mitochondria A search for such a mechanism may extend
the conventional notions on the topogenesis of mito-chondrial proteins
Acknowledgements
The authors are indebted to S A Saveliev for the help in preparing the pYeDP/pCYP11A1 and to A V Galkin for editing the text This work was supported by the Russian Foundation for Basic Research (grants 99-04-48003 and 00-15-97942 to VNL).
References
1 Nabi, N., Kominami, S., Takemori, S & Omura, T (1983) Contributions of cytoplasmic free and membrane-bound ribo-somes to the synthesis of mitochondrial cytochrome P450 (SCC) and P450 (11b) and microsomal cytochrome P450 (C-21) in bovine adrenal cortex J Biochem 94, 1517–1527.
2 Omura, T & Ito, A (1991) Biosynthesis and intracellular sorting
of mitochondrial forms of cytochrome P450 Methods Enzymol.
206, 75–81.
3 Matocha, M.F & Waterman, M.R (1984) Discriminatory pro-cessing of the precursor forms of cytochrome P450scc and adre-nodoxin by adrenocortical and heart mitochondria J Biol Chem.
259, 8672–8678.
4 Ogishima, T., Okada, T & Omura, T (1985) Import and pro-cessing of the precursor of cytochrome P450 (SCC) by bovine adrenal mitochondria J Biochem 98, 781–791.
5 Zuber, M.X., Mason, J.I., Simpson, R.E & Waterman, M.E (1988) Simultaneous transfection of COS-1 cells with mitochon-drial and microsomal steroid hydroxylases: Incorporation of a steroidogenic pathway into nonsteroidogenic cells Proc Natl Acad Sci USA 85, 699–703.
6 Savel’ev, A.S., Novikova, L.A., Drutsa, V.L., Isaeva, L.V., Chernogolov, A.A., Usanov, S.A & Luz ikov, V.N (1997) Synthesis and some aspects of topogenesis of bovine cytochrome P450scc in yeast Biochemistry (Moscow) 62, 779–786.
7 Luzikov, V.N., Novikova, L.A., Whelan, J., Hugosson, M & Glaser, E (1994) Import of mammalian cytochrome P450scc precursor into plant mitochondria Biochem Biophys Res Com-mun 199, 33–36.
8 Cauet, G., Balbuena, D., Achstetter, T., Dumas, D (2001) CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro Eur J Biochem 268, 4054–4062.
9 Savel’ev, A.S., Novikova, L.A., Kovaleva, I.E., Luzikov, V.N., Neupert, W & Langer, T (1998) ATP-dependent proteolysis in mitochondria: m-AAA protease and Pim1 protease exert over-lapping substrate specificity and cooperate with the mtHsp70 system J Biol Chem 273, 20596–20602.
10 Ou, W., Ito, A., Morohashi, K., Fujii-Kuriyama, Y & Omura, T (1986) Processing- independent in vitro translocation of cyto-chrome P450 (SCC) precursor across mitochondrial membranes.
J Biochem 100, 1287–1296.
11 Rojo, E.E., Stuart, R.A & Neupert, W (1995) Conservative sorting of Fo-ATPase subunit 9: export from matrix requires
pH across inner membrane and matrix ATP EMBO J 14, 3445– 3451.
12 Rojo, E.E., Guiard, B., Neupert, W & Stuart, R.A (1998) N-terminal tail export from the mitochondrial matrix: adherence
to the procaryotic positive inside rule of membrane protein topology J Biol Chem 274, 19617–19722.
13 Cullin, C & Pompon, D (1988) Synthesis of functional mouse P450 P1 and chimeric P450 P3–1 in yeast Saccharomyces cerevis-iae Gene 65, 203–217.
14 Wada, A., Mathew, P.A., Barnes, H.J., Sanders, D., Estabrook, R.W & Waterman, M.R (1991) Expression of functional bovine
Trang 8cholesterol side chain cleavage cytochrome P450 (P450scc) in
Escherichia coli Arch Biochem Biophys 290, 376–380.
15 Rech, E.L., Dobson, M.J & Milligan, B.J (1990) Introduction of
a yeast artificial chromosome vector into Saccharomyces cerevisiae
cells by electroporation Nucl Acids Res 18, 1313–1319.
16 Akiyoshi-Shibata, M., Sakaki, T., Yabusaki, G., Murakami, H &
Ohkawa, H (1991) Expression of bovine adrenodoxin and
NADPH-adrenodoxin reductase cDNAs in Saccharomyces
cere-visiae DNA Cell Biol 10, 613–621.
17 Sidhu, R.S & Bollon, A.P (1987) Analysis of a-factor secretion
signals by fusing with acid phosphatase of yeast Gene 54, 175–184.
18 Dibrov, E., Fu, S & Lemire, B.D (1998) The Saccharomyces
cerevisiae TCM62 gene encodes a chaperone necessary for the
assembly of the mitochondrial succinate dehydrogenase (Complex II) J Biol Chem 48, 32042–32048.
19 Usanov, S.A., Chernogolov, A.A & Chashchin, V.L (1990) Is cytochrome P450scc a transmembrane protein? FEBS Lett 275, 33–35.
20 Stuart, R.A & Neupert, W (1996) Topogenesis of inner membrane proteins of mitochondria Trends Biochem Sci 21, 261–267.
21 Chung, B.C., Matteson, K.J., Voutilainen, R., Mohandas, T.K & Miller, W.L (1986) Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in placenta Proc Natl Acad Sci USA 83, 8962–8966.