(Received for publication, June 8, 1995)
From the
We have confirmed by spectral analysis that cytochrome oxidase
is not present in strains of the yeast Saccharomyces cerevisiae having a primary deficiency in cytochrome c, and we have
demonstrated by immunological procedures that such strains lack the
mitochondrially encoded subunits I, II, and III of cytochrome oxidase.
Furthermore, pulse-chase experiments demonstrated that subunit II is
rapidly degraded in vivo. This degradation can be at least
partially suppressed by disruption of the nuclear gene YME1,
which encodes a putative ATP-Zn-dependent protease.
We suggest that the cytochrome oxidase subunits are not properly
assembled in the absence of cytochrome c, and that Yme1 and
possibly other proteases degrade the unassembled mitochondrial-encoded
subunits of cytochrome oxidase.
Eukaryotic cytochrome oxidase, also denoted cytochrome c oxidase and cytochrome aa
, is located
in the inner membrane of mitochondria, where it catalyzes the transfer
of electrons from cytochrome c to molecular oxygen (1, 2, 3) . Cytochrome oxidase is a
multisubunit complex consisting of 3 mitochondrial-encoded subunits,
which form the functional core, and 8 to 10 nuclear-encoded
subunits(4) . In particular, cytochrome oxidase from Saccharomyces cerevisiae consists of a total of 11 subunits:
I, II, and III, encoded by the mitochondrial genes COX1, COX2, and COX3, respectively, and IV, Va, Vb, VI,
VIa, VIb, VII, VIIa, and VIII, encoded by the nuclear genes COX4, COX5a, COX5b, COX6, COX13, COX12, COX7, COX9, and COX8, respectively(5) . (The cytochrome oxidase
subunits I, II, etc., are also denoted CoxI, CoxII, etc.)
Post-translational assembly of all the subunits to a functional complex
requires at least the gene products of COX10 and COX11, which are involved in heme a synthesis(6, 7) , and PET117(8) , PET191(8) , and SCO1(9) , whose functions are unknown. As discussed
below, lack of assembly due to the deletion of COX1, COX2, COX3, COX4, COX10, COX11, or SCO1 leads to degradation of individual
unassembled
subunits(6, 7, 9, 10, 11) . (
)
S. cerevisiae contains two isoforms of
cytochrome c, iso-1- and iso-2-cytochrome c, which
are encoded by the nuclear genes, CYC1 and CYC7,
respectively(12, 13) . Strains completely deficient in
cytochrome c can be produced either by cyc1cyc7
double
mutations (13) or by mutation of the CYC3 gene that
encodes heme lyase, which catalyzes the covalent attachment of the heme
group (14, 15) . Spectral examination of intact cells
revealed that such mutants lacking cytochrome c are also
deficient in cytochrome oxidase, presumably as a secondary effect of
the cytochrome c deficiency(13, 15, 16) . Also,
cytochrome oxidase is exceedingly sensitive to glucose repression in
mutants having trace amounts of cytochrome c, as found with
certain ``leaky'' cyc3 mutations(14) , and
in mutants having low levels of function, as found with certain cyc1
missense mutations. The lack or
diminished levels of cytochrome oxidase was also observed in mutants of Neurospora crassa deficient in cytochrome c(17, 18, 19) .
In this study, we
have used immunological procedures to demonstrate that cytochrome c-deficient cyc1cyc7
mutants lack the mitochondrial-encoded
cytochrome oxidase subunits I, II, and III and have reduced amounts of
the nuclear-encoded subunits IV, V, VI, and VIa. In addition,
pulse-chase experiments demonstrated that subunit II is rapidly
degraded in vivo.
Furthermore, a number of genes encoding, or presumably encoding, mitochondrial proteases were disrupted, and the levels of the cytochrome oxidase subunits were examined. If these proteases are responsible for the degradation of the cytochrome oxidase subunits, the disruptions should act as suppressors in cytochrome c-deficient strains.
The nuclear gene PIM1 encodes
an ATP-dependent protease located in the mitochondrial
matrix(20, 21) . Because Pim1 is in the mitochondrial
matrix and cytochrome oxidase is in the intermembrane space, and
because pim1- mutants become
,
Pim1 was not expected to be the protease acting on the cytochrome
oxidase subunits. On the other hand, YTA10(22) , also
denoted AFG3(23) , encodes a protease that acts on
incompletely synthesized polypeptides in the mitochondrial inner
membrane(24) . YME1(25) and RCA1(26) , also denoted YTA11 and YTA12, respectively(22) , encode putative proteases
related to Afg3 and represent members of a family of ATPases similar to
proposed proteolytic complexes in Escherichia coli and yeast.
However, afg3-
, and rca1-
disruptions are
deficient in the cytochrome oxidase subunits I, II, and III.
Only
the yme1- disruption, but not pim1-
, afg3-
, or rca1-
, increased the level of the
cytochrome oxidase subunits II and III in cytochrome c-deficient strains. These results suggest that the cytochrome
oxidase subunits are not properly assembled in the absence of
cytochrome c, and that Yme1 and possible other proteases
degrade the unassembled subunits I, II, and III.
This degradation of
cytochrome oxidase subunits in strains lacking cytochrome c at
least superficially resembles the degradation of labile forms of
cytochrome c in strains lacking either cytochrome c or cytochrome oxidase, its physiological
partners(27) .
An isogenic series of S. cerevisiae strains, listed in Table 1, was prepared
from the related strains B-8123 (MATacyc1-1011 cyc7-67 ura3-52 lys5-10)
or B-8514 (MATaCYC1cyc7-67 ura3-52 lys5-10)(28) .
All strains of the series lacked iso-2-cytochrome c because of
the cyc7-67 mutation, and each strain contained either
the CYC1
or cyc1-1011 allele,
the YME1
or yme1-
allele and
were either
or
. The yme1-
disruptions, as well as other disruptions, were
prepared as described previously(27) ; the
strains were prepared by growth in the presence of ethidium
bromide(29) . The yme1-
strains were not investigated because they grow slowly or are
nearly lethal(30, 31) .
Figure 1:
Low temperature (-196 °C)
spectrophotometric recordings of a series of isogenic yeast strains.
The strains were grown on 1% sucrose at 30 °C for 3 days, and the
absorption spectra were recorded as described previously (32) .
The peaks of cytochromes a
a
, b, c
, and c are located,
respectively, at 602.5, 558.5, 553.3, and 547.3 nm. Curves A,
B-8514 (CYC1
cyc7-
); B, B-9614 (CYC1
cyc7-
yme1-
); C, B-8123 (cyc1-
cyc7-
); D, B-9621 (cyc1-
cyc7-
yme1-
); E, B-8516 (CYC1
cyc7-
); F, B-9622 (cyc1-
cyc7-
). The peaks at
approximately 577 nm, seen in curves B-F, is due to zinc
protoporphyrin.
Isogenic strains containing disruptions of PIM1, AFG3, or RCA1 had spectral curves very similar to
these strains (data not presented), reflecting
their
phenotype caused by the
disruptions(20, 26, 39) .
Figure 2:
Western blot analysis using monoclonal
antibodies for CoxI, CoxII, and CoxIII, as described under
``Materials and Methods.'' Lanes 1, B-8123 (cyc1- cyc7-
); 2, B-9621 (cyc1-
cyc7-
yme1-
); 3, B-8514 (CYC1
cyc7-
); 4, B-8516 (CYC1
cyc7-
).
Figure 3:
Pulse-chase labeling of CoxII from the
following isogenic strains. A, B-8514 (CYC1 cyc7-
); B, B-8123 (cyc1-
cyc7-
); and C, B-9621 (cyc1-
cyc7-
yme1-
). After
the chase in unlabeled methionine, identical volumes of cell
suspensions were analyzed at the various times, as indicated in minutes
at the top of the figure.
The results presented in this paper can be explained by the protection of one or more of the cytochrome oxidase subunits by cytochrome c. Cytochrome c exhibits high affinity binding to subunit II and low affinity binding to another subunit(43) . The absence of cytochrome c may destabilize CoxII, and possibly CoxI and Cox III, thereby making these, as well as other subunits, susceptible to degradation.
The results
shown in Fig. 2and summarized in Table 1demonstrated
that degradation of subunits II and III, but not subunit I, in a
cytochrome c-deficient strain can be partially suppressed by
the yme1- mutation, which prevents synthesis of the Yme1
presumptive protease. The simplest explanation of these results is that
Yme1 is a protease that, by itself or in a complex with other proteins,
is involved in turnover of unassembled subunits of cytochrome oxidase,
confirming a previous report that turnover of unassembled subunits of
cytochrome oxidase requires a metal/ion-dependent factor (11) .
In addition, Weber et al. (
)observed that CoxII was
degraded in a cox4-deficient strain, and that this degradation
was suppressed by yme1-
. The lack of suppression by yme1-
of the CoxI complete deficiency (Table 1),
and of the possible partial deficiencies of other subunits, suggests
that still other proteases may be involved in the degradation process.
In fact, the turnover of CoxV was not suppressed by yme1-
, pim1-
, afg3-
, or rca1-
(results not presented).
In summary, these
results indicate that cytochrome c protects cytochrome oxidase
subunits from degradation, and our previous results (27) demonstrated that cytochrome oxidase or cytochrome c protects certain labile forms of cytochrome c from degradation, a phenomenon that was previously believed
to occur only with strongly interacting components of protein
complexes.