(Received for publication, July 12, 1995; and in revised form, August 30, 1995)
From the
In this study, a respiration-deficient Chinese hamster cell line
with a defect in succinate dehydrogenase activity is shown to result
from a single base change in a codon in the coding sequence for the
membrane anchor protein C (also referred to as QPs-1). A
premature translation stop results in the truncation of 33 amino acids
from the C terminus. Bovine cDNA encoding this peptide complements the
mutation. There is about 82% identity between these two mammalian
proteins. The gene for C
was mapped on human chromosome
1, and because it is also found on minichromosomes characterized by our
laboratory, we can localize it on the short arm within 1-2
megabases from the centromere.
A series of respiration-deficient Chinese hamster cell mutants
isolated by our laboratory can grow normally in tissue culture as long
as an adequate supply of glucose is available for
glycolysis(1, 2, 3) . Depletion of glucose
leads to rapid cessation of growth and cell death. The mutants were
grouped into seven complementation groups by somatic cell
fusions(1) , and one, represented by mutant CCL16-B9, was
characterized to be almost completely deficient in succinate
dehydrogenase activity (SDH)()(4, 5) . This
enzyme, which links the reactions of the Krebs cycle to the electron
transport chain, is part of a complex of four polypeptides (complex II)
in the inner mitochondrial membrane. The active site for the substrate
is on the flavoprotein (Fp, 70 kDa), while the iron-sulfur protein (Ip,
27 kDa) is believed to link it to two small integral membrane proteins
(C
and C
, 15 and 7-9 kDa,
respectively). A b-type heme group is associated with the membrane
proteins. Electrons from the oxidation of the substrate in the
mitochondrial matrix pass from the Fp via three non-heme iron-sulfur
centers in the Ip ([2Fe-2S], [4Fe-4S],
[3Fe-4S]) to the integral membrane proteins and from there to
ubiquinone. The function of the heme group associated with the membrane
proteins is not completely clear (see (6, 7, 8, 9) for reviews).
All four peptides are encoded by nuclear genes in eukaryotic organisms. Thus, the precursor polypeptides are synthesized in the cytosol and subsequently or concurrently imported into mitochondria. Following processing to their mature forms, the biogenesis of a functional complex II requires covalent attachment of flavin to the largest subunit (Fp)(10) , formation of the three non-heme iron-sulfur clusters in the Ip subunit, and assembly of the heme with the membrane anchor proteins. Although this complex is the smallest and least intricate of the electron transport complexes in the inner mitochondrial membrane, much remains to be learned about the mechanism or pathway of its assembly.
With the Fp-Ip complex dissociated from
the membrane by chaotropic ions, SDH activity can be assayed with
artificial electron acceptors such as tetrazolium ions, phenazine
methosulfate and dichlorophenol-indophenol, or ferricyanide. Based on
the absence of activity in the mutant CCL16-B9, we hypothesized that
the defective protein was either the Ip or the Fp subunit(5) .
It was not known at the time whether these two subunits can assemble
independently of the membrane anchors. Somatic cell hybridization
experiments had shown that CCL16-B9 is complemented by a gene on human
chromosome 1(11) . With probes for the human Ip gene (SDH-2)
the Ip gene was mapped to the location
1p36.1-2(12, 13) . However, the complete bovine
Ip cDNA failed to complement the SDH-deficient Chinese hamster cell
line. ()The somatic cell hybridization experiments also
yielded two independent hybrids in which the only human DNA present was
in the form of a minichromosome containing the centromere of human
chromosome 1, a few megabases of the short arm of chromosome 1, and a
portion of the pericentric heterochromatic region on the long arm of
chromosome 1(11, 14, 15, 16) . The
Ip gene was not present on these minichromosomes(12) .
Two
reports described cloning of cDNA for the membrane anchor protein
C (also referred to as QPs-1)(17, 18) .
Here, we show that the cDNA from bovine heart complements the defect in
the Chinese hamster mutant. The gene for the C
subunit
is on human chromosome 1 and on the smallest minichromosome of the
somatic hybrid Chinese hamster cell line containing an estimated
1-2 megabases of the proximal short arm. Finally, cDNAs were
cloned by RT-PCR from both wild type and mutant hamster cell lines, and
the sequence comparison shows that a single base substitution in the
mutant creates a premature stop codon.
All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) with 5 mM glucose and 10% fetal calf serum. The same medium with galactose instead of glucose (DMEM-GAL) was used to select and maintain respiration-competent cells or hybrids(2, 3) .
The bovine C cDNA probe hybridizes with both hamster and human DNA restriction
fragments even at relatively high stringency (Fig. 1). Multiple
bands are revealed, particularly from the human genome. In the absence
of detailed knowledge about the structure of the gene, the existence of
exons and introns might account for this observation, but the existence
of multiple genes or pseudogenes cannot be discounted. Restriction
enzymes were used in these investigations, which do not cut the bovine
cDNA (XbaI, EcoRI, PstI). Genomic DNAs from
human cells show bands not present in DNA from hybrids containing a
human minichromosome, but clearly, there are bands shared between total
human DNA and the minichromosome. As expected, all hybrids always
contain the DNA fragment of the hamster genome. Results with hybrids
XJM12.1.3, XJM12.2.2, and XEW9.10.4 (not shown) were identical to those
obtained with the XEW8.2.3 hybrid. The data strongly suggest that the
gene for the C
subunit is on the human minichromosome.
Figure 1:
Southern analyses of Chinese hamster,
human, and selected hybrid cell genomic DNA using the bovine C cDNA as a probe. CCL16-B1 and CCL16-B9 are the wild type and
mutant Chinese hamster cell lines, respectively. HT1080 and HELA are
established human tumor cell lines. XJM5.1.1(+) is a hamster/human
hybrid (respiration-competent) containing an intact human chromosome 1;
XJM5.1.1(-) is a respiration-deficient hybrid from which the
human chromosome 1 has been segregated. XEW8.2.3 is a hybrid with the
smallest human minichromosome described by our laboratory(15) .
In the left panel, the DNA has been digested with EcoRI; in the right panel the digestion is with PstI.
DMEM-GAL medium selects for respiration-competent cells. Past experience with hybrid selections involving these phenotypes had shown that complementation of the defective mitochondrial function is not instantaneous, since new functional complexes have to be assembled and accumulated in the mitochondria before complex II levels are adequate to support respiration and oxidative phosphorylation(11, 15) . Thus, a lag period is observed for transfected cultures subjected to direct selection a few days after the transfection. However, after about 2 weeks, cells started to divide in DMEM-GAL. Similarly, in cultures first selected for G418 resistance, the switch to DMEM-GAL medium left many cells to proliferate. In contrast, in cultures that were mock-transfected and in cultures that were transfected with the vector containing an unrelated cDNA and the neo gene, no proliferation was observed after the switch to the DMEM-GAL medium; after a few days, the cells died and became dislodged from the plate. The results also confirm previous observations that the CCL16-B9 mutant does not spontaneously revert to the wild type phenotype(5) .
Figure 2:
Sequence comparisons of two each (out of
four total) C cDNAs from wild type and mutant Chinese
hamster cells. The right nucleotide sequence shows the segment
including the C ⇒ T transition in the antisense strand. In the
sense strand, the corresponding UGG codon (Trp) is converted to the UGA
stop codon.
Figure 3:
Amino acid sequence comparison of the
bovine and Chinese hamster C peptides. Only differences
in the amino acids are indicated in the bovine sequence. The hamster
cDNA is missing the two penultimate codons and the stop codon. The boxed segments represent the three putative transmembrane
regions predicted by the TMAP
algorithm(23) .
Comparison of the bovine and hamster cDNA sequences shows 86%
conservation at the nucleotide level and 82% conservation at the amino
acid sequence level (Fig. 3). By comparison, the Ip peptides are
97% identical in mammals (bovine, human, rat)(24) . The
more rapid divergence for the C
peptide is noteworthy,
but the changes are largely conservative changes and are scattered
throughout the entire peptide.
The failure to obtain the 3`-RACE product with the QPs1(f) primer from the bovine cDNA sequence can also be explained because bovine and hamster sequences differ at the nucleotide that happened to be chosen as the 3`-end of this primer.
Figure 4:
Assays of succinate dehydrogenase
activities in isolated mitochondria from wild type (B1), mutant (B9),
and three clones of mutant cells stably transfected with a vector
expressing the bovine C cDNA (B9C4, B9C5, B9C6). For
details, see ``Materials and Methods.'' The assay also shows
that the activity measured can be inhibited with malonate, a specific
inhibitor of SDH.
In this study, the respiratory defect in a Chinese hamster mutant cell line lacking succinate dehydrogenase activity has been characterized at the nucleotide sequence level. Second, the corresponding human gene has been mapped near the centromere on human chromosome 1.
A single nucleotide substitution in the coding
sequence for one of the membrane anchor proteins of complex II
(C, or QPs-1) creates a premature stop codon and hence
caused the production of a truncated peptide in CCL16-B9 cells. The
mutant peptide is missing 33 amino acids at the C terminus. A structure
of the C
membrane anchor protein has been proposed by Yu et al.(17) . Its predominant features are three
transmembrane segments and a long N-terminal tail extending into the
mitochondrial matrix. The N-terminal domain and possibly a loop of 19
amino acids between the second and third transmembrane segment could
interact with the Ip subunit and could be responsible for the
association of the SDH enzyme with the membrane. The last 20 amino
acids have been postulated to form the third transmembrane segment of
this integral membrane protein(17) . It is also possible that
the entire C-terminal domain interacts with the Ip subunit.
The
insertion of this short hydrophobic peptide into the inner
mitochondrial membrane is not a trivial aspect of the assembly problem.
It remains to be established whether the truncated peptide of the
mutant is also inserted into the membrane, and if so, whether the
topology of the first two transmembrane domains is preserved. From the
hydrophobicity plot alone, the major N-terminal domain should still be
available for interaction with the Ip peptide. Another question is how
membrane insertion of C may be coupled to the assembly
of a heme group shared with the second membrane anchor subunit
(C
, or QPs-2) (7) or possibly attached
exclusively to the C
(QPs-1) subunit (17) .
It is noteworthy that the truncation of the C subunit
also impairs the assembly and or accumulation of succinate
dehydrogenase subunits Ip and Fp. The membrane anchor protein may
interact with the Ip peptide and assist its folding and formation of
the iron-sulfur clusters. Iron ions and labile sulfide must associate
with the Ip peptide, possibly in a stepwise fashion during formation of
the three iron-sulfur clusters. Assembly of a complete complex II
apparently is required for the formation of an active Ip-Fp complex
with three iron-sulfur centers in the Ip subunit ([2Fe-2S],
[3Fe-4S], [4Fe-4S]) and a covalently linked flavin
on the Fp subunit. After maturation of complex II, the Ip-Fp complex
can be dissociated in an active form from the integral membrane
proteins by chaotropic ions(6, 25) . Attempts to
dissociate the Ip and Fp peptides and reconstitute an active SDH
activity have been unsuccessful.
We have argued before from studies with yeast mutants that in the absence of the Fp subunit the Ip subunit does not become stably associated with the inner mitochondrial membrane but is instead rapidly degraded upon import into the mitochondria(26) . Related studies with mutated Ip subunits so far have failed to yield evidence that the Ip and Fp subunits can assemble without first becoming membrane associated (27) . Because simple models of stepwise assembly of complex II have no experimental support, coordinated import and assembly of all four peptides seems to be required for biogenesis of complex II.
The
availability of a mammalian mutant cell line, which can be complemented
with C cDNA should enable systematic studies of
site-directed mutations. Bovine and hamster amino acid sequences differ
by almost 18%, but the substitutions are mostly conservative and
scattered. We observe 30-50% restoration of SDH activity in
complemented cells, with some variation from clone to clone. It is not
yet clear whether reduced activity relative to wild type reflects
sequence differences between the hamster and bovine C
subunits or is due to insufficient expression of the transgene.
In any case, these results also indicate that the C
subunit can limit SDH accumulation. Cloning of the complete
hamster C
cDNA will enable us to make the distinction
between sequence variation and limitation of expression in production
of SDH activity. Moreover, sequence changes in the heme binding region
or N-terminal regions can be explored to resolve the mode of
interaction of C
with the Ip subunit.
The
cytomegalovirus promoter is a relatively strong promoter, and we detect
a transcript in complemented cells that is more abundant than the
endogenous transcript (results not shown). Overexpression of the very
hydrophobic C peptide might be deleterious to the cells
indirectly causing reduced SDH activity.
The gene for the C subunit of complex II maps on the short arm of human chromosome
1. The selection of two independently derived primary hybrid cell lines
(XJM12.1.3, XJM12.2.2) with a human minichromosome containing the
centromere from human chromosome 1 suggested a close linkage between
the complementing gene and the centromere. Even shorter minichromosomes
with the complementing gene could be selected in radiation hybrids.
Southern analyses of human genomic DNA suggest that there are multiple
C
genes or pseudogenes (Fig. 1). The detection of
multiple bands with the bovine cDNA probe could be explained by the
existence of introns in this gene or by restriction sites present in
the human but not the bovine coding sequence. However, the presence of
bands in genomic DNA, which are missing from the minichromosome,
suggests that more than one gene or pseudogene exist in the human
genome. We believe that the locus on the minichromosome (1p12-13)
represents an active gene, since the minichromosome has been repeatedly
selected in somatic cell hybridization and complementation experiments.
Genomic DNA sequences corresponding to the C gene
must be analyzed to clarify the structure and the nature of these
genes. Intron-specific probes and comparisons of whole human genomic
DNA with the minichromosome will distinguish multiple loci. If more
than one active gene exists, one has to address the question of the
expression of these genes in various tissues (isozymes), and their
expression may be related to the regulation of the capacity for
oxidative phosphorylation in different cell types.
Finally, new
questions can be raised about the expression of the recessive phenotype
in the pseudodiploid Chinese hamster cells in culture. A priori one expects two alleles. One of these may be deleted or silenced
by hypermethylation, as demonstrated by us for ornithine
decarboxylase-deficient Chinese hamster ovary cells(28) , and
also in the case of another respiration-deficient Chinese hamster
mutant cell line defective in mitochondrial protein synthesis. ()However, hypermethylated alleles can be de-repressed by
treatment of the cells with 5-azacytidine, and such attempts have
failed with the CCL16-B9 cells. CCL16-B9 cells make normal amounts of
C
transcript when compared to their wild type parents
(results not shown). All four RT-PCR products derived from mutant RNA
had the same mutation, arguing against the presence of two different
mutated transcripts. Therefore, the second allele in these
pseudodiploid hamster cells must be deleted or permanently inactivated.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31241[GenBank].