Download Molecular Characterisation of the 76 kDa Iron

Plant Cell Physiol. 39(4): 373-381 (1998)
JSPP © 1998
Molecular Characterisation of the 76 kDa Iron-Sulphur Protein Subunit of
Potato Mitochondrial Complex I
Allan G. Rasmusson 1 , Volker Heiser 24 , Klaus D. Irrgang3, Axel Brennicke2, Lutz Grohmann 25
1
2
3
Department of Plant Physiology, Lund University, Box 117, S-221 00, Lund, Sweden
Universitdt Ulm, Allgemeine Botanik, Albert-Einstein-Allee, D-89069 Vim, Germany
Max-Volmer-Institut fur Biophysikalische Chemie und Biochemie, Technische Universitdt Berlin, Strasse des 17. Juni 135, D-10623
Berlin, Germany
Genes encoding subunits of complex I (EC 1.6.5.3) of
the mitochondrial respiratory chain vary in their locations
between the mitochondrial and nuclear genomes in different organisms, whereas genes for a homologous multisubunit complex in chloroplasts have to date only been found
on the plastid genome. In potato (Solatium tuberosum L.),
the gene coding for the mitochondrial 76 kDa iron-sulphur
protein is identified in the nuclear genome. The gene is transcribed into polyadenylated mRNA which is most abundant in flowers, and more frequent in tubers than in leaves.
The amino acid sequence is well conserved relative to the
nuclear-encoded 75 kDa and 78 kDa subunits of Bos taurus
and Neurospora crassa, respectively, and to the Paracoccus
denitrificans homologue, most prominently in the region
presumed to carry the iron-sulphur clusters. Polyclonal
antibodies directed against the 78 kDa complex I subunit of
TV. crassa recognise the 76 kDa polypeptide in potato mitochondrial complex I, and additionally a polypeptide of 75
kDa in solubilised stroma thylakoids from spinach chloroplasts. The 32 amino acid residues long presequence of the
potato mitochondrial 76 kDa complex I subunit targets the
precursor polypeptide into isolated potato mitochondria
but not into isolated chloroplasts. These results suggest
that chloroplast stroma thylakoids contain a protein similar in size and antigenicity to, but genetically distinct from,
the mitochondrial subunit.
Key words: Chloroplast — NADH:ubiquinone oxidoreductase — NAD(P)H:plastoquinone oxidoreductase — Plant
mitochondria — Respiratory chain (Complex I) —
Solarium tuberosum.
and is also present in bacteria (Friedrich et al. 1995). In
eukaryotes, complex I is situated in the inner mitochondrial membrane, where it constitutes the first coupling site
for ATP-synthesis in the respiratory chain. The enzyme in
Neurospora crassa has an L-shape with one arm protruding
into the mitochondrial matrix, where NADH is oxidised,
and the other located in the membrane where ubiquinone is
reduced (Leonard et al. 1987). The molecular mass of the
bovine complex is at least 900 kDa, the sum of about 41
subunits (Walker 1992), but only homologues to 14 of the
bovine subunits are present in the Escherichia coli enzyme
(Leif et al. 1995). Since the E. coli complex I contains all
the electron-carrying prosthetic groups, one FMN and five
iron-sulphur clusters, found in the bovine enzyme, it has
been called the minimal form of complex I (Weidner et al.
1993, Leif et al. 1995).
The largest subunit of the bovine complex I, the 75
kDa iron-sulphur protein, is located in the peripheral part
of the enzyme, in close connection to the NADH-binding 51 kDa subunit (Walker 1992, Yamaguchi and Hatefi
1993). Consistently, the E. coli homologues of the bovine
75, 51 and 24 kDa subunits have been isolated as an NADH
dehydrogenase fragment (Leif et al. 1995). Upon disruption of the gene for the 51 kDa subunit in TV. crassa, ironsulphur cluster N-3 disappears (Fecke et al. 1994), whereas
disruption of the gene for the 75 kDa homologue (78 kDa)
removes clusters N-l and N-4 as well as N-3 (Harkness et
al. 1995). EPR analysis of the over-expressed 75 kDa homologue (NQO3) of P. denitrificans showed that the subunit most likely contains the binuclear N-lb and the tetranuclear N-4 iron-sulphur clusters (Yano et al. 1995). Thus,
the 75 kDa subunit appears to connect the moiety of the active site for NADH to the rest of the complex, and transfer
the electrons downstream towards the membrane part.
The proton-translocating NADH:ubiquinone oxidoreductase or complex I (EC 1.6.5.3) has been found in mitochondria of all eukaryotes except some fermentative yeasts,
In plants, mitochondrial complex I has been analysed
in broad bean, red beetroot and potato mitochondria. It
shows large similarities to the complex I of beef heart and
N. crassa with respect to function and polypeptide composition (Leterme and Boutry 1993, Herz et al. 1994,
Rasmusson et al. 1994). The complex I from potato mitochondria contains more than 32 subunits and has a total
molecular mass of about 900 kDa, estimated from the sum
of the apparent molecular masses of the individual subunits (Herz et al. 1994). Apart from the conserved set of
The nucleotide sequence reported in this paper has been submitted to the EMBL under accession number X858O8.
1
To whom correspondence should be addressed.
4
Present address: Max-Planck-Institut fur Molekulare Genetik,
Ihnestrasse 73, D-14195, Berlin, Germany.
5
Present address: Bioinside GmbH, Potsdamer Strasse 18A, D14513 Teltow, Germany.
373
The 76 kDa subunit of complex I
374
seven mitochondrially encoded complex I subunits (ND 16, ND 4L) found in fungi and mammals (Walker 1992),
potato mitochondria also code for the NAD7 and NAD9
subunits, the homologues of the bovine nuclear-encoded 49
and 30 kDa subunits (Gabler et al. 1994, Grohmann et al.
1994). In the protists Acanthamoeba castellanii and Dictyostelium discoideum, genes for homologues of the 75 kDa
subunit of bovine complex I have been found on the mitochondrial genome (Burger et al. 1995, Cole et al. 1995).
The plastid genomes of several plants contain 11 genes
coding for homologues of mitochondrial complex I subunits (Shimada and Sugiura 1991). The genes are expressed
and the protein products localised in the stroma thylakoid
membrane, where they may be subunits of a plastoquinone
reductase involved in cyclic electron transport (Nixon et al.
1989, Berger et al. 1993, Friedrich et al. 1995, Mi et al.
1995). However, no homologue of the bovine 75 kDa subunit is encoded by the plastid genome. Such a subunit
would have to be encoded by the nuclear genome and imported into chloroplasts. A nuclear gene may even encode a
protein targeted to, and active in, both mitochondria and
chloroplast, as has been shown for glutathione reductase
(Creissen et al. 1995). Also for physiological studies of the
nuclear-encoded minimal subunits of plant mitochondrial
complex I, it is necessary to know if homologous polypeptides exist in chloroplasts.
Here we report the characterisation of the nuclear gene
for the 76 kDa subunit of complex I in potato mitochondria, the homologue of the bovine 75 kDa subunit. Furthermore, expression of this gene in different potato plant
organs has been investigated, and the intracellular localisation of the protein analysed.
Materials and Methods
Isolation of organelles and sub-organellar fractions—Mitochondria were isolated from potato tubers {Solatium tuberosum
L. cv. Bintje) from a local market, and complex I was purified according to Herz et al. (1994). For immunological analysis, spinach
chloroplasts were purified (Nakatani and Barber 1977) and fractionated into stroma, thylakoids and envelope (Robinson and
Barnett 1988). Stroma and grana thylakoids were separated by
differential solubilisation according to Berthold et al. (1981) with
the modifications of Volker et al. (1985). Import-competent chloroplasts were isolated from pea leaves according to Schindler et al.
(1987).
Protein analysis—The subunit with apparent molecular mass
of 75 kDa was purified from isolated potato mitochondrial complex I (Herz et al. 1994) by SDS-PAGE, extracted from blots,
digested with endoproteinase Lys-C, fractionated by reverse-phase
chromatography, and peptide-sequenced according to Grohmann
et al. (1994).
Blotted proteins were immunodetected with the ECL system
(Amersham, Little Chalfont, England).
Isolation and sequencing of cDNA clones—Poly(A)+-RNA
was isolated from total potato leaf RNA with oligo(dT)-magnetic
beads (Dynal, Hamburg, Germany), and first-strand cDNA was
synthesised with a preamplification system (Gibco BRL, Life Technologies, Eggenstein, Germany). The first PCR amplifications
were done between primers Homol 1 and Lys C 1. The 1.3 kb product was used as template for a second PCR reaction between
primers Lys C 2 and Lys C 1. Primer Homol 1, 5'-GAYTGYCCIATHTGYGAYCARGG-3', was derived from the peptide sequence of a conserved region in homologues from other species.
Primer Lys C 1, 5'-TTRTCIARRTTIACRTCRTCNGC-3' and
Lys C 2, 5'-AARGAYGCIGTITTYGCNGTNGA-3', were deduced from the peptide sequences of fragments of the purified subunit
of potato (Fig. 1). The PCR amplifications were done with Taqpolymerase (Gibco BRL), following the manufacturers instructions, for 30 cycles: 1 min at 94°C, 2 min at 45°C and 2 min at
72°C. The 193 bp product of the second PCR was randomly labelled, and used to screen a potato (cv. Desiree) cDNA library in
AZAPII (KoBmann et al. 1992) according to Grohmann et al.
(1992).
Sequencing of PCR fragments and cDNA clones was carried
out with a T7 sequencing kit (Pharmacia, Uppsala, Sweden) according to the manufacturers instructions. The cDNA clone was
sequenced on both strands by a combination of subcloning and oligonucleotide primers. Sequences were analysed with the GCG program package, version 8.1 (University of Wisconsin, Madison,
Wi., U.S.A.). The program Pileup was used for amino acid sequence alignments, Gap for calculating similarities between homologues, and Pepstructure for prediction of a-helices in proteins.
Blotting of nucleic acids—Total RNAs were isolated from
potato flowers, leaves, and tubers according to described methods
(Chomczynski and Sacchi 1987, Chang et al. 1993). Poly(A) + RNA was isolated as described above. Northern blotting of total
and poly(A)+-RNA, Southern blotting of endonuclease restricted
potato DNA, and hybridisation to randomly labelled probes was
carried out according to standard procedures (Sambrock et al.
1989).
Organellar import of precursor polypeptides—An [35S]methionine-labelled protein product of the sequenced cDNA
clone in pBluescript II SK was synthesised with a T7/rabbit reticulocyte lysate in vitro transcription/translation system (Amersham). For mitochondrial import 10 p\ translation product were
mixed with 27 /x\ mitochondria (—0.27 mg protein) and 107 /il import buffer (Braun and Schmitz 1995) supplemented with 5 mM
MgCl2 and 2 mM methionine. Import reactions were incubated at
25°C for 30 min. Half of the reaction was incubated with 20 ng
ml" 1 proteinase K at 15°C for 15 min, phenylmethylsulphonylfluoride was added to 1 mM, and after 15 min incubation on ice, mitochondria were reisolated by centrifugation through 0.5 ml of 25%
(w/v) sucrose. Mitochondrial fractions were washed once (0.4
M sucrose, 10 mM potassium phosphate, pH 7.2, 0.1% bovine
serum albumin and 1 mM phenylmethylsulphonylfluoride), resuspended in SDS-PAGE loading buffer, and analysed by SDS-PAGE
(Laemmli 1970). Where used, valinomycin (1 //M final concentration) was added to mitochondria before starting import.
Import into pea leaf chloroplasts was carried out according to
Waegemann and Soil (1991) using 10 /A translation product and
chloroplasts corresponding to 30 fig chlorophyll for each double
(ithermolysin) import reaction.
Results
The 76 kDa subunit of complex I is nuclear-encoded in
potato—The largest complex I subunit, with an apparent
The 76 kDa subunit of complex I
Translation
Start
Mature
Protein
Start
i
HincU
375
500 bp
Lys C 2
Homol 1
AATAAG
Lys C 1
GT-Rich
//men
4,
IIMM
ADAAAAAAAADA
DAVFAAVDTIAK
Fig. 1 Schematic map of the analysed cDNA for the 76 kDa subunit of potato mitochondrial complex I. The reading frame (boxed) is
preceded by an in-frame stop codon (*). The locations of PCR primers and Hindi restriction sites used in the investigation, as well as a
potential polyadenylation signal (AATAAG) with an upstream GT-rich region (24 bp, 88% G or T) is denoted on top. The protein has a
cleaved presequence (white box) before the start of the mature polypeptide (grey box). The deduced amino acid sequences corresponding
to the peptide sequences determined by N-terminal and internal protein sequencing of the purified 76 kDa subunit are given below. An
alanine not detected by peptide sequencing is underlined. The sequences coding for the peptide segments potentially liganding ironsulphur clusters are denoted in black.
molecular mass of 75 kDa, was purified by SDS-PAGE
from isolated potato mitochondrial complex I. The N-terminal sequence (ADAAAAAAAADA) was obtained by direct peptide sequencing, and internal peptide sequences
(DAVFAVDTIAK and FVYLMGADDVNLDK) were obtained after digestion of the protein with Lys-C endoproteinase and separation of the fragments by reverse-phase
chromatography.
With derived degenerated primers, an RT-PCR product was obtained and used as probe to screen a potato
cDNA library. Eight similar cDNA clones were isolated
and partially sequenced. The largest was sequenced in its
full length. The amino acid sequence deduced from the
cDNA matches the peptide sequences from the isolated subunit, apart from an additional Ala residue, not seen in the
protein sequence (Fig. 1). The first ATG triplet is preceded
by an in-frame stop codon verifying cloning of the complete reading frame. No alternative start codon is located
before the start of the mature polypeptide. Thus, a cleaved
peptide of 32 amino acid residues precedes the mature protein which consists of 706 amino acids, with a total molecular mass of 76,475 Da.
Southern analysis with a Hindi fragment of the
cDNA clone (Fig. 1) as probe, corresponding to the most
highly conserved part of the protein, showed single hybridising fragment sizes in total cellular potato DNA digested
with BamUl, EcoRI or Hindlll, whereas no signal was
seen in potato mitochondrial DNA (results not shown).
The analysed cDNA clone has no poly(A) tail but contains
a sequence pattern (AATAAG) 25 nucleotides before the
3' terminus with an upstream GT-rich region (Fig. 1)
similar to polyadenylation signals in the 3' untranslated
regions of other plant mRNAs (Wu et al. 1995). Northern
blots of potato total cellular-, and poly(A)+-RNAs from
flowers, leaves and tubers probed with the Hindi fragment
show expression and in vivo polyadenylation of the mRNA
in all three tissues. The steady-state transcript level is
highest in flowers, and higher in tubers than in leaves, in
both total-, and poly(A)+-RNA (Fig. 2). The results suggest that the 76 kDa subunit of mitochondrial complex I in
potato is encoded by a single nuclear gene, and that the
polypeptide is translated from polyadenylated mRNA,
more abundant in flowers than in tubers and leaves.
Sequence conservation of the 76 kDa subunit—Amino
acid sequence alignment of the mature 76 kDa subunit of
potato to the corresponding complex I subunits from other
species disclosed an N-terminal region of about 270 amino
acid residues to be more conserved than the remaining protein. The N-terminal region contains 59 amino acid residues conserved between all these homologous complex I
subunits. Of the C-terminal 500 amino acid residues only 8
are completely conserved (not shown). The y-subunit of the
NAD + reducing hydrogenase from Alcaligenes eutrophus
lacks the C-terminal part entirely (Tran-Betcke et al. 1990).
Fig. 3 depicts the conservation between the N-terminal
parts of the 76 kDa subunit homologues. Whereas the
mature peptide sequences from most other species align to
a common N-terminus, the potato 76 kDa subunit has an
Poly-A+
Total
F.
L.
T.
F.
L.
T.
Fig. 2 Northern analysis of 76 kDa subunit RNAs in different
potato organs. In order to compare equal relative amounts, 43, 54
and 30//g of flower (F.), leaf (L.) and tuber (T.) total RNA were
loaded per lane, respectively. For each organ, poly(A)+-RNA isolated from the three-fold amount of total RNA was loaded. The
calibration was done by comparing signal intensities when hybridising with a probe against 25S rRNA (not shown).
The 76 kDa subunit of complex I
ITS
St
Bt
He
AC
Dd
ADAAAAAAAADAPSDLPKRHPVGGARVHLPNPEDV Ev FV H T p v K
TATAASNL..
E V P V^BQ S V M
A E E L T iHiK K V SHE . . . A | S A
H X N SFKVMDFQY T B H - . . N K L
MI E F K I N E I E C E I N E E K E D I
MADLKKIKI
THE
HAT IHV
KEY
HSBQITI
HILIIE
Q
IKE
0V0NAEDTRO
V . . . N
. G N
H v
S N SMM
IDE
NFDDES
KDLRPGPEGAPSE
H T«A
S
VBl
SKBL N
A HD Nvr
PESRI
D I
H B E D 1
D Ev
E F Y
P E Q V K E A
EVGIL.DP
S E L
R 1
N B D G V
YBG T
v
Fig. 3 Amino acid sequence alignment of the N-terminal regions of homologues to the potato 76 kDa subunit. For the polypeptides encoded by nuclear genes (St, Bt and Nc) the amino acid sequences of the mature proteins are shown. The conserved cysteine and histidine
residues are marked by asterisks above the sequences, those not conserved in A.eutrophus are in parenthesis. The cysteines of the
putative iron sulphur cluster motif in E. coli (Leif et al. 1995) are marked by dots. The sequences are: St, S. tuberosum (76 kDa subunit);
Bt, B. taunts (75 kDa subunit) (Runswick et al. 1989); Nc, N. crassa (78 kDa subunit) (Preis et al. 1991); Ac, A. castellanii NADU
(Burger et al. 1995); Dd, D. discoideum 80 kDa subunit (Cole et al. 1995); Pd, P. denitrificans NQO3 (Xu et al. 1992); Ec, E. coli NUOG
(Weidner et al. 1993); Ae, A. eutrophus y-subunit of NAD + reducing hydrogenase (Tran-Betcke et al. 1990).
N-terminal extension of about 30 amino acid residues. The
12 Cys and 2 His residues potentially liganding ironsulphur clusters are conserved in the potato sequence. The
postulated sequence motifs for a tetranuclear (CXXCXXCX n CP) and a binuclear (CXXCXnCXXC) iron-sulphur
cluster (Walker 1992) are positioned at amino acid residues
180-231 and 79-135 in the potato sequence, respectively.
The recently suggested motif for a binuclear cluster found
in E. coli (Leif et al. 1995) is not conserved in potato.
The bovine 75 kDa and its homologues are generally
water soluble when isolated (Yano et al. 1995, Takano et
al. 1996). We have analysed the full-length polypeptides
with the TMAP algorithm to predict transmembrane ahelices from multiple sequence alignments (Persson and
Axgos 1994). Putative transmembrane segments were detected neither in the alignment nor in the individual sequences.
Amino acid identity comparisons of the 76 kDa homologues from various species revealed a substantial conservation between the nuclear-encoded subunits of potato, Bos
taurus and N. crassa (Table 1). The three nuclear-encoded
sequences of higher eukaryotes as a group show higher
similarity to the sequence from the bacterium P. denitrificans than to the eukaryotic, mitochondrially encoded sequences from the protists A. castellanii and D. discoideum.
The potato sequence is slightly more similar to the se-
quence of A. castellanii than to that of D. discoideum.
A chloroplast thylakoid protein is recognised by an
antiserum directed against the 76 kDa homologue of
N. crassa mitochondrial complex I—To investigate the
potential presence of the 76 kDa polypeptide, or a homologue of it, in chloroplasts, various protein fractions of spinach leaf chloroplasts were resolved by SDS-PAGE, blotted, and probed with a polyclonal antiserum against the 78
kDa subunit of mitochondrial complex I from N. crassa
(Friedrich et al. 1989). A polypeptide is detected in total
and stroma thylakoid fractions, with an apparent molecular mass of 75 kDa, similar to the immunodetected subunit
in isolated complex I from potato (Fig. 4A). The 75 kDa immunodetected chloroplast polypeptide is observed neither
in the stroma soluble protein fraction nor in the grana thylakoid fraction. Apart from the 75 kDa polypeptide, no protein is detected in the chloroplast fractions, suggesting
a high specificity of recognition by the antibodies. In
Coomassie stained gels only a very faint band could be seen
at 75 kDa, indicating that the antibodies detect minute protein amounts (not shown). The presence of an immunostained polypeptide at 50 kDa in potato mitochondrial complex I (Fig. 4A, lane 5) indicates a partial proteolysis of the
76 kDa subunit during complex I purification. When immunoblotting total protein from isolated potato mitochondria, the 76 kDa subunit is the only protein recognised by
The 76 kDa subunit of complex I
these antibodies (not shown).
Western blot analysis of the chloroplast protein fractions with an antiserum against the NDH-H gene product
of tobacco, the plastid-encoded homologue of the 49 kDa
subunit of bovine complex I, shows a similar distribution
as the 75 kDa polypeptide (Fig. 4B). A polypeptide of 43
kDa is recognised in stroma thylakoids and total thylakoids, but not in grana thylakoids or soluble stroma proteins, consistent with the observations of Berger et al.
(1993).
To control that contaminating mitochondria was not
responsible for the immunoreactive 75 kDa polypeptide,
the chloroplast fractions were tested with an antibody
against the a-subunit of ATPase from maize mitochondria.
The antibody did not react with any of the chloroplast fractions but gave a very strong positive reaction in potato mitochondrial proteins (Fig. AC).
The precursor for the mitochondrial 76 kDa subunit is
imported into mitochondria but not into chloroplasts—
The plastid genome of tobacco contains three reading
377
frames coding for polypeptides of 70-80 kDa (Shimada
and Sugiura 1991), but the encoded proteins have no sequence similarity to the 78 kDa subunit of N. crassa mitochondrial complex I (not shown). This suggests that the immunoreactive polypeptide should be encoded in the
nucleus, either by the gene specifying the 76 kDa subunit of
mitochondrial complex I, or by a separate nuclear gene. If
the gene for the mitochondrial subunit also codes for a pro-
A)
MGLGLLASRALRSSRIIRNSTRTIVSTPELKN-
B)
Import
Prot. K
Valinom.
— +
+
+ +
_ . _ _ _
+
+
97 kDa —
12
3 4 5
-97
-66
-46
69 kDa —
C)
Import
—
Thermol. —
76 kDa
-
ssu
- +
+
p76 -+* mm •—-
B
1
2 3 4
-46
-mSSU
Fig. 5 In vitro import analysis of the 76 kDa subunit into mitochondria and chloroplasts. In (A), the deduced presequence of the
1 2 3 4 5 PC
isolated cDNA for the 76 kDa subunit is shown with positively
charged amino acids denoted above. In (B), [35S]-labelled transla-66
tion products of the cDNA for the 76 kDa subunit were incubated
with isolated potato mitochondria under different conditions and
-46
radiolabelled polypeptides separated on 6-11% SDS-polyacrylamide gels. The different treatments of the translation products
are denoted on top, and the positions of the molecular mass standFig. 4 Western analysis of chloroplast subfractions. Proteins
ards are given on the left side. In (C), the similar experiment was
were separated on 11% polyacrylamide gels, blotted and incubatconducted with isolated pea leaf chloroplasts. On the left, the imed with antibodies against: (A) the 78 kDa subunit from N. crassa,
port analysis for the 76 kDa subunit is depicted, p76; precursor
(B) the NDH-H polypeptide from tobacco, and (C) the a-subunit
protein for the 76 kDa subunit. On the right, the control for
of mitochondrial F,-ATPase from maize. Lane 1, stroma soluble
import competence of the chloroplasts by import of in vitro transproteins (80//g); lane 2, extracts of stroma thylakoids (20 //g Chi);
lated small subunit of Rubisco (SSU) and processing from its
lane 3, grana thylakoids (20/jg Chi); lane 4, total thylakoids (20
precursor form (pSSU) to its mature form (mSSU) is shown. The
fig Chi); lane 5, isolated mitochondrial complex I from potato (3
H% protein in (A), 9/*g in (C)). The positive control (PC) in (C) is different treatments of the translation products are denoted on
top.
potato tuber mitochondria (80//g protein).
The 76 kDa subunit of complex I
378
tein in chloroplasts, the targeting peptide should be able to
direct the precursor protein into both organelles.
The deduced amino acid sequence of the 76 kDa subunit of mitochondrial complex I has a 32 amino acid residues long presequence (Fig. 5A). It is rich in Arg, Ser and
Leu residues, and contains only one acidic amino acid residue close to its C-terminus. The N-terminal 12 amino acid
residues of the presequence are predicted to fold into an amphiphilic a-helix with the positively charged Arg residues localised on the polar side (not shown). Most of these features
of the presequence are common properties of mitochondrial targeting peptides (von Heijne et al. 1989). However,
targeting peptides for mitochondria and chloroplasts are
very heterogeneous, and at the same time share several common features. We therefore considered it necessary to resolve the question by in vitro import experiments with isolated chloroplasts, using mitochondrial import as a positive
control for competence of the translation product to be
recognised by a bona fide molecular interaction.
( +valinomycin), indicating that the targeting sequence is
indeed cleaved off during or after import.
In vitro import analysis of the translation product was
additionally carried out with isolated pea leaf chloroplasts
(Fig. 5C). The precursor of the 76 kDa subunit is bound to
the outer envelope, but is not protected against thermolysin
digestion, and is thus not imported. The import-competence of the isolated chloroplasts was verified by the effective import and processing of the precursor protein of the
small subunit of Rubisco. The chloroplast 75 kDa polypeptide recognised by the antibody against the N. crassa mitochondrial 78 kDa complex I subunit is thus most likely encoded by a gene distinct from the gene encoding the 76 kDa
subunit of mitochondrial complex I in plants.
Discussion
The relationship between the 76 kDa subunit of potato
mitochondrial complex I and homologues in other species
—The primary structure of the largest subunit of mitochondrial complex I has now been determined in representatives
of all eukaryotic kingdoms, including plants. In potato, the
gene is located in the nucleus, and the protein is translated
as a precursor subsequently imported into mitochondria.
The absence of the gene for the 76 kDa subunit in the
recently analysed mitochondrial genome of Arabidopsis
(Unseld et al. 1997) indicates that its localisation in the
nucleus may be a general feature of plants. The nuclear
genes for the 75-78 kDa subunits of potato, B. taurus and
N. crassa are most likely of mitochondrial origin and were
introduced into the cells by the original endosymbiont, as
indicated by the marked amino acid sequence similarity to
the corresponding P. denitrificans subunit (Table 1). The
mitochondrial localisation of the gene homologues in the
protists A. castellanii and D. discoideum (Burger et al.
1995, Cole et al. 1995), further supports the endosymbiont
origin of this gene. The transfer of the gene from the mito-
The precursor of the 76 kDa polypeptide synthesised
in vitro from the analysed cDNA clone (Fig. 1) is imported
into potato mitochondria, as seen from its acquired resistance against degradation by proteinase K (Fig. 5B). In the
presence of the uncoupler valinomycin, the induced protease-resistance is abolished. This suggests that import into
mitochondria is dependent on the membrane potential, as
generally seen for matrix-targeted proteins.
The location of the N-terminus of the isolated mature
mitochondrial subunit in the cDNA (Fig. 1) suggests that
the presequence is cleaved off post-translationally, most
likely by the matrix peptidase. In the import analysis
(Fig. 5B), precursor and mature forms are not completely
separated by SDS-PAGE due to the large size of the subunit in relation to the presequence. However, the protein remaining after protease treatment was repeatedly seen (3
independent experiments) to migrate slightly faster than
the unimported, protease-sensitive precursor polypeptide
Table 1 Amino acid sequence similarities between 76 kDa protein homologues from
different species
Nuclear
encoded
Species
St
Bt
Nc
Ac
Dd
Pd
Bt
Nc
57
55
56
Identity, %
Mitochondrially
encoded
Ac
Dd
44
40
41
37
38
36
38
Bacterial
Pd
Ec
49
49
49
37
35
28
27
28
23
25
27
The mature proteins were compared over the maximum region spanned by all sequences. Abbreviations of species are as in Fig. 3.
The 76 kDa subunit of complex I
chondria to the nucleus, as seen in potato, B. taurus and
N. crassa, has thus most likely occurred after the separation of protists and higher eukaryotes. Unless the transfer
of the gene occurred as separate events, the nuclear location in potato indicates a closer relationship between plants
and other higher eukaryotes. This would be inconsistent
with the proposed affiliation between land plants and
A. castellanii based on the gene contents of the mitochondrial genomes (Burger et al. 1995).
In representatives of eubacteria and all eukaryotic
kingdoms, including the potato mitochondrial 76 kDa subunit, 12 Cys and 2 His residues are found to be completely
conserved (Fig. 3). EPR analysis of potato mitochondrial
complex I detected 3 tetranuclear iron-sulphur clusters and
one binuclear, probably N-lb, a similar cluster conformation as in N. crassa complex I, whereas in B. taurus, P. denitrificans and E. coli also a cluster N-la has been detected
(Lin et al. 1995). The extensive sequence conservation of
the putative iron-sulphur cluster liganding region of the 76
kDa subunit between potato, N. crassa and the other EPRanalysed species is consistent with a localisation of cluster
N-lb in this subunit, as previously suggested (Yano et al.
1995).
Expression of the 76 kDa subunit mRNA—The steadystate mRNA levels of the gene for the 76 kDa subunit
of potato mitochondria are highest in flowers (Fig. 2), as
has been observed for other nuclear-encoded mitochondrial proteins (Huang et al. 1994, Landschiitze et al. 1995).
On the other hand, mRNA levels of the 76 kDa subunit are
higher in tubers than in leaves, even though the Northern
hybridisation experiments are corrected for the elevated
levels of plastid RNA in leaves. Both the matrix processing
peptidase subunit of mitochondrial complex III and the
TCA-cycle enzyme citrate synthase show considerably
higher mRNA levels in leaves than in tubers of potato (Emmermann et al. 1994, Landschiitze et al. 1995). This could
indicate a difference in relative amounts of respiratory enzymes between tissues where different substrates are available for mitochondrial oxidation. Plant mitochondria also
contain a rotenone-insensitive, non-energy-coupled NADH
dehydrogenase competing with complex I for matrix
NADH (Rasmusson and Moller 1991). Differences in expression of complex I subunits may then also reflect a variation in the relative amounts of the two enzymes.
Do chloroplasts contain a homologue of the mitochondrial 76 kDa subunit?—We here observe specific recognition of one polypeptide in spinach leaf chloroplasts by
antibodies against the 78 kDa subunit of N. crassa mitochondrial complex I. This polypeptide has an apparent molecular mass similar to the potato mitochondrial 76 kDa
subunit, and is located in stroma thylakoids as is the NDHH polypeptide (Fig. 4).
The genes of the "minimal" complex I subunits not
present on the plastid genomes correspond to the 24, 51
379
and 75 kDa subunits of bovine complex I (Shimada and
Sugiura 1991). Based on the structural and evolutionary
connection between these subunits and NADH dehydrogenase activity, Friedrich et al. (1995) suggested that the
putative complex I in chloroplasts should contain a different setup of subunits to enable the oxidation of ferredoxin
or NADPH instead of NADH, which is less abundant in
the stroma. Consistent with this hypothesis, the cyanobacterium Synechocystis sp. lacks ndh genes for homologues to
the 24, 51 and 75 kDa subunits (Kaneko et al. 1996). However, NDH proteins encoded by the plastid genome have
been reported to co-elute with NADH, but not NADPH,
dehydrogenase activity upon partial purification of a complex I-type enzyme from pea chloroplasts (Sazanov et al.
1996). This suggests that the complex I of chloroplasts
could have a similar setup of subunits as the mitochondrial
enzyme. The chloroplast enzyme may then contain also the
immunoreactive 75 kDa polypeptide (Fig. 4). However, an
unequivocal answer to this question will be possible only
when the active chloroplast enzyme has been purified to homogeneity and all protein constituents have been characterised.
A possible explanation for the presence of the immunoreacting 75 kDa polypeptide in chloroplasts would be if
the product of the gene for the mitochondrial subunit is
targeted also to chloroplasts. Such dual targeting has been
observed for glutathione reductase (Creissen et al. 1995).
However, the gene for the mitochondrial subunit is not likely to specify also the immunoreactive 75 kDa polypeptide
in chloroplasts. The amino acid presequence deduced from
the isolated cDNA has more properties in common with
mitochondrial targeting peptides whereas the glutathione
reductase presequence shows a bias towards chloroplast
targeting peptides (Creissen et al. 1995). Consistently, the
mitochondrial 76 kDa subunit of potato is in vitro only imported into mitochondria (Fig. 5). The relatively lower
steady-state mRNA levels in leaves additionally imply that
the protein product of the gene for the 76 kDa subunit of
mitochondrial complex I plays no role in chloroplasts.
This study was supported by grants from the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie and
the Deutsche Forschungsgemeinschaft, Bonn, A.G.R. acknowledges a fellowship from the Swedish Council for Forestry and
Agricultural Research. We are grateful to Drs. T. Friedrich (Institut fur biochemie, Heinrich-Heine Universitat Diisseldorf, Germany), C. Leaver (Department of Plant Sciences, University of
Oxford, U.K.) and K. Steinmiiller (Institut fiir Entwicklungs- und
Molekularbiologie der Pflanzen, Heinrich-Heine Universitat
Diisseldorf, Germany) for the gifts of antibodies, Dr. L. Heinz
(Botanisches Institut, Universitat Kiel, Germany) for expert advice on mitochondrial protein import, Dr. W. Schroder (Institut
fiir Biochemie, Frei Universitat Berlin, Germany) for protein sequencing, Dr. J. Soil (Botanisches Institut, Universitat Kiel, Germany) for the chloroplast protein import analysis, and Oliver
Thieck for excellent technical assistance.
The 76 kDa subunit of complex I
380
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(Received September 29, 1997; Accepted January 22, 1998)