Download Prokaryotic features of a nucleus

Eur. J. Biochem. 159,323-331 (1986)
0FEBS 1986
Prokaryotic features of a nucleus-encoded enzyme
cDNA sequences for chloroplast and cytosolic glyceraldehyde-%phosphate dehydrogenases
from mustard (Sinapis alba)
William MARTIN and Riidiger CERFF
Institut fur Botanik, Universitiit Hannover
(Received February 25/June 5, 1986) - EJB 86 0205
Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphatedehydrogenases (GAPDH)
from mustard (Sinapisalba), have been identified and sequenced. Comparison of the deduced amino acid sequences
with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleusencoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic
sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the
hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in
puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to
the eukaryotic enzymes [Eur. J. Biochem. 150,61-66 (1985)l.
It is now well established that plastid and eubacterial genes
share a high degree of sequence homology [I - 61 in agreement
with the endosymbiotic theory of chloroplast evolution (see
[7- 101 for recent reviews). However, most of the chloroplast
components, at least 190 [ill but probably over 300 different
proteins (121, are encoded in the nuclear genome. This raises
the interesting question of whether or not these nuclear genes
are of prokaryotic origin. Nucleus-encoded enzymes of the
photosynthetic Calvin cycle are excellent marker molecules
with which to investigate this question because their primary
structures may be directly compared to the primary structures
of glycolytic isoenzymes located in the cytoplasm of the same
cell.
Chloroplast and cytosolic glyceraldehyde-3-phosphate
dehydrogenases (GAPDH) are especially useful molecular
homology criteria for this important aspect of plant cell evolution because of the large data base available, comprising
GAPDH sequences from seven eukaryotic and three prokaryotic organisms. Using protein-sequencing techniques
Harris et al. established complete amino acid sequences for
the GAPDHs from lobster muscle [13], pig muscle [14] and
yeast [15] and subsequently also for the GAPDHs from the
thermophilic eubacteria Bacillus stearothermophilus [16, 171
and Thermus aquaticus [18]. More recently cloning and DNA
sequencing techniques were used to determine the primary
structures of GAPDH enzymes from yeast [19], chicken [20221, human [23, 241, rat [25, 261 and Drosophila melanogaster
[27] and Escherichia coli [28]. An interspecies comparison of
these GAPDH sequences reveals a high degree of sequence
conservation ranging from 46% (human/Thermus) to 93 %
(human/pig; see Table 1). X-ray cristallographic analyses of
the enzymes from lobster [29 - 311 and B. stearothermophilus
[16] demonstrated that the aminoterminal and carboxyterminal moieties of the GAPDH polypeptide are organized as
two independent three-dimensional units, which were highly
conserved during the divergence of prokaryotes and
eukaryotes: the coenzyme-binding domain, which has a
similar structure in all NAD-dependent dehydrogenases, and
the catalytic domain, which is specific for GAPDH and which
differs in different dehydrogenases [32].
In green plants the classic GAPDH of glycolysis has a
photosynthetic counterpart, which is NADP-dependent and
located inside the chloroplast. Antisera raised against the
chloroplast enzyme do not cross-react with the cytosolic
GAPDH and fingerprints as well as amino acid compositions
are different for the two enzymes [33 - 351. The chloroplast
GAPDH is also exceptional in that it is composed of two
separate subunits A and B, which differ slightly in molecular
mass (A 5 B [35-381. Both subunits are encoded by nuclear
genes [39] and their identities and presumptive functions have
recently been disclosed in our laboratory by molecular cloning
and sequencing techniques [40]: while subunit A of chloroplast GAPDH from pea was found to represent the catalytic
subunit of the enzyme (a ‘true’ GAPDH-like structure), the
cloned partial sequence of subunit B was recognized to be
highly homologous to P-tubulin.
In the present paper we report the molecular cloning and
sequence analysis of cDNAs encoding the catalytic subunits
of chloroplast and cytosolic GAPDHs from mustard (Sinapis
alba). The deduced amino acid sequences are compared with
one another and with the GAPDH sequences from animals,
yeast and bacteria.
Correspondence to R. Cerff, Labontoire de Biologie Molkulaire
Vkgetale, Universite de Grenoble 1 , Boite postale 68, F-38402 St
Martin d’Htres CMex, France
Abbreviotions. bp, base pairs; GAPDH, D-glyceraldehyde-3- MATERIALS AND METHODS
phosphate dehydrogenase; SDS, sodium dodccyl sulfate.
Purification, fractionation and translation of poly(A)-rich
Enzymes. Cytosolic glyceraldehyde-3-phosphatedehydrogenase,
NAD-specific (EC 1.2.1.12); chloroplast glyceraldehyde-3-phosphate mRNAs was done essentially as described previously [3Y411.
dehydrogenase, NADP-dependent (EC 1.2.1.13).
324
Construction of cDNA library
The cDNA was synthesized from size fractionated poly(A)-rich mRNA from light-grown mustard seedlings
essentially according to the protocol of Maniatis et al. [42]
with snapback-primed second-strand synthesis, S 1 nuclease
digestion and C-tailing for annealing into the G-tailed PstI
site of pBR322. Chimeric plasmids were transformed into E.
coli DH 1 by the method of Hanahan [43] with an efficiency
of 3 x lo5 recombinants/pg double-stranded cDNA. Cultures
(2 ml) of single colonies were grown overnight and aliquots
from these were stored in microtiter plates.
Dot-blot hybridizations
From each of three different overnight cultures 0.5 ml
were combined for small-scale plasmid preparation. From
each pooled plasmid preparation 5 pl were denatured and
dot-blotted to nitrocellulose filters. Filters were then prehybridized for 6 h at 42°C in 50% formamide, 1.0 M NaC1,
50 mM Tris/HCl pH 8.0, 100 pg/ml salmon sperm DNA and
2 pg/ml polyadenylic acid. Hybridization was performed for
48 h at 42" C after adding the nick-translated ( x 10' cpm/pg
DNA) 246-base-pair(bp) internal Hind111 fragment of
pGAP30 from chicken [20] or the 800-bp insert of cDNA
clone pP71-11 encoding preA from pea [40]. Filters were
subjected to a final wash at 42°C in 0.3 M NaC1,60 mM Tns/
HCl pH 8.0, 1% SDS and exposed on Kodak X-Omat AR
film. The same blotting and hybridization procedures were
used to identify single positive clones from dots (containing
pools of three). Plasmids corresponding to the strongest hybridization signals were analyzed by hybrid-released translation.
Fig. 1. Identification by hybrid-released translation of clones pS6b and
pS302b encoding cytosolic GAPDH (a, lane I ) and chloroplast
GAPDH (b, lane 1 ) from mustard respectively. M, marker proteins.
The selected mRNAs were translated in 10 pi cell-free wheat germ
system with 5 pCi [35S]methioninefor 60 min. The translation products were submitted to dodecyl sulfate gel electrophoresis and
visualized by fluorography. They have the expected M , values (39)
and can be precipitated with our monospecific antisera (not shown)
of approximately 2 x 10' cpm/pg. Filters were given a final
wash in 1 x NaCl/Cit, 0.1% SDS at 68°C and autoradiographed for 12 h with Kodak X-Omat AR film.
Hybrid-released translation
For this procedure 50 pg plasmid DNA was bound to a
0.25-cm nitrocellulose filter, which was then washed for 3 h
in 5 x NaCl/Cit and baked for 2 h at 80°C. Prehybridization
was performed for 1 h at 52°C in 65% formamide, 0.4 M
NaCl, 10 mM Pipes pH 6.4 and 80 pg/ml polyadenylic acid.
The prehybridization solution was replaced with 120 pl/filter
of 65% formamide, 0.4 M NaCI, 10 mM Pipes pH 6.4, 0.1%
SDS and 0.25 mg/ml poly(A)-rich mRNA from light-grown
mustard seedlings. After 6-h incubation at 52" C, filters were
washed six times for 2 min at 60°C in 2 x NaCl/Cit, 0.5%
SDS, twice for 1 min at 20°C in 2 mM EDTA pH 7.9 and
once for 5 min at 50°C in 2 mM EDTA pH 7.9. The mRNA
was eluted for 8 0 s at 100°C in H 2 0 . The eluate was precipitated with ethanol and translated in a cell-free wheat germ
system.
Colony hybridizations
Colonies were grown on Schleicher and Schiill BA 85
filters over agar medium with tetracycline (10 pg/ml) for 48 h and transferred to agar plates containing chlorarnphenicol
(200 pg/ml) for amplification overnight. Filters were then
transferred sequentially to Whatman 3 MM paper, soaked
with the following solutions, and incubated for the time indicated: 0.5 M NaOH, 10 min; 1.0 M Tris/HCl pH 7.4,
10 min; 1.5 M NaCI, 0.5 M Tris/HCI pH 7.4, 5 min; 0.3 M
NaCI, 5 min. Dry filters were baked for 2 h at 80°C and
hybridized as described [42] to the PstI inserts of cDNA clones
pS6b and pS302b, each nick-translated to a specific activity
DNA sequence determination
Both strands of clones pS198c and pS84b were sequenced
according to Maxam and Gilbert [44].Fragments were labeled
at the terminal PstI sites with terminal transferase and
[32P]dideoxy-ATP( x 5500 Ci/mmol) or at the 3'-recessed
termini with Klenow polymerase and [32P]deoxynucleotides
('filling in' reactions).
RESULTS
Cloning of cDNAs and identification of specific clones
The construction of the present cDNA library was
performed with pBR322 and size-fractionated poly(A)-rich
mRNA from light-grown mustard seedlings [41]. The cDNA
synthesis and the construction of the recombinant plasmids
(see Materials and Methods) were done in collaboration with
H. Sommer in the laboratory of H. Saedler (Max-PlanckInstitut fur Ziichtungsforschung, Cologne). About 1200 transformants were analyzed. Identification of specific clones,
corresponding to chloroplast (subunit A) and cytosolic
GAPDHs, was achieved in four consecutive steps. (a) Dot
blots were first screened with heterologous probes from
chicken (cytosolic GAPDH) and pea (chloroplast GAPDH,
subunit A) respectively. (b) Single clones from aparent positive
dots were then analysed by hybrid-released translation and,
if positive by this criterion (see Fig. 1a and b), (c) submitted
to partial sequence analysis. (d) Positive clones, as defined by
criteria (b) and (c), were then used as homologous probes to
325
rescreen the library by colony hybridization. The positive
clones with the longest inserts were then selected for complete
sequence analysis.
We found the cytosolic GAPDH with the heterologous
chcken probe pGAP30, kindly provided by H. H. Arnold,
Medical School, University of Hamburg.
Screening of the dot blots was performed with an internal
Hind111 fragment, which is 246 bp long and which comprises
codons 225-305 of the catalytic domain [20]. Among the
15-20 possible candidates we found just one clone (pS6b,
approx. 350 bp long), which was positive according to criteria
(b) and (c).
The heterologous pea probe, clone pP71-11, harbors an
800-bp fragment of preA cDNA previously cloned in our
laboratory and identified by hybrid-released translation [40].
For initial isolation of chloroplast GAPDH cDNAs, pea was
best suited due to the strong light induction of preA and
preB mRNAs in this species [39], providing a simple basis
for differential hybridization in screening procedures. But
average insert length in the pea library necessitated a second
cDNA cloning, for which mustard was chosen since amino
acid compositions, fingerprint data and a partial aminoterminal peptide sequence for subunit A of chloroplast GAPDH
had previously been determined for the mustard enzymes [34,
411. With the heterologous probe pP71-11 we identified the
mustard clone pS302b, whose insert is about 700 bp long and
which was found to be positive according to criteria (b) and
(c).
Screening of our cDNA library with the two homologous
probes pS6b (cytosolic GAPDH) and pS302b (chloroplast
GAPDH) by colony hybridization led to the identification of
two separate groups, which each contained seven strongly
cross-hybridizing members. From each group the clone with
the longest insert was selected, pS198C for cytosolic and
pS84b for chloroplast GAPDH, and submitted to nucleotide
sequence analysis according to Maxam and Gilbert [MI.
Primary structures of the GAPDH messages
and amino acid sequences
Both strands of clones pS198C (cytosolic GAPDH) and
pS84b (chloroplast GAPDH, subunit A) have been sequenced. In Fig. 2 the coding strands for cytosolic (sequence
1) and chloroplast (sequence 2) GAPDH are aligned
according to the standard scheme for GAPDH enzymes [45].
Clone pS198c (cytosolic GAPDH) is 1106 nucleotides
long. A 7-nucleotide 5’-non-translated sequence is followed
by the complete coding region of 1017 nucleotides, comprising
339 codons including initiation and termination codons. The
3‘-non-translated region is 82 nucleotides long. It contains
the presumptive polyadenylation signal AATAAG at position
+ 51 and terminates with a poly(A) tail of 15 adenosine
nucleotides.
Clone pS84b (chloroplast GAPDH) contains 836
nucleotides and starts at codon 100. The 3’-non-translated
region is 132 nucleotides long, contains the polyadenylation
signal AATAAA at position + 108 and ends with an
uninterrupted stretch of 7 adenosine nucleotides, the apparent
beginning of the poly(A) tail. The two clones show 55%
nucleotide homology with respect to their coding sequences
starting at codon 101. They have no homology in their 3‘-noncoding regions.
The protein translated from clone pS198c contains 337
residues and presently represents the longest GAPDH known
(see also Fig. 3). It starts with the strongly polar peptide Ala-
Asp-Lys-Lys in positions - 3 to 0. The presence of these four
additional aminoterminal residues could not be confirmed
directly, because the aminoterminus of the native enzyme is
blocked [41]. The enzyme also differs from all other eukaryotic
GAPDHs in that it contains two insertions, probably Lys53A and Glu-68A (see Fig. 3). It has a calculated M , of 36768,
which is somewhat smaller than expected from its electrophoretic mobility in sodium dodecyl sulfate gels, suggesting a
M , of approximately 39000 [34]. The calculated amino acid
composition is in rough agreement with the previously determined experimental values [34], except that Cys was overestimated (7 instead of 2 residues) and Val underestimated (26
instead of 37 residues).
The amino acid sequence of mustard chloroplast GAPDH
is not yet complete. The first 17 aminoterminal residues have
previously been determined by automatic Edman degradation, with two, uncertainties, but probably arginines, in
positions 10 and 13 [41]. The native subunit starts with a
methionine in position 0 and the subsequent aminoterminal
peptide is clearly homologous to the other GAPDH enzymes
(see Fig. 3). Clone pS84b translates into a polypeptide
comprising 70% (residues 101- 333) of chloroplast GAPDH
(subunit A) from mustard. Among these 233 carboxyterminal
residues there are only 114 (49%) which are identical with the
cytosolic GAPDH from the same species (boxed regions in
Fig. 2).
DISCUSSION
Plants have parallel pathways of sugar phosphate metabolism located on either side of the chloroplast envelope. As a
consequence, many of these metabolic reactions are catalyzed
by pairs of plastid/cytoplasmic isoenzymes, which can be
separated on the basis of their charge differences [46].
Although it is now generally accepted that the majority of the
chloroplast-located isoenzymes are encoded in the nucleus
[47, 481, their origin and evolution are not yet understood.
There are basically three different possibilities which have
been discussed previously by Bogorad [49] and elaborated
further more recently by Weeden [47]. (a) The genes for a
given pair of plastid/cytoplasmic isoenzymes may be the result
of an ancient duplication event in the early plant eukaryotes.
(b) A second hypothesis suggests that the genes of plastid
isoenzymes may be of prokaryotic origin and that their present nuclear location would be the result of gene transfer from
an endosymbiotic plastid ancestor to the nucleus of a primitive
eukaryotic ‘host’. (c) A third possibility is that plastid isoenzymes are posttranscriptional or posttranslational modifications of the cytoplasmic forms.
Although chloroplast and cytosolic GAPDHs are not
isoenzymes in the strict sense of the word, because they differ
in their pyridine nucleotide requirements, the two nucleusencoded enzymes have clearly homologous sequences (Fig. 2).
In the following the seven eukaryotic and three prokaryotic
sequences published will be used as a data base to analyse the
molecular origin of the two plant GAPDHs according to the
three alternative possibilities mentioned above.
Sequence homologies between the GAPDHs of chloroplasts
und thermophilic bacteria
In Fig. 3 all sequences known to the present day are
aligned and compared to mustard cytosolic GAPDH, which
326
a -3
l a asp l y s lys0
1'
1.
2.
2'
Cytosol: clone pS198c
Chloroplast: clone pS84b
I'
1.
2.
i l e lys i l e g l y i l e asn g l y phe gly arg i l e g l y arg l e u val a l a arg Val i l e leu g l n arg asn asp Val
ATT AAG ATC GGA ATC AAC GGT TTC GGA AGA ATC GGT CGT TTG GTG GCT AGA GTT ATC CTT CAG AGG AAC GAT GTT
,GTTTCGAA
10
fi GCT
GAC AAG AAG
20
2'
1'
30
40
50
glu leu Val a l a Val asn asp pro phe i l e t h r t h r g l u t y r met t h r t y r met phe l y s t y r asp s e r Val h i s
21 .
GAG CTC GTC GCT GTT AAC GAT CCC TTC ATC ACC ACC GAG TAC ATG ACG TAC ATG TTT AAG TAT GAC AGT GTT CAT
2'
1.
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2.
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1.
2.
2'
53A
60
68A
70
gly gln t r p lys h i s asn glu leu l y s Val l y s asp glu l y s t h r leu leu phe gly glu l y s pro Val t h r Val phe g l y
GGT CAG TGG AAG CAC AAT GAG CTC AAG GTG AAG GAT GAG AAA ACA CTT CTC TTC GGA GAG AAG CCT GTC ACT GTT TTC GGC
80
90
100
i l e arg asn pro glu asp i l e pro t r p gly glu a l a gly a l a asp phe Val Val g l u s e r t h r gly Val phe t h r
ATC AGG AAC CCT GAG GAT ATC CCA TGG GGT GAG GCC GGA GCT GAC T T T GTT GTT GAG TCT ACT GGT GTC TTC ACT
,GTG
1'
1.
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138A
I'
1.
2.
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140
lys
--- ser asp leu asn
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AGC
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CAT GAA GAT ACC
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ser
-------
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300
a l a l y s a l a gly i l e a l a leu
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TCT TCT CTC ACA ATG GTT ATG
2.
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s e r s e r leu t h r met Val met
1'
21..
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2.
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330
t10
t20
+30
+40
t50
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i l e his met - - - s e r l y s a l a s t o p
ATT CAT ATG --- TCC AAG GCC TAA - - - AACGCTGAAG ATCTACAATG ATGTAATGGT GTCTTAATTT GTGGTTTTCG U A T T T
GAC ATT GTT GCC AAT AAC TGG AAG TGA AGTAAGACAC AACTTTTGAT GTCTTTTCTT TAACAGTTTT ATATATGATT CGGAATGTAG
asp i l e Val a l a asn asn t r p l y s stop
+m
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+110
+120
..
..
CTTTGGGA' ;cn
AATTGTAGfT CCCGAGTTTA TGTATTTGTG TTCTACAATT TTATAGTAAT AAACTTTATT CAAACA7Cn
Fig. 2. Coding sequences and deduced amino acid sequences of cDNAs for cytosolic ( 1 . 1') and chioropiast ( 2 , 2') GAPDH from mustard. The
numbering system used is that of Hams and Waters [SS]. Regions of amino acid homologies between the two enzymes are boxed. The initiation
codon ATG and the polyadenylation signals AATAAG and AATAAA are underlined. Both strands were sequenced according to Maxam and
Gilbert [44]
is the only sequence written in full (line 1). For the other
enzymes, including chloroplast GAPDH subunit A (sequence
12), only amino acids not identical to the reference sequence
are indicated, thereby creating distinct patterns of mutations
and homologies throughout taxonomic groups. Since recent
authors [23, 251 have used their own numbering systems, it
should be emphasized that in Fig. 3 the numerical order of
Harris and Waters [45] is used to permit comparisons with
most other papers throughout the literature. At the end of
the amino acid sequences in Fig. 3 the homologies between
glycolytic and chloroplast GAPDHs and relative to all other
sequences have been listed for the.polypetide 101 - 334. It can
327
be seen that the divergence between cytosolic GAPDH from
mustard and its photosynthetic counterpart (only 49% homology) is at least as large as between the GAPDH sequences
of eukaryotes and thermophilic bacteria in general (see also
Table 1). It may be argued that this difference reflects the
different metabolic functions of the two plant enzymes in
photosynthesis and glycolysis respectively. This, however, is
probably not the primary reason, because photosynthetic
GAPDH subunit A shows the highest homology, that is 64%,
with the enzyme from B. stearothermophilus, a non-photosynthetic moderate thermophile bacterium, while the two glycolytic enzymes from Bacillus and mustard share only 52% of
their residues (50% if the full-length sequences are compared,
see Table 1).
Among the 120 residues which are different for the two
mustard enzymes there are 47 positions (39%) where the
photosynthetic enzyme is identical with Bacillus GAPDH.
Among the 86 residues which are different for the photosynthetic and the Bacillus enzymes there are only 13 positions
(1 5%) where the photosynthetic GAPDH is identical with its
cytosolic counterpart. It appears, therefore, that chloroplast
and cytosolic GAPDHs from higher plants are orthologous
rather than paralogous proteins. Differences in orthologous
proteins reflect the phyletic divergence of different organisms,
while paralogous proteins originated by gene duplication and
subsequent divergent evolution in the same genome [50].
The data in Fig. 3 suggest that cytosolic and chloroplast
GAPDH belong to two separate GAPDH subfamilies, the
eukaryotic subfamily (sequences 1-9) and the Thermusl
Bucillus/chloroplast subfamily (sequences 10 - 12) respectively. The surprising fact that E. coli GAPDH (sequence 9)
belongs to the eukaryotic subfamily will be discussed below.
The carboxyterminal part of GAPDH, starting at position
101,contains 124 sites at which all sequences from eukaryotes
and E. coli are identical. In 46 out of these 124 positions one
or both thermobacterial sequences are mutated. Among these
46 ‘bacteria-specific’ mutation sites there are 30 (65%) which
are also mutated in chloroplast GAPDH, 20 of which (67%)
are homologous to one or both of the thermobacterial
sequences. In addition to this the sequences of chloroplast
and thermophilic bacteria also share an insertion Lys-l22A
and a deletion in the boxed S-loop region (Ser-189 for bacteria,
Pro-188 for chloroplast), insertion Lys-122A being also present in E. coli. There are only 9 mutation sites within sequence
101 - 334 which are unique for chloroplast GAPDH, that is
where all sequences except chloroplast GAPDH are identical
(Thrrmus: 12, Bacillus: 1). These positions are: 175
(Val-Thr), del-188 (Pro), 191 (Lys-tArg), 204 (Ile-Val),
228 (MetdIle), 246 (Leu-tVal), 265 (Gly-+Lys),273 (TyrwVal) and 294 (Ala+Ser).
The dichotomy of known GAPDHs into subfamilies is
particularly evident in amino acid positions 178- 201. These
residues comprise the so-called S-loop region of the catalytic
domain. The four S-loops form the core of the GAPDH
tetramer, most of their residues being internal and making
important interactions with the coenzyme and the other subunits [16, 171. Eukaryotes and thermophilic bacteria have very
different Sloop regions, which are highly conserved within
the respective subfamilies. It is remarkable to what extent
the ‘bacterial character’ of this functionally and structurally
important peptide has been conserved in the chloroplast
GAPDH (see boxed region 178- 200 in Fig. 3). However, two
of the changes unique for chloroplast GAPDH, del-188 (Pro)
and 191 (Lys-tArg), occur in this region. Since chloroplast
GAPDH is the only GAPDH which uses NADP as coenzyme
and which can form a heterotetramer AzB2 [38, 401, these
changes may be significant.
Fig. 3 also discloses some interesting features of the
cytosolic GAPDH from mustard. It can be clearly seen that
the NAD-binding domain (residues - 3 to 148) as a whole is
considerably less conserved than the catalytic domain (residues 149-331) as previously also shown for the E. coli
enzyme [28]. In Table 1 total homologies and domain
homologies for pairwise comparisons of all GAPDH sequences, are given. For example, the GAPDH sequences of
mustard cytosol and yeast are in total 68% homologous, while
the two domains share 54% and 80% of their residues. Similar
differences are found when the enzyme from mustard cytosol,
yeast or E. coli is compared with any of the other eukaryotic
sequences (values boxed by continuous lines), while differences in domain homologies are relatively moderate in comparisons between the non-vertebrate and vertebrate sequences
(values boxed by dashed lines) and in all comparisons involving the Bacillus enzyme. Differences are small or absent in
all comparisons involving T. aquaticus. Within the eukaryotic
subfamily the non-vertebrate enzymes from lobster and
Drosophila represent a notable exception to this rule of
differential domain conservation: the two enzymes share 78 ?Lo
and 75% sequence homology with respect to their NADbinding and catalytic domains. To summarize these
considerations one can conclude that divergent evolution of
the two domains has been especially strong along the lineages
leading to the enzymes of yeast, higher plants and E. coli.
The region most heavily altered in all GAPDH enzymes
so far investigated is the peptide 51 - 70 of cytosolic GAPDH
from mustard, which is part of an external loop of the NADbinding domain [16]. The two insertions Lys-53A and Glu68A occur in this extremely polar region, containing 9 charged
residues ( 5 lysines) in mustard and 3-8 in the other 10
sequences. Finally, as might be expected, the longest stretch
of fully conserved amino acids is found surrounding the catalytically active Cys-149. This homology block comprises residues 145- 156 and is identical in all GAPDH enzymes with
the exception of Cys-tSer in position 153 for T. aquaticus.
E. coli and the GAPDH sequence paradox
While the amino acid sequence of nucleus-encoded
chloroplast GAPDH subunit A is related to those of
thermophilic bacteria, E. coli has recently been shown to
contain a GAPDH which is more similar to the eukaryotic
enzymes [28]. The homology between E. coli GAPDH and
the eight eukaryotic sequences, especially with respect to the
catalytic domain (residues 149- 331), is indeed surprising (see
Fig. 3 and Table 1)and Branlant and Branlant [28]interpreted
this finding in terms of a divergent evolution of the
thermobacterial enzymes as a result of selection for stability
and catalytic efficiency of the enzymes in organisms growing
under high-temperature conditions. In view of the present
data and keeping in mind that higher plants are mesophilic
organisms, the data concerning E. coli GAPDH require reevaluation. Sequence similarities, especially those involving
hundreds of amino acids as in the present comparisons, probably reflect a common historical background rather than
adaptations to similar selection pressures [51]. We, therefore,
suggest the alternative possibility that the coding part of the
GAPDH gene found in E. coli represents, in fact, the descendant of a reverse-transcribed GAPDH mRNA of an
ancient and unknown eukaryotic host. There is ample evidence for a reverse-transcriptase-mediated pseudogene pro-
328
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Fig. 3
K
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V V
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A
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A E
A
K
A A E
P
P
P
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P Q
P
E M
Q A A
Q
H Q
Q
F
D E E
C
C
5
C
N S N S H
N D S T H
N S
T H
N
S
H
S
V
I
L S
T H
N
E V C T
V N A
V N A
V N A
G N M X X
D G K M
G
D M
L A A A E P
L
A A A E
E
F R D A A E K E
V F A
V
V I A
A
E I
A
S
E P L
D V C D E P L
A N
T
H
a
A
V E L V L R
G V
A A Y I f i A
G L
A D I V A N N W K
S
V
V
V
L Z E I
M
P H
T I
R N Y N
S T V
T I
V
R C S D V
Mustard M u s t a r d
31 0
320
330
cytosol chloropl.
S D N F V K L V S W Y D N E W G Y S T R V V D L I I H M - S K A
100% 49 %
P K
Y
Y E
I A A
77 %
50 %
N
I
Y
N
M A Y
A
E
73 %
49 %
N
H
I
F
N
M V
A
E
75 %
49 %
N
H
F
N
I
M A
A
E
74 %
49 %
N
H
F
N
MY
A
E
75 %
49 %
K T
V
F
1
L K
Q K V D S A
7 4 % 48 %
F
N
K
I
$
1
73
%
K Y
Q
D
41 %
N
T
N K
L
A
I K
7 4 5.
49 5.
A E
Y E
F A E
G
G
G
G
T
T
T
T
4 9 % 56 %
5 2 % 64 %
4 9 % 100 %
9
L T K
L S T M V I
S S L T M V M
( r e s . 101-334)
1. c y t . GAPDH
2. y e a s t
3. r a t
4.
5.
6.
7.
;:
pig
human
chicken
lobster
g o g l a
10. Thernus
1 1 . EXiTIGs
12. m
H
329
Table 1. Identities matrix of full-length G A P D H sequences showing the number of sites as the percentage occupied by identical amino acids in
pairwise comparisons (see Fig. 3)
Values above the diagonal: total homologies. Values below the diagonal: homologies between NAD-binding domains (‘numerators’, residues
- 3 to 148) and catalytic domains (‘denominators’, residues 149 - 334) respectively. Values boxed by continuous lines: cytosolic GAPDHs
from mustard, yeast and E. coli as compared to one another and relative to the animal sequences. Values boxed by dashed lines: GAPDHs
from lobster and Drosophila as compared to the vertebrate sequences. All values for the E. coli enzyme (except the comparisons of E. coli
with mustard, rat and Drosophila) were taken from [28]. Column 12 and ‘numerator‘ values in row 12 have been omitted because the sequence
of chloroplast GAPDH is not full-length. The identities matrix for the partial sequence 101 - 334 is shown at the bottom of Fig. 3
3.
4.
5.
6.
7.
8.
9.
10.
11.
1. M u s t a r d
c y t oso 1
66
67
67
67
68
66
64
45
50
2. Yeast
65
65
63
66
66
65
69
50
53
1.
3. Rat
4. P i g
2.
56
-
54
74
--
92
93
92
71
75
65
47
49
56
55
74
93
-
--
93
92
72
77
68
47
51
--
91
70
75
67
46
51
94
90
92
--
73
77
68
48
52
68
75
65
7 1 1 -74
75 I
76
64
48
52
--
66
48
53
52
55
55
53
52
54
7 F n i t T r n r a 7 - i - --
49
58
--
60
46
51 48
55
57
55
54
60
57
62
--
--
-55
-67
74
7r
92
5 . Human
91
92
51
56
-
76
74
93
95
6. Chicken
55
-
53
76
90
77
!n
89
-
7. L o b s t e r
60
75
56
71
r---I
69
172
--------
I
I
8. Drosophi 1a
9. E. c o l i
56
55
76
47
m
73
57
79
~
~
70
80
70
78
67
72 I 82
83
10. Thermus
42
45
46
-
48
48
55
48
11. B a c i l l u s
45
55
--49
57
45
49
46
55
56
12. Mustard
chloroplast
56
12.
46
46
82 ! 75
48
47
49
49
m
48
Fig. 3. Amino acid sequence alignment of 12 GAPDHproteins from 11 different organisms, as specified at the bottom of thefigure. Amino acid
sequences are aligned to maximize homology according to Hams and Waters [45]. All sequences are compared to cytosolic GAPDH of
mustard (sequence I), which is the only sequence written in full. For the other enzymes, including chloroplast GAPDH from mustard (sequence
12),only amino acids not identical to the reference sequence are indicated. The first and last residues of each sequence are indicated irrespective
of homology. Boxed sequence 178 -200 designates the S-loop region of bacterial GAPDHs. The two mustard enzymes (sequences 1 and 12)
have been compared with one another and relative to all other sequences with respect to residues 101 -334 and the homology values are
tabulated in two columns after the corresponding C termini (see also Table 1). Sources of sequence information: yeast [19], rat and Drosophila
[25], pig and lobster [451, human [23], chicken [21], E. coli [28], Thermus and Bacillus [18]
330
duction in mammals [52, 531. Therefore, if horizontal
gene transfer plays a role in evolution, as there are reasons to
believe (see above and [54, 55]), it cannot be seen why a
transfer from eukaryotes to bacteria should be a ‘forbidden’
route of information flow. A gene transfer from eukaryote
to prokaryote has previously been suggested to explain the
occurrence of the copper
zinc-containing, eukaryotic,
superoxide dismutase in Photobacter leiognathi, the bioluminescent bacterial symbiont of the ponyfish [56]. Interestingly superoxide dismutase is also the subject of a possible gene transfer in the reverse prokaryote-to-eukaryote
direction since the iron-containing, prokaryotic enzyme was
found in three higher plant families out of 43 investigated [57].
We do not want to carry this discussion any further at
the present time and would rather wait until more GAPDH
sequence information from bacteria, especially cyanobacteria,
the free-living descendants of the presumptive ‘ancestors’ of
chloroplasts (see [7 - lo]), is available and until the structure
of the GAPDH gene from chloroplasts has been elucidated.
For the time being we believe that our data are compatible
with the endosymbiotic theory of chloroplast evolution and
in particular with the gene-transfer concept, mentioned above
as hypothesis (b). Since the glycolytic GAPDH of B. stearothermophilus is more closely related to the photosynthetic than
to the glycolytic enzyme of higher plants it may finally be
hypothesized that the photosynthetic enzyme (subunit A)
diverged from the glycolytic enzyme of eubacteria long after
the separation of the eubacterial and eukaryotic lineages.
Since the Calvin cycle originated more than two billion years
ago [58] this separation would have occurred very early in the
history of life, in agreement with modern concepts of cell
evolution [59, 601.
+
We thank H. Sommer (Max-Planck-Institut fur Zuchtungsforschung, Cologne) for teaching us how to clone and sequence; H. H.
Arnold (University of Hamburg) for the gift of the GAPDH clone
from chicken; J. Collins, H. J. Hauser, H. Blocker and colleagues
(Gesellschaft fur Biotechnologische Forschung, Braunschweig) for restriction enzymes, radioisotopes and for the use of the computer;
Yvette Guyenon for her help in the preparation of the manuscript.
The financial support of the Deutsche Forschungsgemeinschaft (Ce 1/
11-3) is gratefully acknowledged.
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