THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 266,No. 35, Issue of Decemher 15, pp. 24044-24047,1991
Printed in U.S.A.
Glucose Increases the Expression of the ATP/ADP Translocator and
the Glyceraldehyde-3-phosphate Dehydrogenase Genesin Chlorella*
(Received for publication, April 29, 1991)
Christian Hilgarth, Norbert Sauer, and
Widmar Tanner
From the Lehrstuhl fur Zellbiologie und Pflanzenphysiologie, Universitat Regensburg, Universitatsstrosse 31,
W-8400 Regensburg, Germany
A presumably full-length cDNA clone of the mito- of glucose-treated Chlorella cells was hybridized to radiolachondrial ATP/ADP translocator (AAT) of Chlorella beled total cDNA from glucose-treated or untreated control
expres- cells, a clone coding for the glucose transporter was isolated,
kessleri has been isolated and sequenced. The
sion of theAAT gene is highly increased in the presence
sequenced (6),and itsfunction proven by heterologous expresof D-glucose (14 mM).At least nine more genes are sion in Schizosaccharomycespombe (7). However, several more
activated when autotrophically grown Chlorella cells genes were found to be expressed to a much higher degree or
switch to heterotrophic growth. Among these is the even exclusively after glucose treatment. One of these was the
HUPl gene coding for the hexose transporter (Sauer, mitochondrial ATP/ADP translocator gene (AAT), and anN., Caspari, T., Klebl, F., and Tanner,W. (1990)Proc.
Natl. Acad. Sci. U.S. A. 87, 7949-7952) and, as also other one was the cytoplasmic glyceraldehyde-3-phosphate
dehydrogenase gene (GAPDH).’ A full-length cDNA clone of
shown in this paper, the glyceraldehyde-3-phosphate
the AAT gene has been sequenced and compared with the
dehydrogenase(GAPDH)gene.When
glucose or the
nonmetabolizable analogue 6-deoxyglucose
is added to coresponding sequence of maize, the only AAT plant sequence
the cells, an increased expression of GAPDH or AAT known so far. The regulation of expression of both the AAT
is observed after 10 or 30 min, respectively. Hexose and theGAPDH gene has been studied.
uptake mutants (HUP1-) do not respond to sugars in
MATERIALS AND METHODS
this way, which indicates that either the inducer has
to be internalized or that the
HUPl translocator is part
Plant Material-The strain of C. kessleri and the conditions for
of the signal transduction mechanism.
autotrophic culture were described previously (8, 9).
Although carbon autotrophy is the most typical feature of
plant cells, they do have the potential to use and live on
organic carbon sources. This is known for a great number of
algae (l), but it is also the case with all higher plants. Plants
can be considered as physiological mosaics with their nongreen tissues like those of roots, stems, and flowers leading a
more or less completely carbon heterotrophic life. The fact
that either of these two opposing metabolic pathways may
dominate within a plant cell raises questions on the responsible regulatory mechanisms. Although it is known that light
is the major regulating signal for the expression of various
genes required to be active in photosynthesis (2), little is
known at the molecular level on how cells, even in the presence of light, turn to heterotrophy. Certainly sugars play an
important role in preparing cells for this switch by increasing
the expression of certain genes and decreasing that of others
(3).
The unicellular green alga Chlorella kessleri is able to grow
both photosynthetically and on glucose in the dark. Addition
ofglucose or nonmetabolizable glucose analogues tothe
growth medium of C. kessleri leads to the rapid induction of
a hexose transporter (4) and toa strongincrease in theactivity
of two amino acid uptake systems (5). When a cDNA library
* This work wassupported by funds from the Deutsche Forschungsgemeinschaft (SFB 43) and from the Fonds der Chemischen Industrie.
The costs of publication of this article were defrayed in part by the
payment of page charges. This articlemust therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
The nucleotide sequence(s)reported in thispaper has been submitted
to the GenBankTM/EMBL Data Bank withaccessionnumber(s)
M76669.
Induction-Out of the autotrophically grown culture a sample was
shock-frozen (control-autotroph). The rest was harvested, washed
with distilled water, and preincubated at a cell density of 100 pl of
packed cells/ml for 10 min at 27 “C in 25 mM sodium phosphate
buffer, pH 6.0. The total time in the dark before sugar was added
amounted to 40 min. Then glucose or 6-deoxy-D-glucose wereadded
(14 mM); to a control sample an equal amount of buffer was added.
The cells were subsequently shaken in the dark for the times indicated. For RNA time course experiments the sugar concentration was
held constant throughout the experiment by adding, every 15 min,
the amount of glucose used up from a concentrated stock solution.
The induction of hexose uptake was tested as described previously
(10).
Northern Blot Analysis-Cells for the Northern time course were
shock-frozen immediately at thetime points indicated. Total cellular
RNA wasisolated as described by Bell et al. (11). 1.2% formaldehydeagarose gels were prepared according to Maniatis et al. (12), 25 pg of
total RNA were loadedper lane. Northern blots were probed with 3’specific cDNA fragments of the corresponding genes.
The conditions for all hybridizations were 50% formamide, 0.1%
sodium dodecyl sulfate, 2 X SSC, 1 X Denhardt’s, 100 pg/ml salmon
sperm DNA, 42 “C (12). Filters were washed at a maximal stringency
of42 “C with 0.1 X SSC, 0.1% sodium dodecyl sulfate. Following
autoradiography mRNA was quantitated by scanning densitometry.
Screening of a cDNA Library for AATIGAPDH-A X g t l O cDNA
library from glucose-treated C. kessleri cells (6) was screened differentially to obtain glucose-inducedtranscripts. After comparison with
available data libraries, the clones pTF47 and pTF35 were identified
as GAPDH and AAT fragments, respectively. pTF47 exhibited 82.0%
identical amino acids as compared with the respective fragment of
the GAPDH from Drosophila, and pTF35 had 32.7% idential amino
acids as compared with the respective fragment of the AAT sequence
from maize (data not shown).The library was rescreened with pTF35,
coding only for the 3’-end of the AAT, to find a full-length cDNA
clone. Out of 50 positive clones, pCHCll with a 1636-base pair insert
was the longest AAT cDNA clone obtained. To get the full cDNA
The abbreviation used is: GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
24044
Increased Expressionof ATPIADP Translocator andGAPDH Gene
24045
gene (16-18). The AAC2 gene of S. cerevisiae is the main gene
of this family in yeast; the degree of identity as compared
with the other two yeast genes is not significantly different
(19).
Control of AAT Gene Expression-There is a detectable
level of constitutive AAT mRNA in autotrophically grown
RESULTS
Chlorella cells (Fig.3A). This level is about 10-fold higher
TheAAT cDNA Sequence of Chorella-The protein se- after a 60-min treatment of the cells with D-ghcose. Whereas
quence predicted from the presumably full-length cDNA clone the gene of the hexose carrier (HUP1) is equally well expCHCll is 339 amino acids in length and is shown in Fig. 1. pressed when cells were treated with glucose or the nonmeOf the two ATG codons at theN terminus, the first one has tabolizable sugar analogue 6-deoxy-D-glucose, the AAT
been assumed to be used as the start codon, since in more mRNA level is only increased 2-3-fold with 6-deoxy-~-glucose
than 90% of 79 plant mRNAs tested (14) this hasbeen shown (Fig. 3A). A detailed time course of induction by glucose is
to be the case. In thiscase, however, the Chlorella AATprotein shown in Fig. 4; 30 min after glucose had been added to the
has a much longer N terminus than other ATP/ADP trans- cells a clear response can be noticed. A new maximal steady
locators (Fig. 2). On the other handSaccharomyces cereoisiae state of AAT mRNA is reached after about 60 min. A control
cells possess three ATP/ADP translocator genes and the N gene (pTF30)not changing in expression (6) shows that
termini of the corresponding gene products differ by about 40 comparable amounts of RNA have been blotted.
Control of GAPDH Gene Expression-OnecDNA
clone
amino acids. With either ATG start site the deduced protein
obtained in the differential screening showed high similarity
does not possess a typical signal sequence.
The cDNA sequence contains the predicted algal polyade- to published GAPDH sequences. The Chlorella gene possessed
nylation signal TGTAA (15). Two cDNA clones showed an the highest percentage of identical amino acids (>55%) with
11 base pair distance between the TGTAA polyadenylation the cytoplasmic GAPDHs of tobacco, corn, and mustard (20signal and the poly(A) tail, whereas in the full-length clone 22). When the expression of the corresponding Chlorella gene
pCHCl1 the poly(A) tail starts immmediately behind the was studied with a 3'-specific probe in glucose treated and
TGTAA (Fig. 1,arrow). pCHCl1 is assumed to be full length control cells, the dataof Figs. 3A and 4 were obtained. Again
due to thehomology of the position of the startATG in this a strong increase in expression due to glucose treatment was
clone and other AAT clones (Fig. 2).
found. Control cells were incubated under identical conditions
The amino acid sequence of the Chlorella gene possesses a but without glucose. The increase in the amountof GAPDHhigh degree of identity (Fig. 2) with the AAC2 gene of S. mRNA from photosynthetically grown cells to cells at "time
cereukiae (48.7%),the gene of Zea mays (50.7%),and surpris- 0" is due to the anaerobiosis plus darktreatmentduring
ingly the highest degree of identity (64.9%) with the bovine harvesting (40 min). Whereas this increased level of mRNA
sequence of the 5'-end, 36 base pairs from another clone (pCHC16)
were added (Fig. 1).
Nucleic Acid Sequencing-Sequencing was performed according to
Sanger et al. 1977(13). Bal-31-nuclease deletions provided a complete
sequence of pCHC11. For sequence analysis we used the software of
the University of Wisconsin Genetics Computer Group.
12
15
79
112
FIG. 1. cDNA and deduced amino
acid sequenceof the AAT-gene.The
discussed start points for translation
and
the polyadenylation signal for green algae (15) are underlined. The presented
sequence was composed from three individual cDNA clones: the full-length
clone pCHC 11, the clone with the longest 5'-untranslated end (pCHClG), and
the clone with the longest 3"untranslated end (pTF35). The arrow marks the
start of the poly(A) tail in pCHC11.
I15
179
212
a15
279
312
of ATPIADP Translocator
and
GAPDH
Increased
Expression
24046
50
1
Consensus
Chlorella k.
Zea mays
saccharomyces
8 0 s bovis
.......... ..............................
.......... ..........
.......... ......
.......... ........................
F..D...GC.
MLSSALYQQA CLSCLLRASA UGPQTPFIAS PKETQADP?IA FVKDLLAGGT
..MQTPLCAN APAEKCG.KN FMIDFXUCGV
MSSN AQVKTPLPPA PAPKKES..N FLIDFLUCCV
SDQALS FLKDFLAGGV
51
Consensus
Chlorella k.
Zea mays
Saccharomyces
80s bovis
1
..A..KTA..
ACAISKTAVA
SMVSKTlUA
SAAVAKTAAS
AAAISKTAVA
P1ERVKLL.Q
PIERVKLLLQ
PIERVKLLIQ
PIERVKLLIQ
PIERVKLLLQ
1
.Q
100
......K. ......Y.Gl ..C..R....
TQDSNPMIKS GQV.PRYTG1 VNCFVRVSSE
NQDE..UIKS GRLSEPYKGI VDCFKRTIKD
NQDE..MLKQ GTLDRKYACI LDCFKRTATQ
VQHA.S..KQ ISAEKQYXGI IDCWRIPKE
150
.G..S..RG. .ANV.RYFPT QA.NFAFKD. .K..F.....
F.
Consensus
QGVASFWRCN LANWRYFPT QAFNFAFKDT IKCLFP.KYS PKTDFWRFFV
Chlorella k.
ECFSSLWRGY TANVIRYFPT QALNFAFKDY FKRLFNFKKD RD.CYWKWFA
Zea mays
saccharomyces ECVISFWRGN TANVIRYFPT QALNFAFKDK IKAMFC..FK KEECYAKWFA
8 0 s bov1s
QGFLSFXRGNLANVIRYFPTQALNFAFKDKYKQIFZCVDRHKQFXRYFA
101
Control
1
Glc-Induction
n
0.
g
-2
3
0 30 60 120 rnin
0 2.5 5 103OM90 120rnin
***-
AAT
om
GAPDH
........
151
Gene
-
200
....
Consensus
.NLASGC.AG A.SL..VY.L
D.ARTRLA..
K....R.F.GL.....
VNLASCGLAC AGSLLIVYPL DFARTRLAAD VGSGKS...R EFTCLVDCLS
Chlorella k.
ZeamaysGNLASCCAACASSLFFVYSLDYARTRLANDAKAAKGGGERQFNCLVDVYR
Saccharomyces CNLASGGMC ALSLLFVYSL DYARTRLAAD SKSSKKGCAR QFNGLIDVYK
8 0 s bovis
GNLASGCAACATSLCFVYPLDFARTRLA..ADVCKGAAQREFTGLGNCIT
201
250
K..K..G... LY.GF..S..
GI..YR..YF C.YD..K...
Consensus
Chlorella k.
KWKRGCPUA LYQGFGVSVQ GIIVYRCAYF CLYDTAKGVL FKDERTANFF
Zea maysKTLKSDCIAGLYRCFNISCVCIIVYRCLYF
G L Y D S I K P WL T G N M D N F F
saccharomyces KTLKSDGVAG LYRCFLPSW GlWYRCLYF GUYDSLKPLL LTGSLEGSFL
Ros bovls
KIFKSDGLRGLYOGFNVSVQCIIIYRAAYFGVYDTAKC.M
LPDI’KNVHII
..........
25 I
300
....
......... ..
T
A...SYP. DTVRRR.UM. S...C....Y
D.....
Consensus
chlorella k.
AKWAVAQAVT ACACVLSYPF DTVRRRLnnQ S...GGERQY NGTIDCWRKV
ASFALLWLIT NGACLASYPI DTVRRRMUMT S...GEAVKY KSSLDAFQQI
Zes mays
Saccharomyces ASFLICWWT TGASTCSYPL DTVRRRHMHT S. ..GQAVKY DCAFDCLRKI
0 0 s bovls
V R W I A Q T V T AVACI.VSYPF DTVRHUHHUQSCRKGADIMYTGTVDCWRKI
301
...
..
347
................
EG....F KC...N.LR.
GA.V...Y D
Consensus
Chlorella k.
AQQECUKAFF KCAWSNVLRC ACGAFVLVLY DEIKKFINPN AVSSASE
Zea mays
LKKECPKSLF KGACANILRA IAGAGVLSGY DQLQILFFGK KYCSCCA
Saccharomycas VAAEGVGSLF KGCGANILRG VAGAGVISUY DQLQMILFCK
KFK....
80s bovis
AKDECPKAFFKGAWSNVLRGMGGAFVLVLYDEIKKFV..
........
FIG. 2. Comparison of the amino acid sequences
of the AAT.
The translated protein sequence of the Chlordla AAT is more than
48% identical with those of higher plants (17), yeast (16), and mammals (18). Marked are the conserved lysines of the binding site
for
adenine nucleotides (17, 30) and the cystein residue responsible for
total activity (17, 31). The AAC2 gene from the AAT family of S.
cereuisiae has been taken for comparison (19).
Control
FIG. 4. Time course of increased expression caused by glucose. Northern blot analysis of total RNA
( 2 5 pg/lane) from rclls
grown photoautotrophically in inorganic medium and light/C02 atmosphere (control-autotroph), incubated in the dark without (control) and with glucose (Clc-Induction). The control gene (pTF.70)of
unknownfunctionhas
been shown previously not to change in
expression when cells are treated with sugars ( 6 ) .
deoxyglucose give rise to internal concentrations of20mM.
In wild type cells this concentration suffices for induction of
two sugar inducible amino acid uptake systems (5). whereas
it fails to induce these amino acid uptake systems in HUPcells. This finding indicates that besides the inducing sugar
an intact transporter may have to be present for induction
also (3, 23).
DISCUSSION
The addition of glucose to autotrophicallygrown C. kpssleri
cells gives rise to a highly increased expression of a number
of genes. This has now definitely been shown for HUP1, the
glucose transporter gene (6), the mitochondrial ATP/ADP
translocator, and the cytoplasmic GAPDH gene. From previous results ( 5 ) this seems also highly likely for the arginine
transporter (substrates are Arg, Lys, His) and the proline
transporter (substrates are Pro, Ala, Gly, Ser). Furthermore,
.)(I).)
HUP
additional clones for five different transcripts were obtained
so far by the screening procedure outlinedabove, which also
showa markedlyincreasedexpression
in thepresence of
glucose;
these
clones
have
not
been
identified
asyet. Thus, at
@
GAPDH
Y
Chlorella
least 10 genes are activated when an autotrophic
FIG.3. A, expression of glucose-regulated genes in C. kessleri wild cell switches to heterotrophic growth and glucose seems tobe
type. Northern blot analysis af total RNA (25 pgllane) probed with the key signal even for an increased uptake
of organic nitrogen
9”specific cDNA fragments. RNA extracted from cells incubated in
( 5 ) . The 10-fold increase in the expression of the A A T gene
the dark without sugar ( K , , &), with glucose for 1 h (Clc,,,,) or 6may be related to anincreased demand of mitochondrial ATP
deoxyglucose for 1 (6DC6J or 2 h (6DG,,,). R, lack ofincreased
most
C. k s s k r i mutant HUP-. when C-heterotrophy gets started and photosynthesis
expression of glucose-regulated genes in the
likely becomes less dominant. That the changein expression
Northern blot analysis of poly(A) RNA (5 pg/lane). The blots show
a comparison ofRNAsisolated
from HUP- mutantcells atthe
isnotcaused by the“lightoff‘
signal but indeed by the
and 60 min later (Glcw).
moment of glucose addition(KO)
presence of the sugaris clearly seen in the corresponding dark
control (Fig. 4).Similarlytheincreased
expression of the
disappears completely after 60-120 min in thecontrol, a GAPDH gene reflects the increased rate of glycolysis under
strong positive response due to the presence of glucose is C-heterotrophic conditions (24). Thus, a monosaccharide or
observed within 10 min after the addition of sugar and this even a nonmetabolizable sugar analogue serves as signal for
a considerable change in cellular metabolism. Genes coding
high level of mRNA is maintained throughout the experiment.
for proteins which are responsible for key reactions of subThe Glucose Respome Requires an Intact HUPl Gene-A
Chlorella mutant selectedfor resistanceagainstthetoxic
strate supply (HUP), the flow-through glycolysis (GAPDH),
and energy supply (AAT) are
positively regulated in this way.
glucose/mannose analogue 2-deoxyglucose (23)hasmost
likely a defectiveH U P l gene (6). When this mutant
is treated
A 20-fold increase caused by sucrose in the mRNA of a
with glucose the increase in expression of the AAT geneor of cytosolic GAPDH has also been reported fortobacco (25).
the GAPDHgene cannot be observed (Fig.3 R ) . Although this and a positive anaerobiosis effect on expression of the same
may indicate that the sugar has to be internalized to act as gene has been previously observed forcorn (26).The fact that
inducer,anotherexplanationismademore
likely by the the corresponding gene in Chlorella is regulated in an analoprevious observation (23) that HUP- cells flooded with 6- gous way (Fig. 4) encourages theview that Chlnwlla can serve
Increased Expression of ATPIADP Translocator and GAPDH Gene
as a model to study regulatory events in plantcells during the
switch from C-autotrophic to C-heterotrophic growth.
It has long been known that the addition of glucose to
Chlorella cells also inhibits a number of photosynthetic reactions (glucose bleaching(27)). These observations with Chlorella and the corresponding classicwork carried out with
Euglena (28) is furthermore supported by the recent finding
that in corn mesophyll cells the expression of seven genes
responsible for photosynthetic reactions is repressed by glucose (29).
The fact that the nonmetabolizable 6-deoxygIucoseas well
as glucose act as inducer for three genes described herein
implicates the sugar itself and not some metabolite of it as
the primary signal. It is true, however, that atleast for AAT
and GAPDH expression, glucose definitely ismore potent
(Fig. 3A); this suggests an additional signal, which may be
derived from glucose metabolism.
The results obtained with the HUP- mutants suggest that
either the hexoses and non-metabolizable analogues have to
be taken up to be active as inducer or thatthe glucose
transporter protein in the plasmamembrane may be part of a
signal transduction pathway. Evidence for the second possibility has beendiscussedin the result section as well as
elsewhere (3, 23).
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