Because of photorespiratory metabolism, leaf mitochondria contain much more GDC than root Keywords alternative splicing; glycine decarboxylase; H-protein; photorespiration Correspondence
Trang 1substrate properties for the P-protein of glycine
decarboxylase
Dirk Hasse1, Stefan Mikkat2, Martin Hagemann1and Hermann Bauwe1
1 Department of Plant Physiology, University of Rostock, Germany
2 Core Facility Proteome Analysis, Medical Faculty, University of Rostock, Germany
Introduction
Several thousand plant genes are known to produce
multiple transcripts, but the precise function of most
of the alternatively encoded proteins is not known [1]
Alternative splicing has also been observed for
H-pro-tein, a component of the ubiquitous multi-enzyme
sys-tem glycine decarboxylase (GDC, EC 1.4.4.2) [2,3]
GDC is essential for photorespiratory and one-carbon
metabolism, and the vital function of this enzyme
complex is indicated by the fact that its inactivation is
fatal to plants [4] and animals [5]
The GDC H-protein (GLDH) has no catalytic
activ-ity itself but interacts via its lipoyl arm one after the
other with the three other GDC subunits, P-, T- and
L-protein As a result of the GDC reaction cycle,
which requires the coenzymes NAD+ and
tetrahydro-folate, methylene-tetrahydrofolate and NADH are generated and CO2and NH3are released [6]
Plant H-proteins are often encoded by small multi-gene families, and the individual H-proteins suppos-edly fulfil various functions in plant metabolism [7–9] Some plants, however, harbour only one H-protein gene, and we have previously reported that C4 species
of the genus Flaveria produce two H-proteins in an organ-dependent manner by alternative 3¢ splice site selection [2,10] The alternatively encoded H-protein, FtGLDHAA, harbours two additional alanine residues very close to its N-terminus, and is by far the most dominant H-protein type in leaf mitochondria of these plants Because of photorespiratory metabolism, leaf mitochondria contain much more GDC than root
Keywords
alternative splicing; glycine decarboxylase;
H-protein; photorespiration
Correspondence
H Bauwe, Department of Plant Physiology,
University of Rostock,
Albert-Einstein-Straße 3, D-18059 Rostock, Germany
Fax: +49 381 498 6112
Tel: +49 381 498 6110
E-mail: hermann.bauwe@uni-rostock.de
Website: http://www.biologie.uni-rostock.de/
pflanzenphysiologie
(Received 7 July 2009, revised 5 September
2009, accepted 25 September 2009)
doi:10.1111/j.1742-4658.2009.07406.x
Several thousand plant genes are known to produce multiple transcripts, but the precise function of most of the alternatively encoded proteins is not known Alternative splicing has been reported for the H-protein subunit of glycine decarboxylase in the genus Flaveria H-protein has no catalytic activity itself but is a substrate of the three enzymatically active subunits, P-, T- and L-protein In C4 species of Flaveria, two H-proteins originate from single genes in an organ-dependent manner Here, we report on differ-ences between the two alternative H-protein variants with respect to their interaction with the glycine-decarboxylating subunit, P-protein Steady-state kinetic analyses of the alternative Flaveria H-proteins and artificially pro-duced ‘alternative’ Arabidopsis H-proteins, using either pea mitochondrial matrix extracts or recombinant cyanobacterial P-protein, consistently dem-onstrate that the alternative insertion of two alanine residues at the N-ter-minus of the H-protein elevates the activity of P-protein by 20% in vitro, and could promote glycine decarboxylase activity in vivo
Abbreviations
GDC, glycine decarboxylase; GLDH, GDC H-protein; GLDH AA, alternative H-protein; LplA, lipoate–protein ligase.
Trang 2mitochondria [11,12], but the relevance of alternative
splicing for cellular metabolism in these organs is not
known An inspection of published structural data
[13,14] revealed that the N-terminus of the H-protein
is close to its lipoyl arm (Fig S1) Hence, structural
alterations to this region could possibly provide GDC
variants that are fine-tuned for different metabolic
environments
In this study, we examined whether the changes to
the N-terminus of Flaveria H-protein alter its reactivity
with P-protein, the actual glycine-decarboxylating
sub-unit of GDC These experiments revealed that
P-pro-tein showed significantly higher activity with the
alternative H-protein than with the normal H-protein
This was also found to occur with P-protein from
Pisum sativum (pea) leaf mitochondrial matrix extracts
as a source of native plant P-protein and with
recom-binant P-protein of the cyanobacterium Synechocystis
sp strain PCC 6803 (referred to simply as
Synecho-cystis below) Further support came from experiments
with an engineered ‘alternative’ H-protein from
Ara-bidopsis thaliana, a C3 plant This artificially modified
H-protein also showed higher activity with P-protein
in comparison with the native H-protein Hence, both
natural and artificial N-terminal insertion of two
ala-nyl residues result in higher P-protein activities in vitro,
and could increase GDC activity in vivo
Results and Discussion
The two alternative H-proteins from the C4 plant
Flaveria trinervia, FtGLDH and FtGLDHAA, were
overexpressed in Escherichia coli and obtained as
apparently pure recombinant proteins (Fig 1) To be
functional, H-protein must be lipoylated at a specific
lysine residue [12,15] While complete lipoylation can
be achieved by the addition of lipoic acid to the E coli
growth medium in some expression systems [16], we
observed that lipoylation of H-protein was still
incom-plete in our system (Fig S2) Therefore, all
recombi-nant H-proteins used in our study were treated with
E coli lipoate–protein ligase A (LplA, EC 2.7.7.63)
[17] Complete lipoylation was verified by native
poly-acrylamide electrophoresis in combination with use of
lipoate-specific antibodies, and further confirmed by
MALDI-TOF MS (Fig 2)
Leaf mitochondria contain large amounts of all
GDC subunits and provide a convenient source of
native P-protein Under in vitro assay conditions,
P-protein dissociates from the other GDC subunits
and its activity becomes sensitive to the addition of
extra H-protein [18,19] Likewise, addition of 50 lm
recombinant FtGLDH to assays containing matrix
extracts prepared from purified pea leaf mitochondria resulted in an approximately 50-fold stimulation of the glycine–bicarbonate exchange rate [20] relative to the rate measured in the absence of exogenous H-protein (Table 1) Similar to previously reported data [21,22], the glycine saturation kinetics were hyperbolic, with a
Kmof 6 mm (Fig S3)
We next examined the reactivity of the two F triner-viaH-proteins in this system (Table 1) H-protein satu-ration followed Michaelis–Menten kinetics for both FtGLDH and FtGLDHAA The Km values for H-pro-tein were between 16 and 20 lm, and hence somewhat larger than published values for H-proteins from other sources [18,23] This difference could be due, at least
in part, to the small amounts of pea H-protein intro-duced into the assay with the mitochondrial extracts Notably, the Vmax values were significantly higher with FtGLDHAA than they were with FtGLDH, indicating that alternative splicing could produce a more efficient H-protein This possibility was examined in a more precisely defined assay using recombinant cyanobacte-rial P-protein
As eukaryotic P-proteins cannot yet be produced by recombinant DNA technology, we used the P-protein from the cyanobacterium Synechocystis, which is structurally very similar to eukaryotic P-proteins Moreover, recombinant Synechocystis P-protein is enzymatically active and can be produced in large enough quantities (lane 6 in Fig 1) The enzymatic activities obtained with recombinant plant H-proteins
as substrates were in a similar range to those measured with Synechocystis H-protein, which served as an inter-nal control (Table 1) This extends an earlier report
on the use of chicken H-protein as a substrate for the P-protein from Arthrobacter globiformis [3,24] In
Fig 1 SDS–PAGE with recombinant H-proteins from Flave-ria trinervia, Arabidopsis thaliana and Synechocystis, and recombi-nant P-protein from Synechocystis M, size markers (kDa); lane 1, FtGLDH; lane 2, FtGLDH AA ; lane 3, AtGLDH1; lane 4, AtGLDH1 AA ; lane 5, Synechocystis H-protein; lane 6, Synechocystis P-protein.
Trang 3comparison with Synechocystis H-protein, the plant
H-proteins showed lower affinities for the prokaryotic
P-protein; however, similar to our experiments with
mitochondrial matrix extracts, the P-protein activity
with saturating FtGLDHAA was about 20% higher
than that with saturating normal FtGLDH
In order to determine whether this finding can be
generalized beyond Flaveria H-proteins, we engineered
a splice variant of the H-protein AtGLDH1 from the
C3 plant A thaliana (AtGLDH1AA) This artificial
‘alternative’ H-protein differs from the naturally occur-ring Arabidopsis H-protein only by the insertion of two additional alanyl residues near the N-terminus Kinetic analysis of the these two H-proteins fully con-firmed our findings with the two Flaveria H-proteins, i.e the AtGLDH1AA-saturated specific reaction rate was significantly higher than that with the normal AtGLDH1 (Table 1) This corroborated our view that the alternatively spliced Flaveria H-protein is a better substrate for P-protein, and indicated that this improvement is exclusively caused by a slight modifica-tion to its N-terminus
It should be noted that our method for producing recombinant H-proteins leaves a small extension of four amino acid residues at the N-terminus of each recombinant protein after removal of the His tag (Fig S4) The kinetic differences between the normal and alternative H-proteins, either from Flaveria or from Arabidopsis, were very consistent Nonetheless, to fully exclude the possibility that this artificial extension could bias our results, all four plant H-proteins were re-cloned into the tagless expression vector pET-3a The biochemical parameters obtained with these tagless H-proteins were almost identical to those for the respective thrombin-cleaved H-proteins (Table 1) This confirmed that the higher Vmax values measured
Fig 2 Correct structure of H-proteins was confirmed by
MALDI-TOF MS, with differences between theoretical and measured
molecular masses always below 1 Da The signal at m ⁄ z 14 230
corresponds to the singly charged ion of lipoylated mature
Flave-ria trinervia H-protein GLDH (mass shift of 188 Da relative to
unli-poylated H-protein) H-protein FtGLDH AA shows the expected mass
increase of 142 Da, which is due to the insertion of two extra alanyl
residues Lipoylated mature Arabidopsis thaliana H-protein GLDH1
also shows the expected signal at m ⁄ z 14 417 Da The Arabidopsis
H-protein variant AtGLDH1 AA was produced by mutagenesis, and
the two extra alanyl residues result in the calculated mass increase.
Small satellite peaks and shoulders are artefacts that originated
during sample preparation or the desorption ⁄ ionization process.
Table 1 Kinetic parameters for Pisum sativum and Synechocystis P-protein with H-protein from Flaveria trinervia, Arabidopsis thaliana
or Synechocystis as substrate H-proteins carrying an N-terminal extension of four amino acids, which remain after thrombin cleav-age of the His tag, are labelled ‘+4’ Values are means ± SE from three (mitochondrial matrix P-protein) or four (purified Synechocys-tis P-protein) independent experiments.
Km (H-protein) (l M )
Vmax (nmolÆCO 2 Æmg)1Æmin)1)
Mitochondrial P-protein
Synechocystis P-protein
Trang 4with the alternative H-proteins, both the naturally
occuring FtGLDHAA and the artificial AtGLDH1AA,
are indeed exclusively due to the insertion of two
additional alanyl residues into the N-terminus of the
H-protein
In conclusion, the substrate properties of the
alter-natively encoded Flaveria H-protein FtGLDHAAin the
P-protein reaction of GDC differ significantly from
those of the normal Flaveria H-protein This was
found in two independent assay systems, using either
mitochondrial matrix extracts or recombinant
cyano-bacterial P-protein It was further substantiated by
analysis of an engineered ‘alternative’ Arabidopsis
H-protein Hence, several lines of independent
experi-mental evidence consistently demonstrated that
alternative 3¢ splice site selection results in a more
efficient H-protein
As fas as the physiological relevance of this effect is
concerned, several other important variables are still
unknown and require further research From a
methodical point of view, the GDC concentration is
extremely high in leaf mitochondria but very much
lower in mitochondria from heterotrophic tissue
Although root mitochondria have not yet been
exam-ined for this feature, H-protein is present at levels of
approximately 1 mm in the matrix of pea leaf
mito-chondria, which is 7-fold higher than the concentration
of P-protein [12] and 40-fold higher then the Kmvalues
determined in vitro This could indicate that H-protein
is possibly saturating in the leaves of C3 plants, but
unfortunately in vitro assays are very difficult to
per-form at such high protein concentrations Our assay
conditions may be closer to the situation in C4 leaf
mitochondria, which contain distinctly less GDC, and
even closer to that of root mitochondria
Furthermore, it is not known whether alternative
splicing of H-protein pre-mRNA is a particular feature
of Flaveria C4 species or also occurs in other C4
plants The genomes of maize (Zea mays, http://www
maizesequence.org) and Sorghum bicolor [25] do not
indicate the presence of alternative H-proteins in these
monocot C4 plants, and sequence data for other
dicot-yledonous C4 plants are not yet available Our data
show that the alternative H-protein enhances P-protein
activity by about 20% in vitro Although this is not a
very large effect, it could have significance for both C3
and C4 plants in vivo For C3 plants, this may be
assumed from effects on photosynthesis observed after
antisense suppression of P-protein in potato, which
indicated co-limitation of leaf metabolism by GDC
activity [26] In C4plants, the photorespiratory carbon
flux is low, but nevertheless essential [27] C4 plants
hence require less GDC than C3 plants However, the
photorespiratory flux can considerably increase under conditions of low water supply and high temperature [28] Under such conditions, which occur intermittently
or may even prevail in many habitats typical for C4 plants, the use of alternative H-protein could counter-act limitations to photosynthetic⁄ photorespiratory metabolism Possible enhancement effects on T- and L-protein remain to be examined, but could add to a larger overall activity enhancement
Finally, alternative splicing of H-protein pre-mRNA
is regulated organ-dependently in C4 Flaveria species [2] It appears that C4 plants very often harbor only one H-protein gene (Zea mays, C4 Flaveria) or two H-protein genes (Sorghum bicolor), whereas up to four H-protein genes occur in C3 plants [7,9] Interestingly, transcript analyses revealed that at least one member
of these gene families is preferentially expressed in photosynthesizing tissues [7,8] This is why some researchers differentiate between two classes of H-pro-teins: class I H-proteins, which are preferentially expressed in tissue with high photorespiration, and class II H-proteins, which are more strongly involved
in one-carbon metabolism of non-photorespiratory tis-sues [9] Similar to this, alternative H-proteins could possibly allow fine adjustment of GDC to the parti-cular metabolic requirements in various organs of C4 plants
Experimental procedures
Overexpression constructs
Overexpression constructs for Synechocystis P-protein (SwissProt P74416) and H-protein (Swissprot P73560) have been described previously [29] Coding regions of the mature F trinervia H-proteins [2] FtGLDH and FtGLDHAA (SwissProt P46485) were ligated into the expression vectors pET-28a (tagged) and pET-3a (tagless) (Merck, Darmstadt, Germany) via the NdeI and BamHI restriction sites
For the A thaliana H-proteins AtGLDH1 (At2g35370, SwissProt P25855) and AtGLDH1AA, cDNA was prepared from 2.5 lg of purified leaf RNA (Nucleospin RNA plant kit, Macherey-Nagel, Du¨ren, Germany) using the Revert-Aid H Minus cDNA synthesis kit (MBI Fermentas, St Leon-Rot, Germany) The coding region of the mature H-protein AtGLDH1 was PCR-amplified using the gene-specific primers (with underlined restriction sites) 5¢-CATAT GTCCACAGTTTTGGA-3¢ (sense, for native AtGLDH1), 5¢-CATATGTCCACAGCTGCAGTTTTGGA-3¢ (sense, for the artificial AtGLDH1AA) and 5¢-GGATTCCCTAGTGA GCAGCATCT-3¢ (antisense), and the Elongase enzyme mix (Invitrogen, Karlsruhe, Germany) The PCR product
Trang 5was ligated via pGEM-T (Promega, Mannheim, Germany)
into pET-28a, and recloned into pET-3a as described above
for F trinervia H-protein overexpression constructs
The E coli lplA gene (SwissProt P32099) [17] was
ampli-fied by PCR (sense primer, 5¢-CATATGTCCACATTAC
GCCTGCT-3¢; antisense primer, 5¢-AAGCTTCTACCTTA
CAGCCCCCG-3¢) The sense and antisense primers were
extended by NdeI and HindIII sites (underlined),
respec-tively After initial cloning of the PCR amplificates into
pGEM-T, coding sequences were excised and ligated into
corresponding cloning sites of pET-28a
Recombinant proteins
Synechocystis P-protein was produced essentially as
described previously [29] Major modifications to this
earlier procedure comprised addition of 200 lm
pyridoxal-phosphate and 15 mm 2-mercaptoethanol to all buffers
E coli strain BL21 (DE3) cells overexpressing
H-pro-teins were induced using 1 mm
isopropyl-b-d-thiogalacto-pyranoside for 16 h at room temperatures in 2YT medium
supplemented with 0.4 mm lipoic acid, harvested, and
soni-cated in 20 mm Bis⁄ Tris (pH 6) Cleared lysates were then
loaded onto a Q-Sepharose column (GE Healthcare,
Munich, Germany), and eluted using a linear buffered
0–0.5 m NaCl gradient Fractions containing H-protein
were pooled, concentrated, and further purified on a
Seph-acryl S-100 column (GE Healthcare) equilibrated in the
same buffer
E coli LplA was overexpressed and purified by metal
affinity chromatography similar to Synechocystis P-protein
but with 10% glycerol added to all buffers The enzyme was
stored at)20 C in a buffer containing 10 mm Tris ⁄ 10 mm
Mops (pH 7.5), 0.5 mm MgSO4, 50% glycerol and 5 mm
2-mercaptoethanol Prior to use, it was activated for at least
30 min by the addition of 200 mm 2-mercaptoethanol
For complete lipoylation, recombinant H-proteins (at
0.6 mgÆmL)1, corresponding to approximately 50 lm) were
incubated at 30C for 3 h in 10 mm Tris ⁄ 10 mm Mops
(pH 7.5), 5 mm MgSO4, 5 mm ATP, 5 lm E coli LplA and
200 lm lipoic acid (which is saturating for LplA) LplA
was removed by chromatography through Q-Sepharose
and Sephacryl S-100 columns as described above Purified
H-proteins were used immediately for kinetic assays, or
flash frozen in liquid nitrogen after addition of 20% v⁄ v
glycerol and stored at)80 C
MALDI-TOF MS
The purity of recombinant proteins was assessed by SDS–
PAGE [30], and the molecular identity of all H-proteins
including completeness of lipoylation was further verified
by MALDI-TOF MS Sample aliquots were desalted using
ZipTip pipette tips containing C18 reverse-phase medium
(Millipore Corp., Bedford, MA, USA) and applied to a
polished steel MALDI target by mixing with an equal volume of saturated a-cyano-4-hydroxycinnamic acid in 35% acetonitrile⁄ 0.1% trifluoroacetic acid Data were acquired in linear mode using a Reflex III mass spectro-meter (Bruker Daltonics, Bremen, Germany) For exact determination of the mean mass, the protein sample was mixed with an aliquot of protein standard I (Bruker Daltonics) to allow internal calibration, resulting in a mean mass error of < ±1 Da at the investigated mass range
Matrix extracts from pea leaf mitochondria
Mitochondria were prepared from young garden pea plants and purified as described previously [31] on self-generating density gradients Matrix extracts were prepared by lysis in four freeze–thaw cycles, followed by centrifugation at
44 000 g for 30 min to remove mitochondrial membranes The supernatant was concentrated using Vivaspin col-umns with a 10 kDa exclusion limit (Sartorius, Go¨ttingen, Germany), and adjusted to a protein concentration of 3.5 mgÆmL)1 All steps were performed at 4C
Determination of P-protein activity
The recombinant P- and H-proteins were equilibrated in
20 mm sodium phosphate (pH 7.5) using pre-packed PD-10 columns (Sephadex G-25 M; Pharmacia, Freiburg, Germany) After determination of protein concentration [32], P-protein activity was assayed using the glycine–bicar-bonate exchange reaction [20] Assays with Synechocystis P-protein contained 100 mm sodium phosphate (pH 6.0), 0.1 mm pyridoxalphosphate, 2 mm dithiothreitol, 20 mm glycine, 30 mm NaH14CO3 (2.5 lCi), 2.5 lg P-protein (0.04 lm P-protein dimer) and 0–250 lm H-protein in a total volume of 300 lL [29] Assays with mitochondrial matrix extracts contained 50 mm sodium phosphate (pH 6.0), 0.1 mm pyridoxalphosphate, 2 mm dithiothreitol,
30 mm glycine, 30 mm NaH14CO3 (2.5 lCi), 9 lg matrix protein and 0–100 lm H-protein in a total volume of
150 lL H-protein concentrations varied as indicated in the figures Reactions were run at 30C in the linear response range for 20 min (varying glycine) or 30 min (varying H-protein) Rates obtained without substrate were sub-tracted, and kinetic parameters were calculated by nonlin-ear regression analysis using the software package graphpad prism (GraphPad Software, San Diego, CA, USA) All kinetic data are means ± SD from three or four analyses over the full substrate range
Acknowledgements
Financial support to D.H by the Landesgraduier-tenfo¨rderungsprogramm Mecklenburg-Vorpommern is gratefully acknowledged
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Supporting information
The following supplementary material is available: Fig S1 Three-dimensional structure of pea H-protein showing the N-terminus and the position of the lipoyl arm
Fig S2 Overexpression and effects on H-protein of
E colilipoate–protein ligase A
Fig S3 Glycine saturation kinetics of pea mitochon-drial matrix P-protein
Fig S4 N-terminal sequences of thrombin-cleaved recombinant H-proteins and mass spectrometric verifi-cation of the recombinant Arabidopsis H-proteins This supplementary material can be found in the online version of this article
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