FEBS 23804
FEBS Letters 475 (2000) 213^217
Protein phosphorylation/dephosphorylation in the inner membrane of
potato tuber mitochondria
Andrë Struglicsa;1 , Kenneth M. Fredlundb;2 , Yuri M. Konstantinovb;3 , John F. Allena;4 ,
Ian M. MÖllerb; *
a
Plant Cell Biology, Lund University, P.O. Box 7007, S-220 07 Lund, Sweden
b
Plant Physiology, Lund University, P.O. Box 117, S-221 00 Lund, Sweden
Received 6 May 2000
Edited by Vladimir Skulachev
Abstract Inside-out inner mitochondrial membranes free of
matrix proteins were isolated from purified potato tuber
(Solanum tuberosum L.) mitochondria and incubated with
[QQ-32 P]ATP. Proteins were separated by SDS^PAGE and
visualized by autoradiography. Phosphorylation of inner membrane proteins, including ATPase subunits, was strongly
inhibited by the phosphoprotein phosphatase inhibitor NaF. We
propose that an inner membrane phosphoprotein phosphatase is
required for activation of the inner membrane protein kinase.
When prelabelled inner membranes were incubated in the absence
of [QQ-32 P]ATP, there was no phosphoprotein dephosphorylation
unless a soluble matrix fraction was added. This dephosphorylation was inhibited by NaF, but not by okadaic acid. We conclude
that the mitochondrial matrix contains a phosphoprotein
phosphatase that is responsible for dephosphorylation of inner
membrane phosphoproteins. ß 2000 Federation of European
Biochemical Societies. Published by Elsevier Science B.V. All
rights reserved.
Key words: Protein phosphorylation; (Plant) mitochondrion;
Inner membrane; Matrix; Protein kinase;
Protein phosphatase; Protein kinase phosphatase
1. Introduction
Protein phosphorylation/dephosphorylation by protein kinases and protein phosphatases is a ubiquitous mechanism
in eukaryotes and prokaryotes for modulating the activity of
intracellular proteins in response to extracellular signals [1].
In mitochondria, two well-characterized enzymes, the Ksubunit of pyruvate dehydrogenase and the K-subunit of
branched-chain 2-oxoacid dehydrogenase complex, both located in the matrix are under the control of phosphorylation/dephosphorylation [2]. Four inner membrane phospho*Corresponding author. Fax: (46)-46-222 4113.
E-mail: ian_max.moller@fysbot.lu.se
1
Present address: Active Biotech Research AB, P.O. Box 724,
S-220 07 Lund, Sweden.
2
Present address: Novartis Seeds AB, P.O. Box 302, S-261 23
Landskrona, Sweden.
3
Present address: Laboratory of Plant Genetic Engineering, Siberian
Institute of Plant Physiology and Biochemistry, P.O. Box 1243,
Irkutsk 664033, Russia.
4
Present address: Department of Plant Biochemistry, Lund
University, P.O. Box 117, S-221 00 Lund, Sweden.
Abbreviations: IO-SMP, inside-out submitochondrial particles (inner
membranes); TCA, trichloroacetic acid
proteins have been identi¢ed by N-terminal sequencing: the
18 kDa AQDQ subunit of complex I [3]; the 17 kDa subunit
IV of cytochrome c oxidase [4]; and the 22 kDa NP-subunit
and the 28 kDa L-subunit of the F0 F1 -ATPase [5]. The phosphorylation of a number of unidenti¢ed proteins has also been
demonstrated in bovine heart mitochondria [6,7] and in potato tuber mitochondria [5,8^10].
When inside-out submitochondrial particles (IO-SMP) from
plant mitochondria are incubated with [Q-32 P]ATP, more than
20 proteins are phosphorylated mostly on serine/threonine
residues [11]. The basic properties of the phosphorylation,
speci¢city, time course, pH dependence, Km (ATP), have
been characterized and two putative kinases/kinase subunits
of 16.5 and 30 kDa identi¢ed [11]. In the present study, we
show that two distinct phosphoprotein phosphatases are involved in the protein phosphorylation/dephosphorylation in
IO-SMP, one bound to the mitochondrial inner membrane
and another, soluble, in the matrix.
2. Materials and methods
Highly puri¢ed intact mitochondria free from plastids (amyloplasts)
and peroxisomes were puri¢ed from potato tubers (Solanum tuberosum L. cv. Bintje or Ukama) [12]. The mitochondria were subfractionated and inside-out inner mitochondrial membrane (IO-SMP)
vesicles lacking any substrates or matrix proteins were prepared as
in [10]. The matrix fraction was concentrated in an Amicon 8010
Stirred Ultra¢ltration Cell with a 10 kDa cut o¡ ¢lter (Amicon YM
10).
Protein phosphorylation assays were carried out in a volume of 50
Wl containing 250 Wg IO-SMP proteins [11]. Final concentrations in
the reaction mixture were as follows: 0.3 M sucrose, 50 mM HEPES^
KOH (pH 7.5), 5 mM MgCl2 , 0.1 mM CaCl2 and 0.2 mM (0.4^1.0 Ci
mmol31 ) [Q-32 P]ATP. After 2^15 min incubation at room temperature
(22^23³C), the reaction was stopped in either of two ways: (A) by
addition of sample bu¡er [13] and thereafter boiling at 100³C for
2 min, or (B) by addition of trichloroacetic acid (TCA) (6.5% w/v
¢nal concentration), and after 30 min at room temperature the samples were pelleted by centrifugation at 13 000Ug for 6 min, resuspended in 100 Wl 50 mM HEPES^KOH (pH 7.5) and pelleted again.
The washed pellets were then resuspended in the sample bu¡er.
For the dephosphorylation assay, inside-out inner mitochondrial
membrane proteins were ¢rst phosphorylated using [Q-32 P]ATP as
described above. The membranes were then pelleted (1 min at
100 000Ug) with an Airfuge (Beckman, rotor A-100) and the supernatant containing excess ATP was discarded. The membranes were
resuspended in 50 mM HEPES^KOH (pH 7.5), 5 mM MOPS (pH
7.2), 0.3 M sucrose, 125 mM mannitol, 7 mM MgCl, 0.1 mM CaCl2
and 0.4 mM EDTA in the presence or absence of 400 Wg matrix
proteins in a total volume of 50 Wl. After incubation for 1^60 min,
the inner membranes were pelleted as above and the supernatant,
which contained matrix proteins, was removed. TCA was added to
the inner membranes to a ¢nal concentration of 6.5% (w/v) and after
30 min at room temperature proteins were pelleted at 13 000Ug for
0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 0 1 4 - 5 7 9 3 ( 0 0 ) 0 1 6 8 0 - X
214
6 min. The pellet was resuspended in 100 Wl 50 mM HEPES^KOH
(pH 7.5) bu¡er and treated as above before gel electrophoresis.
SDS^PAGE was performed according to [13] on a 10^15% T
(C = 2.7%) gradient gel with a Protean II xi Slab Cell (Bio-Rad) ap-
A. Struglics et al./FEBS Letters 475 (2000) 213^217
paratus. Gels were stained with Coomassie brilliant blue R250, destained and dried. The phosphoproteins were visualized in two ways:
(1) the gels were placed on a phosphorimager plate (Molecular Dynamics) for 2^5 days. The plate was then screened in a phosphorim-
Fig. 1. The e¡ect of NaF on the phosphorylation of mitochondrial inner membrane proteins. (A) Coomassie-stained gel identical to that used
in B. (B) Autoradiograph of IO-SMP proteins which had been phosphorylated for 2 min in the absence or presence of NaF at di¡erent concentrations following separation using SDS^PAGE. Lane 1, no NaF; lane 2, 1 mM NaF; lane 3, 2 mM NaF; lane 4, 5 mM NaF; lane 5, 10 mM
NaF; lane 6, 25 mM NaF; lane 7, 50 mM NaF. Lane 8 shows molecular mass standards (in kDa) visualized using a 35 S marker pen on the
Coomassie-stained gel before autoradiography. Phosphoproteins discussed in the text are indicated by arrows and size in kDa. (C) Relative inhibition of 32 P incorporation at di¡erent NaF concentrations given as the average value for all detected phosphoproteins in IO-SMP. Results
from B.
A. Struglics et al./FEBS Letters 475 (2000) 213^217
ager (Molecular Dynamics SI) and the data analyzed with a Microsoft
Windows NT version 3.1 program. (2) The gels were placed on a
screen with autoradiographic ¢lm (Hyper ¢lm MP, Amersham) for
5^7 days at 380³C. Protein concentration was determined by use of
Coomassie brilliant blue G250 according to the instructions from BioRad using IgG as the standard.
All experiments were carried out at least twice, and only consistently reproducible results are presented.
3. Results and discussion
Incubating IO-SMP vesicles with [Q-32 P]ATP in the presence
of divalent cations results in phosphorylation of more than 20
proteins as judged by SDS^PAGE and autoradiography
[5,11]. In the experiment shown in Fig. 1, the involvement
of phosphatases was investigated by incubating the IO-SMP
in the presence or absence of the phosphatase inhibitor NaF.
The Coomassie-stained gel showed equal loading of all lanes
(Fig. 1A). A comparison with the autoradiograph (Fig. 1B)
showed that the polypeptide and phosphoprotein patterns
were quite di¡erent. The labelling time was only 2 min, the
time giving half-maximal phosphorylation [11] to maximize
the sensitivity to inhibition, resulting in somewhat less 32 P
incorporation than in subsequent experiments (cf. Fig. 2A).
NaF at concentrations of 10 mM and above inhibited 32 P
incorporation into all bands strongly (Fig. 1B). The phosphorylation of all the proteins was reduced by 75% on average
at 25 mM NaF (Fig. 1C). Speci¢cally, NaF reduced the labelling of the two F0 F1 -ATPase subunits of 22 and 28 kDa [5],
the prominent, but as yet unidenti¢ed, 16 kDa polypeptide, as
well as the 41 kDa K-subunit of the pyruvate dehydrogenase
(sometimes associating with the IO-SMP, [8]) (Fig. 1B).
To test whether the e¡ect of 25 mM NaF on phosphorylation was due to increased ionic strength, we included 25 mM
NaCl instead in a control experiment but it had no e¡ect on
32
P incorporation into the inner membrane proteins (results
not shown). Okadaic acid, which is a phosphatase 1 and 2A
inhibitor, used in mammalian systems [14], had no e¡ect on
protein phosphorylation of the inner membrane of potato
tuber mitochondria even at a concentration of 1 WM (results
not shown).
NaF has been demonstrated to be an e¡ective phosphoseryl
and phosphothreonyl protein phosphatase inhibitor [15] in
C
Fig. 2. The e¡ect of the matrix fraction on the time-dependent dephosphorylation of inner membrane phosphoproteins. A: Autoradiograph of inner membrane phosphoproteins subjected to dephosphorylation for di¡erent times in the absence (lanes 2^6) or
presence (lanes 8^12) of the matrix fraction. An inner membrane
fraction was ¢rst phosphorylated for 15 min, then the ATP/
[Q-32 P]ATP was removed by centrifugation and the labelled inner
membranes were incubated at room temperature for di¡erent times
to become dephosphorylated. Lane 1, molecular weight standards
(in kDa) visualized as in Fig. 1; lane 2, 1 min without matrix fraction; lane 3, 2 min without matrix fraction; lane 4, 7 min without
matrix fraction; lane 5, 15 min without matrix fraction; lane 6, 60
min without matrix fraction; lane 7, molecular weight standards (in
kDa); lane 8, 1 min with matrix fraction; lane 9, 2 min with matrix
fraction; lane 10, 7 min with matrix fraction; lane 11, 15 min with
matrix fraction; lane 12, 60 min with matrix fraction. The arrow indicates the position of phosphoproteins discussed in the text. The
Coomassie-stained gel showed equal loading of proteins and identical polypeptide patterns in all lanes. B: Dephosphorylation pro¢le
of the 16 kDa phosphoprotein. Results from A.
215
plant chloroplasts [16], and in mammalian [17] and plant
[18] mitochondria. In these systems, NaF usually enhances
32
P incorporation into proteins by inhibiting protein phosphatases whose activity compete with that of the protein kinases.
Since NaF inhibits protein phosphatases and not protein kinases, the decrease in 32 P incorporation in the presence of
NaF (Fig. 1) was unexpected. We suggest that phosphorylation of the inner membrane proteins is dependent on a protein kinase that is activated by a protein phosphatase. The
possible mechanism will be discussed later.
Protein phosphatase activity was investigated as follows:
the inner membrane proteins were phosphorylated using labelled ATP. After 15 min of labelling, to achieve maximum
labelling of the phosphoproteins, the ATP was removed and
216
Fig. 3. Autoradiograph of the mitochondrial inner membrane phosphoproteins subjected to dephosphorylation under di¡erent conditions. Lanes 1^3, the ATP/[Q-32 P]ATP was removed after 10 min
phosphorylation and the samples were further incubated for 60 min
with the following additions: lane 1, matrix fraction; lane 2, bu¡er
only; lane 3, matrix fraction+25 mM NaF. Lane 4, control sample
labelled for 10 min with no subsequent incubation. Molecular mass
standards are indicated at the left side of the ¢gure (in kDa).
the incubation continued. There was no detectable dephosphorylation of the phosphoproteins even after a 60 min chase
(Fig. 2A, lanes 2^6). Upon addition of the matrix fraction to
inner membrane vesicles containing 32 P-labelled inner membrane proteins, dephosphorylation of all phosphoproteins in
the inner membrane, including the 22 and 28 kDa subunits of
the F0 F1 -ATPase, was observed (Fig. 2A, lanes 8^12). As an
example, the time course of dephosphorylation of the 16 kDa
inner membrane phosphoprotein, when treated in the presence
or absence of a concentrated matrix fraction, is shown in Fig.
2B. The half-time of dephosphorylation of the 16 kDa phosphoprotein in the presence of matrix proteins was about
6 min. When NaF was added together with matrix proteins
to prelabelled inner membrane proteins, the dephosphorylation was prevented (Fig. 3), while okadaic acid had no such
e¡ect (result not shown). When a heat-denatured matrix fraction was added to phosphorylated inner membrane proteins
lacking ATP, there was no detectable dephosphorylation
(results not shown). There was no detectable proteolysis of
32
P-labelled IO-SMP proteins, and the protease inhibitors
phenylmethylsulphonyl £uoride and E64 (L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane) had no e¡ect on the dephosphorylation pattern of IO-SMP proteins (results not
shown). These ¢ndings suggest that a phosphoprotein phos-
A. Struglics et al./FEBS Letters 475 (2000) 213^217
phatase in the mitochondrial matrix is responsible for the
dephosphorylation of the inner membrane phosphoproteins.
There are at least two possible explanations for the NaF
inhibition of protein phosphorylation in mitochondrial inner
membranes (Fig. 1): (i) The substrates for the protein kinase
are already phosphorylated when the IO-SMP fraction is prepared, and an active phosphatase is needed to dephosphorylate the IO-SMP proteins before they can be labelled with
[Q-32 P]ATP.
(ii) The protein kinase itself is inactive in the phosphorylated form and it is either phosphorylated in vivo or becomes
phosphorylated upon addition of ATP. It will then require an
active protein kinase phosphatase for activation of the protein
kinase and phosphorylation of its substrates.
Since the substrate phosphoprotein phosphatase is located
in the matrix and not in the inner membrane (Fig. 2), then
there must be another phosphatase that is inhibited when
NaF is added to inner membranes resulting in a decrease of
32
P incorporation into IO-SMP proteins (Fig. 1). Therefore
the decrease in 32 P incorporation must be the result of an
inhibition of a kinase phosphatase in the IO-SMP. This supports the second proposal above: the protein kinase is in a
phosphorylated and inactive form at the start of the experiment or when ATP is added to the membranes. Activation of
a kinase then requires an active phosphatase.
Sommarin et al. [18] and Petit et al. [9] showed that total
32
P incorporation into intact mitochondria reached a maximum incorporation after 1^2 min. A time course experiment
on phosphorylation of inner membrane proteins showed that
the maximum 32 P incorporation was reached only after 15^23
min [11]. The di¡erences between these results could be explained by the fact that in intact mitochondria, an equilibrium
between kinase activities and phosphatase activities is rapidly
reached. In the inner membrane vesicles where the substrate
phosphatase is missing, an equilibrium is not reached, and
thus it takes much longer to reach maximum 32 P incorporation.
The half-time for the dephosphorylation of prelabelled inner membrane phosphoproteins by a phosphatase in the matrix was 6 min when 250 Wg IO-SMP proteins was incubated
with 400 Wg matrix proteins in a total volume of 50 Wl (Figs. 2
and 3). The relative amounts of inner membrane and matrix
proteins are close to that in intact mitochondrion (I.M.
MÖller, A.G. Rasmusson and K.M. Fredlund, unpublished).
However, the actual concentration of matrix proteins in the
assay (8 mg ml31 ) is more than an order of magnitude below
that in the intact mitochondrion where it may be as much as
500 mg ml31 [19], a concentration it is not possible to reach in
vitro. Thus, the potential rates of dephosphorylation in vivo
may well be much higher than those estimated here.
4. Conclusions
b
b
Plant mitochondria contain a protein kinase phosphatase in
the inner membrane which dephosphorylates, and thereby
activates, the intrinsic protein kinase.
Plant mitochondria contain a protein phosphatase in the
matrix which can dephosphorylate all the phosphoproteins
on the inner matrix surface of the inner membrane.
Acknowledgements: We are grateful to Christina Nilsson for excellent
technical assistance, and to the Swedish Natural Science Research
A. Struglics et al./FEBS Letters 475 (2000) 213^217
Council for research grants to I.M.M. and J.F.A., the Swedish Council for Planning and Coordination of Research (FRN) to J.F.A. and
to the Swedish Institute for a grant to Y.M.K.
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