(Received for publication, May 30, 1995; and in revised form, January 10, 1996)
From the
A protein kinase was located in the cytosol of pea mesophyll
cells. The protein kinase phosphorylates, in an ATP-dependent manner,
chloroplast-destined precursor proteins but not precursor proteins,
which are located to plant mitochondria or plant peroxisomes. The
phosphorylation occurs on either serine or threonine residues,
depending on the precursor protein used. We demonstrate the specific
phosphorylation of the precursor forms of the chloroplast stroma
proteins ferredoxin (preFd), small subunit of
ribulose-bisphosphate-carboxylase (preSSU), the thylakoid localized
light-harvesting chlorophyll a/b-binding protein (preLHCP),
and the thylakoid lumen-localized proteins of the oxygen-evolving
complex of 23 kDa (preOE23) and 33 kDa (preOE33). In the case of
thylakoid lumen proteins which possess bipartite transit sequences, the
phosphorylation occurs within the stroma-targeting domain. By using
single amino acid substitution within the presequences of preSSU,
preOE23, and preOE33, we were able to tentatively identify a consensus
motif for the precursor protein protein kinase. This motif is
(P/G)X(R/K)X
(S/T)X
(S*/T*),
were n = 0-3 amino acids spacer and S*/T*
represents the phosphate acceptor. The precursor protein protein kinase
is present only in plant extracts, e.g. wheat germ and pea,
but not in a reticulocyte lysate. Protein import experiments into
chloroplasts revealed that phosphorylated preSSU binds to the
organelles, but dephosphorylation seems required to complete the
translocation process and to obtain complete import. These results
suggest that a precursor protein protein phosphatase is involved in
chloroplast import and represents a so far unidentified component of
the import machinery. In contrast to sucrose synthase, a cytosolic
marker protein, the precursor protein protein kinase seems to adhere
partially to the chloroplast surface. A
phosphorylation-dephosphorylation cycle of chloroplast-destined
precursor proteins might represent one step, which could lead to a
specific sorting and productive translocation in plant cells.
Chloroplasts and mitochondria contain their own genome; however,
the vast majority of their proteins is encoded in the nucleus and
synthesized in the cytosol. In general these proteins carry
NH-terminal targeting domains which direct them to the
proper organelle in a posttranslational event. Whereas some knowledge
about the components and mechanisms which are involved in the
recognition and translocation of the precursors on the organellar
surface has accumulated (Hirsch et al., 1994; Schnell et
al., 1994; Gray and Row, 1995; Soll, 1995), almost nothing is
known about the events which take place in the cytosol, after the
precursor emerges from the ribosome and before binding to the
chloroplast. A loosely folded precursor, however, seems to be a
prerequisite for membrane translocation. In all import systems
aggregation of highly hydrophobic membrane proteins and premature
folding has to be prevented. To attain or maintain such an
import-competent, soluble conformation, some (Waegemann et
al., 1990) but not all proteins need the help of cytosolic factors in vitro (Pilon et al., 1992). A 70-kDa heat shock
protein (hsc70) (
)was found to be involved in the transport
of proteins to different destinations, e.g. mitochondria,
endoplasmic reticulum, and chloroplasts (Deshaies et al.,
1988; Zimmermann et al., 1988; Waegemann et al.,
1990). It was also shown that precursor proteins readily interact with
hsc70 during translation (Beckmann et al., 1990). Whereas this
interaction seems a rather general phenomenon (Ellis and van der Vies,
1991), two cytosolic factors have been described which act as specific
chaperones for mitochondrial precursors. These factors, called
presequence binding factor and mitochondrial import stimulation factor,
have been purified from rabbit reticulocyte lysate and rat liver
cytosol, respectively, and were shown to recognize mitochondrial
presequences and stimulate their import into the organelle (Murakami et al., 1992; Hachiya et al., 1993, 1995). Such
cytosolic factors which interact specifically with the organellar
targeting domain could be even more important in a plant cell, because
the plastids represent an additional compartment to which the cytosolic
synthesized precursors have to be properly routed. Whereas the
mitochondrial presequences share an overall similar framework, i.e. positively charged residues, amphiphilic
-helical structure
in plants, fungi, and mammals (von Heijne, 1986; Hartl et al.,
1989), the plastid-directing transit sequences are much more
heterogenous in length and secondary structure. The only common
features of the chloroplast transit domains are an uncharged
NH
-terminal region, a characteristic stromal processing
site, and a particularly high content of serine and threonine residues
(Karlin-Neumann and Tobin, 1986; von Heijne and Nishikawa, 1991; von
Heijne et al., 1989). In order to understand the series of
events which ultimately yield a specific and productive routing of
precursor proteins to plastids, we have started an investigation on
cytosolic components which could be involved in these processes.
Here we report the specific phosphorylation of several chloroplast precursor proteins within the stroma-targeting portion of the transit sequences, but not of their mature forms. Phosphorylation seems to inhibit import of precursor proteins into chloroplasts. Mitochondrial or peroxisomal precursors are not recognized by the protein kinase. The protein kinase, which belongs to the serine/threonine kinase family, is a plant-specific enzyme. It was shown to be located in the cytoplasm, but it also adheres to the chloroplast outer envelope.
With the exception of preLHCP/LHCP plasmids which
were transformed into the E. coli strain JM101 all plasmids
were introduced in BL21De3 E. coli cells for expression.
Expression and isolation of the overproduced proteins was done as
described by Paulsen et al.(1990), with the exception that
preF was expressed at 30 °C instead of 37 °C.
PreF
and preFd were expressed with a C-terminal
His-Tag.
Exchange of serine 22 for alanine in preOE23 yielding cpreOE23-M22-S/A was done by recombinant PCR (Innis et al., 1990). For the two primary PCR runs cpreOE23/pet 3c was used as template. For the first amplification T7-promotor-primer (= outside primer) was used as forward primer. The sequence for the reverse primer containing the point mutation (A into C) was 5`-CTTGGCGTTGAGCTAAGGTTCTAG-3` (= inside primer). For the second amplification, the outside primer sequence was 5`-GATCAGATCTGATATCATC-3`, resembling a region in the multiple cloning site of pet 3c. As an inside primer resembling the complementary sequence of the inside primer of the first primary PCR, including the respective point mutation (T into G), we used 5`-CTAGAACCTTAGCTCAACGCCAAG-3`. The two primary PCR products were purified by gel electrophoresis and polyethyleneglycol precipitation. The secondary PCR which was performed by mixing the two primary PCR products, which overlap in the inside primer sequence, with the above described outside primers produce full-length cpreOE23 containing the mutation serine 22 into alanine. This PCR product and pet 3c were digested with NdeI and EcoRV and ligated. The clone cpreOE23-M22-S/A was controlled by sequencing and transformed into BL21De3 cells to produce preOE23-M22-S/A.
Exchange of threonine 21 for alanine in preOE33 was also done by recombinant PCR as decribed above. The two outside primers were identical with those of the preOE23 mutation. As inside primers we used 5`-GCGTAGCAACGCATTGCAGC-3` and 5`-GCTGCAATGCGTTGCTACGC-3`, respectively, containing the mutations (A into G) and (T into C). The template for the two primary amplifications was cpreOE33/pet 3c. The PCR product of the secondary amplification was digested with NdeI and SacI and ligated into appropriate digested pet 24c vector. The obtained clone was controlled by sequencing and transformed into BL21De3 cells to produce preOE33-M21-T/A.
PreSSU-M54-R/D was also created by recombinant PCR. As outside primers we used T7-promotor primer and T7-terminator primer. We used the two complementary inside primers 5`-CGGCGGAGATGTGCAATGCATGC-3` and 5`-GCATGCATTGCACATCTCCGCCG-3` containing the mutation AGA into GAT and TCT into ATC, respectively. The template DNA for both primary amplifications was tobacco cpreSSU/pet 11c. The PCR product of the secondary amplification was digested with NcoI and NsiI and ligated into appropriate digested wild type cpreSSU/pet 21d. The obtained clone was controlled by sequencing and was transformed in E. coli BL21De3. The protein (preSSU-M54-R/D) was expressed with a C-terminal His-Tag.
Figure 1:
Overexpression and phosphorylation of
proteins from different plant cell compartments. A, isolated
bacteria-expressed proteins were subjected to SDS-PAGE and stained with
Coomassie Blue. Mature Fd was isolated from spinach leaves according to
Yasunobo and Tanaka(1980). B, the purified proteins, which
were recovered from inclusion bodies, were dissolved in 8 M urea and subjected to in vitro phosphorylation in the
presence of cytoplasm (20 µg of protein) and
[-
P]ATP. PreMDH and MDH were purified from E. coli as soluble proteins and were also adjusted to 8 M urea prior to the phosphorylation reaction. An autoradiogram is
shown.
Each of the above described proteins was subjected
to an in vitro phosphorylation assay with
[-
P]ATP in the presence of cytoplasmic
proteins isolated from pea mesophyll protoplasts (Fig. 1B). All chloroplast precursors examined, i.e. preSSU, preFd, preLHCP, preOE23, and preOE33 became
phosphorylated (indicated by > in Fig. 1B). Their
mature forms or the intermediate processed form of OE23 (iOE23),
however, were not able to serve as a substrate for the cytosolic
protein kinase activity, indicating that the phosphorylation occurred
in the stromal targeting domain of the transit sequence. The cytoplasm
used in the phosphorylation assays is likely to contain several protein
kinases (Ranjeva and Boudet, 1987). We observed the phosphorylation of
several endogenous proteins in the absence of added precursor proteins (Fig. 1B, lane 1). The pattern of endogenous
phosphorylation varied with the different batches of cytoplasm used,
while the precursor protein protein kinase activity seemed more stable.
This could be due to the long isolation procedure of a cytosolic
fraction from protoplasts (see ``Materials and Methods'') and
to different lengths of storage. The endogenous phosphoproteins were
similar to those described by Hracky and Soll(1986). To ascertain that
this phosphorylation is specific for chloroplast precursor proteins and
not a general event, other precursor proteins carrying
NH
-terminal presequences but which are destined for
different locations inside the plant cell were also subjected to in
vitro phosphorylation. As shown in Fig. 1B the
plant mitochondrial preF
could not be phosphorylated.
The peroxysomal preMDH and also the mature MDH were phosphorylated only
very weakly. We conclude that the radioactive label is probably
incorporated into the mature part of MDH. Altogether these data
demonstrate that a protein kinase is localized in cytoplasm enriched
from pea mesophyll cells which is able to specifically phosphorylate
precursor proteins destined for the chloroplast compartment. The
phosphorylation seems to occur exclusively in the stroma-targeting
domain of the presequence.
Figure 2: Phosphoamino acid analysis of the phosphorylated chloroplast precursor proteins. Precursor proteins were subjected to in vitro phosphorylation with wheat germ extract (10 µg of protein) and separated by SDS-PAGE. After electroelution and precipitation the proteins were hydrolyzed in 6 N HCl and subsequently analyzed by high voltage electrophoresis on silica gel thin layer chromatography plates. The positions of the phosphoamino acid standards phosphoserine (P-Ser) and phosphothreonine (P-Thr) are indicated beside the respective autoradiogram. A, preSSU; B, preFd; C, preOE23; D, preOE33; E, preLHCP.
To determine the phosphorylation
sites exactly and to find a putative consensus motif in the chloroplast
transit peptides phosphorylated preSSU was treated with the protease
trypsin. This resulted in one radioactive labeled fragment (not shown).
Sequencing of this peptide revealed that this fragment represents
indeed a part of the preSSU transit sequence from tobacco. The fragment
comprises amino acid 31-39 of preSSU. Three serines are present
in this fragment which could serve as potential phosphate acceptors (Fig. 3). Two of these serines were exchanged for alanine by in vitro mutagenesis. The mutations affected serine 31 and
serine 34 which were exchanged either separately resulting in the
clones cpreSSU-M31-S/A and cpreSSU-M34-S/A or
simultaneously yielding cpreSSU-M31/34-S/A (Fig. 3).
The three mutated cDNAs were subsequently expressed in E. coli (Fig. 4A, lanes 1-4, C)
and subjected to in vitro phosphorylation as before (Fig. 4A, lanes 1-4, P). The phosphorylation experiments of the
mutated precursor proteins resulted in a reduced
P
incorporation into preSSU-M31-S/A (Fig. 4A, lane
2,
P) and no incorporation into
preSSU-M34-S/A and preSSU-M31/34-S/A (Fig. 4A, lanes 3 and 4,
P). From these
data we conclude that serine 34 is the actual phosphoacceptor in
tobacco preSSU, whereas serine 31 might function in the recognition or
binding of the kinase.
Figure 3: Amino acid sequence of chloroplast precursor proteins and the introduced mutations. Amino acids are listed in the single-letter code. A, the marked box in preSSU-WT indicates the phosphorylated tryptic fragment which was sequenced. The amino acids which are involved in the formation of the putative consensus motif are underlined. TTD refers to the thylakoid transfer domain, mature refers to the mature part of the respective protein. B, a putative consensus motif for chloroplast precursor protein protein phosphorylation is shown. The asterisk marks the phosphorylation site. n represents a variable amino acid spacer.
Figure 4:
Determination of the phosphorylation site
in several chloroplast precursor proteins. A, wild-type preSSU (lane 1) preSSU-M31-S/A (lane 2), preSSU-M34-S/A (lane 3), or the double mutant preSSU-M31/34-S/A (lane
4) were either subjected to SDS-PAGE and Coomassie Blue staining
as indicated by the letter C at the bottom of the figure or to in vitro phosphorylation in the presence of cytoplasm as
indicated by a P (autoradiogram) at the
bottom of the figure. B, Coomassie Blue staining of wild-type
preOE23 (lane 1) or the mutant preOE23-M22-S/A (lane
2) in part C and the phosphorylation of the respective
protein in part
P. C, Coomassie
Blue staining of overexpressed wild-type preOE33 (lane 1) and
mutant preOE33-M21-T/A (lane 2) in part C and the
phosphorylation of the respective protein in lane 1 and 2 of part
P. D, phosphorylated
preOE33-M21-T/A was excised from the gel and subjected to phosphoamino
acid analysis. The positions of the phosphoamino acid standards
phosphoserine (P-Ser) and phosphothreonine (P-Thr)
are indicated beside the autoradiogram.
With the knowledge of this phosphorylation
site we analyzed other transit sequences for homologies. A similar
motif was found in preOE23. The respective serine 22 was exchanged for
alanine resulting in cpreOE23-M22-S/A ( Fig. 3and Fig. 4B, lane 2, C). The
phosphorylation experiment using the mutated protein clearly shows that
preOE23-M22-S/A is not phosphorylated any more indicating that serine
22 is indeed the phosphoacceptor amino acid in the pea preOE23 transit
peptide (Fig. 4B, lane 2, P). A similar sequence homology was found in
preOE33 (Fig. 3). From the phosphoamino acid analysis we knew
that this protein is labeled on a threonine residue. Therefore we
exchanged threonine 21 for alanine resulting in cpreOE33-M21-T/A (Fig. 3). The overexpressed protein (Fig. 4C, lane 2, C) was subjected to
the kinase assay (Fig. 4C, lane 2,
P). To our surprise the mutated protein was only
slightly less phosphorylated than the wild-type precursor (Fig. 4C, compare lane 1,
P and 2,
P). This observation could
be due to three reasons. First, the mutation did not affect the
phosphoacceptor amino acid. Second, threonine 21 acts as
phosphoacceptor group, but the kinase can also use another residue with
a lower efficiency if threonine 21 is not available. And third, with
the exchange of threonine 21 into alanine a new phosphorylation site
was created which could also be used by the kinase with reduced
efficiency. In order to address this question we analyzed the
phosphoacceptor amino acid in preOE33-M21-T/A by acid hydrolysis and
thin layer chromatography. As shown in Fig. 4D the
mutated preOE33-M21-T/A was now exclusively phosphorylated on a serine
in contrast to the wild type (Fig. 2D). From these data
we conclude that threonine 21 is the phosphoacceptor group in pea
preOE33. We cannot decide now whether the kinase is able to switch to
serine phosphorylation or whether a new phosphorylation motif was
created.
Altogether these results suggest a consensus motif in the chloroplast transit peptides which could serve as a putative recognition site for the cytosolic precursor protein protein kinase. We postulate that the motif consists of a turn-promoting residue (P/G) followed by a basic amino acid (R/K), an hydroxylated group (S/T) and finally by the actual phosphoacceptor amino acid (S/T) (Fig. 3). The spacing between this residue seems to be variable (see Fig. 3and ``Discussion'').
Figure 5:
Dephosphorylation of preSSU is required
for complete translocation into pea chloroplasts. A, PreSSU (lanes 1 and 2) and preSSU-M54-R/D (lanes 3 and 4) were incubated with a partially purified kinase
from wheat germ in the presence of 10 µCi of
[-
S]ATP
S (specific activity, 3000
Ci/mmol) prior to the chloroplast import experiment.
S-Labeled preSSU (lanes 5 and 6) and
preSSU-M54-R/D (lanes 7 and 8) were treated as above
except that unlabeled ATP was used. Import experiments (Waegemann and
Soll, 1991) were done in the presence of 3 mM ATP and
chloroplasts equivalent to 10 µg of chlorophyll for 5 min at 25
°C. After completion of the import reaction, organelles were either
not treated (odd numbers) or treated with the nonpenetrating
protease thermolysin (even numbers). All other manipulations
are indicated on top of the figure. B, PreSSU was
labeled as above either with [
-
P]ATP (lanes 1-4) or with
[
-
S]-ATP
S (lanes 5-8),
or
S-labeled preSSU was phosphorylated with unlabeled ATP (lanes 9-12 as above). Lanes 13-16,
S-labeled preSSU-M34-S/A was incubated with unlabeled ATP
and partially purified kinase from wheat germ extract. The pretreated
precursors were added to intact purified pea chloroplasts, and import
was assayed in the absence or presence of 50 mM NaF as
indicated on top of the figure. All other manipulations were
as above and are indicated at the top of the figure. The
positions of P-TimA and P-TimB in lane 12 are indicated by arrowheads, respectively.
Different experimental approaches were used to test
this idea (Fig. 5, A and B). First we wanted
to know at what stage during import dephosphorylation of phosphorylated
preSSU (P-preSSU) occurred, i.e. early during translocation at
the envelope membranes or in the stroma before or during processing. To
elucidate this a preSSU processing mutant was constructed and
synthesized from the tobacco cDNA clone in analogy to a preSSU mutant
described for pea (Archer and Keegstra, 1993). Due to an amino acid
exchange (M54-R/D) close to the stromal processing site, this precursor
is impaired in normal processing but yields an intermediate form
(iSSU), which is about 2 kDa smaller than the precursor form. Import
efficiency of the mutant preSSU is similar to wt-preSSU (Fig. 5A, lanes 5-8). The intermediate
processing site in preSSU-M54-R/D is most likely
NH-terminal of the phosphorylation site in preSSU, which is
at amino acid position 34. We reasoned that, if removal of the
phosphate from preSSU would occur via or after processing, iSSU-M54-R/D
should still be phosphorylated. To test this we used
[
-
S]ATP
S to label both wt-preSSU and
preSSU-M54-R/D. ATP
S is accepted as a phosphate donor by protein
kinases but thiophosphorylated proteins are not or much more slowly
dephosphorylated by protein phosphatases (McGowan and Cohen, 1988;
MacKintosh, 1993). This seems also true in plants as thiophosphorylated
preSSU is not dephosphorylated by a soluble protein extract from pea
chloroplasts over a 60-min period (not shown). (
)
S-Labeled wt-preSSU is imported into
intact chloroplasts and processed, and the mature form appears
protease-protected inside the organelles (Fig. 5A, lanes 5 and 6). The mutant
S-labeled
preSSU-M54-R/D is imported and processed with similar efficiency as the
wt-preSSU, only processing yields an intermediate form (iSSU) (Fig. 5A, lanes 7 and 8). This
intermediate form is also detected in the experiment using wt-preSSU
but to a much lesser extent. In parallel assays nonradioactive preSSU
and preSSU-M54-R/D, both labeled by phosphorylation with a partially
purified kinase fraction from a wheat germ lysate and
[
-
S]ATP
S prior to import into
chloroplasts, were used. The thiophosphorylated forms of preSSU and
preSSU-M54-R/D accumulated at the chloroplast surface in comparison to
preSSU or preSSU-M54-R/D phosphorylated by ATP, presumably because
complete translocation was inhibited or slowed down by the
thiophosphate group in the precursor proteins. No labeled iSSU or
iSSU-M54-R/D was detectable (Fig. 5, lanes 1-4)
inside chloroplasts, although the thiolabeled precursor cannot be
dephosphorylated in the time course (5 min) of the import experiment
(see above) (not shown). This indicates that the thiophosphorylated
precursor had not yet reached the stroma for processing. In contrast,
the chloroplast-bound preSSU and preSSU-M54-R/D yield two new distinct
thiophosphorylated translocation intermediates (P-TimA and P-TimB) upon
protease treatment (Fig. 5A, lanes 2 and 4). In addition the thiophosphorylated precursor forms of both
proteins seem to be more protease-protected than the controls (Fig. 5A, lanes 6 and 8). P-TimA and
P-TimB are recovered in the envelope membrane fraction and not in the
soluble chloroplast protein (not shown). These data indicate that
dephosphorylation of preSSU might be necessary for complete
translocation into the stroma, i.e. if dephosphorylation is
hindered by introduction of a thiophosphate group, the import process
comes to an early stop shortly after binding.
To obtain further
experimental evidence for this notion, we conducted import experiments
in the presence of NaF, a broad range protein phosphatase inhibitor (Fig. 5B). Unlabeled wt-preSSU was phosphorylated by
[-
P]ATP and a partially purified kinase
fraction from wheat germ extracts (see ``Material and
Methods'').
P-Labeled preSSU bound to chloroplasts in
the absence or presence of NaF. Protease treatment of organellar bound
precursors yielded
P-labeled P-TimA and P-TimB. In the
absence of NaF,
P-labeled translocation intermediates
accumulate to about 10-20% of the NaF level (compare Fig. 5B, lanes 2 and 4). When we used
thiophosphorylated preSSU in a standard import assay in the presence of
NaF, binding of preSSU to chloroplasts increased, compared to that in
the the absence of NaF (Fig. 5B, lanes 5 and 7). Upon protease treatment P-TimA and P-TimB and protease
protected precursor protein were detected in the absence (Fig. 5B, lanes 5 and 6) of NaF. In
the presence of NaF the amount of protease protected preSSU and P-TimA
and P-TimB increased similar to the increased amount of binding (Fig. 5B, lanes 7 and 8). The
detection of P-TimA and P-TimB in the absence (Fig. 5, A and B) or presence (Fig. 5B, lanes
1-8) of NaF indicates that NaF per se does not
deviate the precursor from its normal import to a bypass pathway. This
is corroborated by earlier findings (Flügge and
Hinz, 1986; Schindler et al., 1987). In a parallel experiment
we used
S-labeled wt-preSSU which had been incubated prior
to the import with unlabeled ATP and partially purified kinase. In the
absence of NaF, import occurred normally (Fig. 5B, lanes 9 and 10), while in the presence of NaF almost
no import was detectable, and bound preSSU accumulated again (Fig. 5B, lane 11). Subsequent protease
treatment resulted in the occurrence of P-TimA and P-TimB (Fig. 5B, lane 12, indicated by an arrowhead). The difference in labeling intensities of P-TimA
and P-TimB in Fig. 5B, lanes 8 and 12, is due to (i) differences in the specific activity of the
labeled educts, i.e. [
-
S]ATP
S
and
S-labeled preSSU and (ii) substoichiometric
phosphorylation of preSSU in vitro, i.e. the import
reactions presented in Fig. 5B, lanes
9-12, contain two different precursor proteins, one which is
phosphorylated and one which is not. The nonphosphorylated wt-preSSU
subpopulation probably gives rise to the additional translocation
intermediates (Tim1-4) seen in Fig. 5B, lane
12. The yield of Tim1-4 in these experiments varied,
probably due to the different ratio of phosphorylated to
nonphosphorylated preSSU (a typical result out of five repeats is shown
in lanes 9-12). Tim1-4 are most likely identical
to deg 1-4, which have been described as translocation
intermediates in the preSSU import pathway into chloroplasts for the
reticulocyte lysate-synthesized precursor protein (Waegemann and Soll,
1991). To test this we used the nonphosphorylatable mutant
preSSU-M34S/A subjected it to a mock phosphorylation treatment and
assayed its import characteristics in the absence or presence of NaF.
In the absence of NaF, preSSU-M34S/A imports normally and is processed
to the mature form (Fig. 5B, lanes 13 and 14). In the presence of NaF, preSSU accumulates at the
chloroplast surface, and almost no import occurs. After a protease
treatment, translocation intermediates appear that are not related to
phosphorylation, namely Tim1-4 (Fig. 5B, lanes 15 and 16). Translocation intermediate
homologues to P-TimA and P-TimB are not generated to a significant
amount from preSSU-M34S/A. From these data we conclude that a
phosphorylated chloroplast precursor protein cannot be imported into
the organelle, but that dephosphorylation is required sometime after
binding but before translocation.
Figure 6:
Localization of the protein kinase
activity. A cytoplasm-enriched fraction from pea mesophyll cells (20
µg of protein) was incubated in the absence (lane 1) or
presence (lane 2) of overexpressed preSSU with
[-
P]ATP. A protein kinase assay was
performed with isolated intact pea chloroplasts (equivalent to 15
µg of chlorophyll) in the absence (lanes 3 and 5)
or presence (lanes 4 and 6) of preSSU. After
completion of phosphorylation, the organelles were reisolated by
centrifugation. Either the pellet, containing the intact chloroplasts (P) or the supernatant (S) was subjected to SDS-PAGE
and autoradiography. Intact chloroplasts were pretreated without (lanes 7 and 8) or with (lanes 9 and 10) thermolysin (Th) (10 µg of Th/100 µg of
chlorophyll) as indicated in the figure. After termination of the
digestion with a 2-fold molar excess of
-macroglobulin, the
organelles were reisolated by centrifugation and subjected to in
vitro phosphorylation in the presence (lanes 8 and 10) or absence (lanes 7 and 9) of preSSU.
After completion of the reaction, the assays were divided into pellet
and supernatant by centrifugation. The autoradiogram of resulting
supernatants (S) is shown. In vitro phosphorylation
with chloroplast supernatant, which was prepared as described under
``Materials and Methods,'' was performed in the absence (lane 11) or in the presence (lane 12) of preSSU. The
position of preSSU is indicated by an arrowhead in each
autoradiogram.
To test the specificity of the association of this largely cytosolic enzyme with the chloroplasts, we analyzed the distribution of sucrose synthase, a cytosolic marker enzyme (Stitt and Steup, 1985), with that of the protein kinase activity in isolated cytoplasm and in chloroplasts. Quantification of the kinase activity and sucrose synthase activity in the chloroplast supernatant and in isolated cytoplasm shows that both fractions were able to phosphorylate preSSU, but sucrose synthase activity could be detected exclusively in the cytoplasm (Fig. 7). Therefore we conclude that the protein kinase is a cytosolic enzyme which might specifically interact and adhere to the chloroplast surface. The nature of this interaction remains to be elucidated.
Figure 7:
Protein kinase activity is associated with
chloroplasts. Protein kinase activity of cytoplasm or chloroplast
supernatant (35 µg of protein, respectively) was determined by
performing an in vitro phosphorylation experiment in the
presence of preSSU and [-
P]ATP. Radioactive
labeled preSSU was quantified in both reactions by laser densitometry
of the autoradiograms. Sucrose synthase activity was measured in
identical amounts (70 µg of protein) of both fractions as described
under ``Materials and Methods.'' Maximal enzymatic activity
of the protein kinase and sucrose synthase in the cytoplasm was set to
100%.
Most chloroplast proteins are synthesized in the cytosol and
have to be imported into the chloroplasts. The import is mediated by an
NH-terminal transit sequence which contains all of the
information necessary for translocation in vitro. However, the
transit sequences of the different chloroplast proteins are very
heterogenous in length and also in secondary structure. Attempts to
dissect the presequences into functional domains, which are important
for certain steps in protein import, have been successfully conducted
for some precursor proteins, i.e. preSSU (Reiss et
al., 1989) and preFd (Pilon et al., 1995). But
unfortunately these motifs do not fit to the presequences of all or
most chloroplast precursors. On the way to find such a common motif, we
addressed the question whether a specific phosphorylation in the
transit sequence could be such a motif. Here we have reported on the
phosphorylation of five different overexpressed chloroplast precursor
proteins. Each of the proteins, i.e. preSSU, preFd, preLHCP,
preOE23, and preOE33, was labeled in the transit sequence. Precursors
containing a bipartite presequence, i.e. preOE23 and preOE33,
are phosphorylated in the stromal targeting domain of the transit
peptide as indicated by the fact that iOE23 could not serve as a
substrate for the precursor protein protein kinase. Other precursor
proteins with NH
-terminal cleavable presequences, i.e. mitochondrial preF
or peroxysomal preMDH, were
not phosphorylated or like the MDH in the mature part of the protein.
We are currently trying to overexpress more mitochondrial precursor
proteins from plants to broaden the basis for this notion.
Phosphoamino acid analysis showed that the phosphorylation of the chloroplast transit sequences occurs on serine or threonine residues. For preSSU, preOE23, and preOE33, the phosphorylation site was determined exactly. Comparison of these sequences revealed a loose motif for a consensus sequence which is defined by a turn-promoting residue (P/G) followed by a spacer, a basic amino acid (R/K), a hydroxylated residue (S/T), a spacer, and the actual phosphoacceptor amino acid (S*/T*). The length of the spacer can vary from protein to protein. The kinase belongs to the serine/threonine protein kinase family and was shown to be a cytoplasmic enzyme which might associate loosely with chloroplasts. The precursor protein protein kinase was detected in phylogenetically different plants, the dicotyledonous pea versus the monocotyledonous wheat and in developmentally very different stages, i.e. pea leaf mesophylls cells and wheat germ embryos. To date it is not clear whether the chloroplast precursors are phosphorylated in the cytoplasm during translation or before binding to the organelle, or if they are phosphorylated at the chloroplast surface during initial phases of chloroplast-precursor interaction (Keegstra et al., 1989).
Import studies using
the nonphosphorylatable preSSU-M34-S/A revealed that the mutated
proteins are imported into chloroplasts as efficient as the wt-preSSU,
indicating that phosphorylation of the transit sequence is not
prerequisite for the translocation in vitro. However, if
phosphorylation of plastid-destined precursor proteins would be
complete during or after translation in vivo, then
dephosphorylation by a protein phosphatase could be a regulatory step
in the import process, i.e. a phosphorylated precursor would
not be translocated into chloroplasts. This is not without precedent in
posttranslational protein translocation. It has been reported that the
nuclear import of several proteins, e.g. lamin B,
SV40 T-antigen, and yeast transcription factor SW15 (Hennekes et
al., 1993; Jans et al., 1991; Moll et al., 1991;
Rihs et al., 1991), is inhibited by phosphorylation of a
serine or threonine residue near the nuclear localization signal. Only
upon dephosphorylation are these proteins imported into the nucleus. To
evaluate such a possibility, we used two approaches. First,
thiophosphorylated precursor proteins were used for import. The
thiophosphate group is not or only very slowly released from the
phosphoprotein by all known protein phosphatases (McGowan and Cohen,
1988; MacKintosh, 1993). In addition we used preSSU-M54-R/D, a
precursor form that imports normally but is aberrantly processed. The
processing site of preSSU-M54-R/D is most likely
NH
-terminal to the phosphorylation site; however, no
thiophosphorylated imported iSSU-M54-R/D was detectable in standard
import assays. The thiophosphorylated preSSU-M54-R/D bound normally to
intact chloroplasts and became partially protease-protected, indicating
that it had moved into the translocation machinery. The phosphorylated
translocation intermediates P-TimA and P-TimB are different in size to
translocation intermediates (deg 1-4) described before for
wt-preSSU, which was synthesized in a reticulocyte lysate system
(Waegemann and Soll, 1991). The translocation intermediates deg
1-4, which are identical to Tim1-4, thus seems to originate
from the nonphosphorylated precursor form, e.g. after
dephosphorylation (this study, Fig. 5B, lanes 12 and 16) (Waegemann and Soll, 1991). Tim1-4 are most
prominent in import experiments in the presence of preSSU-M34-S/A, the
nonphosphorylatable mutant, corroborating this idea.
Furthermore,
P-TimA and P-TimB are detected from a wt-preSSU phosphorylated either
by [-
S]ATP
S or by phosphorylation with
[
-
P]ATP. The chloroplast-bound
[
P]preSSU yields P-TimA and P-TimB upon protease
treatment, demonstrating that the thiophosphate group did not result in
an artificial translocation intermediate. P-TimA and P-TimB are also
detected from organellar bound
S-labeled preSSU
phosphorylated with unlabeled ATP in the presence of NaF upon protease
treatment. NaF is a protein phosphatase inhibitor, which had been shown
before to reversibly inhibit preSSU import into chloroplasts
(Flügge and Hinz, 1986; Schindler et al.,
1987). Since in vitro phosphorylation of precursor proteins is
substoichiometric, P-TimA and P-TimB appear in combination with the
nonphosphorylated Tims. Hence it is not yet possible to experimentally
follow the dephosphorylation of preSSU and the concurrent appearance of
SSU. It remains to be established that a cycle of precursor
phosphorylation-dephosphorylation is a general and essential part of
the import process in vivo. Other possibilities have also to
be considered, e.g. this process could represent a regulatory
phenomenon, which is activated only under certain developmental or
environmental conditions to control precursor protein import into
plastids.
The precursor protein protein phosphatase is most likely
localized in the outer envelope membranes since P-TimA and P-TimB are
membrane-bound early translocation intermediates. The precursor protein
protein phosphatase would represent a new yet to be identified
component of the chloroplast import machinery. Preliminary experiments
to classify the protein phosphatase using specific inhibitors failed.
Calyculin and microcystin, potent inhibitors of PP1 and PP2A
(MacKintosh and MacKintosh, 1994), were without influence on preSSU
import. NaMoO (40 mM) had the same inhibitory
effect as NaF, while vanadate (1 mM) (Hunter, 1995) was
without influence.
To test for PP2B- or PP2C-specific
activities is more complex in a crude system (Shenolikar, 1994) and was
not tried in this study.
NaF seems to inhibit an additional step in the import pathway downstream to dephosphorylation of the precursor protein since preSSU-M34-S/A is not completely imported in the presence of NaF. Precursor import into chloroplasts requires nucleoside triphosphates at various steps in the translocation pathway, e.g. for binding (Olsen et al., 1989) or for pulling the precursor across the membrane by the ATP-dependent action of hsc70 (Stuart et al., 1994). Furthermore, key components of the chloroplast outer envelope import machinery, i.e. OEP86 (Hirsch et al., 1994) and OEP34 (Seedorf et al., 1995; Schnell et al., 1994), are prominent phosphoproteins of this membrane. Their activity might be regulated by a phosphorylation-dephosphorylation cycle (Soll, 1995), which could also be influenced by NaF and result in the inhibition of complete preSSU-M34-S/A translocation into chloroplasts. One of these events or one yet to be identified process seems also be inhibited by NaF and impairs import.
Protein phosphatases do not merely reverse the effect, which is provoked by phosphorylation, but are highly regulatory enzymes themselves (Mumby and Walter, 1993; Hunter, 1995). Thus, the function(s) of a precursor protein protein phosphatase might be to regulate or modulate precursor uptake in dependence of the chloroplast import competency, e.g. energy or redox status. Another possibility might be the developmental regulation of import into chloroplasts during chloroplast differentiation and maturation. It has been reported that mature chloroplasts from fully expanded pea leaves loose their competence to import precursor proteins, while chloroplasts from young, rapidly expanding leaves showed highest import rates (Dahlin and Cline, 1991). Another possibility would be that the precursor protein protein kinase is not always in an activated mode in vivo, i.e. a nonphosphorylated precursor could short-circuit the protein phosphatase. It is clear, that much remains to be learned on the exact purpose of this regulatory circuit in protein import into chloroplasts.
In conclusion phosphorylation of chloroplast destined precursor proteins in the cytosol in combination with their dephosphorylation at the chloroplast envelopes might represent one step in a cascade of events which ultimately lead to specific sorting and translocation to plastids in plant cells.