(Received for publication, September 2, 1994; and in revised form, October 24, 1994)
From the
Protein isoprenylation in vivo is demonstrated using
spinach seedlings labeled with [H]mevalonate.
This report provides evidence for the occurrence of a large number of
isoprenylated proteins in plants. Seedlings, without roots, were
labeled quantitatively through the cut stem. Mevinolin treatment of the
seedlings resulted in increased incorporation of radiolabel into
proteins. Approximately 30 labeled bands could be detected after
autoradiography of SDS-polyacrylamide gel electrophoresis-separated
polypeptides, ranging in molecular mass from 6 to 200 kDa. Methyl
iodide hydrolysis resulted in the release of covalently bound farnesol,
geranylgeraniol, phytol, and some unidentified isoprenoid compounds
from mevalonate-labeled proteins. It was found that all cellular
fractions contained some isoprenylated proteins, although most were
located in the mitochondria and nuclei. Subfractionation of the nucleus
revealed that the majority of isoprenylated proteins in this
compartment were components of the nuclear matrix. The results
demonstrate that in vivo labeling of a complex organism can be
performed using a plant system in order to study protein isoprenylation
and distribution of modified proteins in different cellular
compartments.
Isoprenylation of proteins is a common cellular event, which
until recently has not been studied in plants. It has been estimated
that as much as 2% of total mammalian cell protein is isoprenylated,
which corresponds to 60-80 different proteins/cell(1) .
Proteins previously shown as being isoprenylated include small
GTP-binding proteins(2, 3, 4) , nuclear
lamins (prelamin A and lamin B)(5, 6) , and
-subunits of heterotrimeric G-proteins(7) .
Recently, a large amount of interest has been focused on protein isoprenylation since it was established that several members of the Ras superfamily were modified in this manner(2, 3, 4) . These proteins regulate a variety of functions including control of cell growth and differentiation, cytokinesis, and membrane trafficking(8) .
So far, three different prenyl protein
transferases have been identified: farnesyl protein transferase (9) and geranylgeranyl protein transferases I (10) and
II(11) . These enzymes catalyze the transferal of the isoprene
moieties from isoprenyl pyrophosphates (farnesyl pyrophosphate,
C, or geranylgeranyl pyrophosphate, C
),
produced in various cellular compartments(12, 13) , to
at least 1 cysteine residue near the carboxyl terminus of the substrate
protein by the formation of a thioether linkage. Farnesyl protein
transferase and geranylgeranyl protein transferase I recognize a
specific sequence of amino acid residues, here referred to as a
-CX
X
X
-box,
where X
and X
could be any
amino acid residues. The last residue of this motif (X
) appears to direct which isoprene pyrophosphate
should be attached. Serine, methionine, glutamine, cysteine, and
alanine signal for farnesylation (see (14) ) whereas leucine
and phenylalanine are targets for geranylgeranylation(10) .
Geranylgeranyl protein transferase II activity is dependent on an as
yet unidentified internal sequence, as well as a carboxyl-terminal
motif, designated -CC, -CXC or -CCXX, which contains
2 cysteines(11) . There have been several reports of
protein-bound isoprenoids other than farnesol and geranylgeraniol,
including dolichyl phosphate (15) and isopentenyl
adenine(16) , although the nature of the covalent binding in
these cases and the enzymes catalyzing these modifications remain to be
identified.
Protein isoprenylation is a ubiquitous event, occurring
in organisms as diverse as yeast and humans. However, although the
chemical mechanism of protein isoprenylation is now beginning to be
unraveled, little is known about the extent of this post-translational
modification or its specific physiological function. Many studies have
been performed in yeast and mammalian cell cultures utilizing
radiolabeled precursors of the mevalonate pathway for the tagging of
isoprenylated proteins. Studies of the isoprenylation process by these
methods in more complex systems or in whole organisms have proved
limited. In a recent investigation(17) , it was found that
pieces of etiolated spinach leaves, incubated in a
[H]mevalonate solution, incorporated the label
into protein-bound farnesol, geranylgeraniol, phytol, and longer chain
polyprenols, although only one radiolabeled band was identified by
SDS-PAGE. (
)This work established that plants possess
isoprenylated proteins that may be studied in an appropriate in
vivo system. Cell cultures of tobacco also show incorporation of
[
C]mevalonate into a protein-associated
form(18) . Additionally, the occurrence of plant protein
isoprenylation has been implied by the results of several recent
studies. For example, farnesyl- and geranylgeranyl protein transferase
activities have been found in cell extracts of Atriplex
nummularia, and isoprenylation has been performed in vitro with ANJ1 (a higher plant homologue of the bacterial molecular
chaperone DnaJ) as a substrate(19) . Farnesyl protein
transferase
-subunit has recently been cloned and sequenced from Pisum sativum(20) . Small GTP-binding proteins of the
Ras superfamily have been cloned and sequenced from Nicotiana
plumbaginifolia(21) , Zea mays(22) , and Arabidopsis thaliana(23) and shown to contain
carboxyl-terminal motifs corresponding to those found in mammalian
isoprenylated proteins. In an in vitro system, a small
GTP-binding Ras-related protein from P. sativum could be
geranylgeranylated(24) .
The results reported in this article demonstrate that isoprenylation of plant proteins occurs in vivo, and a system has been established to study the mechanism of this process. The data provide evidence that in plant cells isoprenylation of proteins occurs extensively, and isoprenylated proteins are located in many cellular compartments, in particular the mitochondria and nuclei.
Labeling of spinach proteins with
[S]methionine (Amersham) was essentially as
described above. Seedlings were fed with 1 µCi/plant in spinach
medium, and labeling continued for 6 h in the presence or absence of
mevinolin. Plants were given an appropriate 20-h pretreatment prior to
labeling in spinach medium with or without mevinolin.
The supernatant from the initial centrifugation
(5,000 g, 90 s) was spun again for 3.5 min at 20,000
g to pellet the crude mitochondrial fraction. The
remaining supernatant was centrifuged at 146,000
g for
1.5 h to separate the microsomal and cytosolic fractions. The crude
mitochondrial fraction was resuspended in 250 mM sucrose, 10
mM MOPS (pH 7.2), 1 mM EDTA and purified on a
discontinuous Percoll gradient(31) . All samples were
resuspended in double distilled water and immediately precipitated by
the addition of 10% trichloroacetic acid. Proteins were extracted with
organic solvents, as described above, prior to electrophoretic analysis
or scintillation counting. Nuclei were further fractionated according
to the method described by Wolda and Glomset (6) and after Long et al.(32) . Basically, soluble nuclear proteins and
the nuclear membrane were removed by incubation with 0.2% Triton X-100,
DNA- and RNA-bound proteins were released by digestion with DNase and
RNase (at 250 and 150 µg
ml
, respectively),
and histones were removed by a 1.6 M NaCl wash.
Figure 1:
Time
dependence of [H]mevalonate labeling of spinach
seedling proteins. An autoradiograph of lipid-extracted protein samples
separated by SDS-PAGE from spinach seedlings labeled in vivo with 100 µCi of [
H]mevalonate/plant is
shown. Labeling was performed for 10, 21, and 42 h, respectively, in
the presence of 30 µM mevinolin. Homogenization,
extraction, solubilization, SDS-PAGE, and autoradiography were carried
out as described under ``Experimental Procedures.'' The gel
was loaded with 90 µg of protein/lane. The position of the
molecular mass markers is indicated on the right of the
figure.
Labeling of proteins with
[H]mevalonate was found to be related to both the
concentration of radiolabel supplied to the plants (data not shown) and
the time of labeling (Fig. 1). Weakly labeled bands could be
seen after 5 h of exposure to the radiolabeled substrate (not shown).
Labeling of proteins was found to be increased with time up to 21 h and
then to decline (Fig. 1). The data shown suggest that a
metabolic product(s) of mevalonate is covalently attached to plant
proteins during labeling in vivo. This experiment was
performed using 100 µCi of
[
H]mevalonate/seedling. The radiolabeled bands
detected by autoradiography are, therefore, more intense here than in
the following experiments where only 50 µCi/plant was utilized. By
feeding spinach seedlings [
H]mevalonate (50
µCi/plant) in this manner it was possible to incorporate 500-1500
dpm
µg
total protein. The percentage of
supplied [
H]mevalonate incorporated into proteins
using this spinach system was estimated to be 0.05-0.1%.
Fig. 2shows that the protein-associated radiolabel cannot be extracted from any individual protein bands during the solvent washing steps. It was observed that the extensive extraction procedure carried out was not necessary to remove noncovalently attached lipids in the plant system used since SDS-PAGE is an excellent additional purification step. However, in order to determine the exposure time required for detection of labeled proteins, extractions were performed to allow an accurate estimation of protein-bound radioactivity by scintillation counting prior to electrophoresis. In addition, omitting solvent extractions results in a large quantity of radiolabel in the low molecular mass region of the gel (Fig. 2, lane 1), possibly obscuring any labeled bands in this area. The results in Fig. 2also show that all labeled bands observed are due to covalently bound, rather than loosely associated, lipid moieties derived from mevalonate.
Figure 2:
Solvent extraction of spinach proteins
labeled in vivo with [H]mevalonate.
Seedlings were labeled with 50 µCi/plant in the presence of 30
µM mevinolin for 24 h. At the end of this time, the
samples were homogenized. Solvent extractions were performed as
described under ``Experimental Procedures,'' and samples were
sequentially solubilized for SDS-PAGE and autoradiography. Lane
1, acetone-insoluble material; lane 2,
chloroform:methanol-insoluble material; lane 3,
ethanol-insoluble material; lane 4,
chloroform:methanol:water-insoluble
material.
The radiolabeling is demonstrated to be associated specifically with protein, as all radiolabeled bands are digested upon incubation with trypsin (Fig. 3). The appearance of tryptic fragments still carrying a radiolabeled moiety can be observed.
Figure 3:
Autoradiograph of a trypsin digest of
[H]mevalonate-labeled spinach proteins. Seedlings
were labeled (50 µCi/plant) as described (see ``Experimental
Procedures''). Samples were homogenized, lipid-extracted, and
solubilized for SDS-PAGE. Prior to electrophoresis, one sample (+)
was digested with trypsin (100 µg
ml
) for 1
h at 37 °C. The undigested control sample(-) was incubated
without trypsin under identical conditions. The positions of molecular
mass markers are indicated.
The extent of labeling was found to be dependent on a
preincubation with mevinolin (Fig. 4A), a competitive
inhibitor of hydroxymethylglutaryl-CoA-reductase(33) . Without
mevinolin preincubation, labeling of proteins was feasible (275
dpmµg
protein). However, a pretreatment
with mevinolin was observed to increase the level of incorporated
radiolabel, possibly due to a build up of unmodified protein precursors
(see (34) ) and a decrease in the internal pool of free
isoprenyl groups to be attached during this time. It was found that a
24-h pretreatment with the inhibitor resulted in a doubling of the
incorporation of [
H]mevalonate as compared with
the control (no preincubation). If mevinolin was excluded from the
labeling solution and no mevinolin preincubation treatment was given to
the plants, an identical pattern of labeled bands could be observed
after electrophoresis and autoradiography as that shown in Fig. 1and Fig. 2, although levels of incorporation were
reduced further (data not shown).
Figure 4:
Effect of mevinolin on the metabolic
labeling of proteins in spinach seedlings. A, graph showing
the radiolabeling of spinach proteins in vivo with respect to
the time of preincubation of the seedlings with 30 µM mevinolin. Seedlings were preincubated for the indicated period
with mevinolin and then labeled for 24 h with 50 µCi/plant
[H]mevalonate as described under
``Experimental Procedures.'' Samples were homogenized,
lipid-extracted, and solubilized. Labeling level was assessed by
scintillation counting of the solubilized material. Results shown are
the mean of three replicates ± S.E. B, seedlings were
preincubated and labeled with [
S]methionine (1
µCi/plant) in the presence or absence of 30 µM mevinolin. The preincubation and labeling times were 20 and 6 h,
respectively. Samples were homogenized, acetone-precipitated,
solubilized, and separated by SDS-PAGE. The gel was loaded with 90
µg of protein/well, and labeled bands were detected by
autoradiography.
Interestingly, mevinolin treatment
of the seedlings appears to result in a significant increase in total
protein synthesis, as can be observed by pretreatment and
[S]methionine labeling of seedlings in the
presence or absence of mevinolin (Fig. 4B). The pattern
of radioactive bands produced by labeling with
[
H]mevalonate ( Fig. 1and Fig. 2)
was found to be very different from that observed after labeling under
similar conditions with [
S]methionine (Fig. 4B). This illustrates that the radioactivity
associated with proteins after labeling with
[
H]mevalonate was due to association of a product
of mevalonate with specific proteins, rather than degradation of the
radiolabel to precursors of amino acid biosynthesis. This degradation
pathway is known as the mevalonate shunt(35) . We have also
observed in a separate experiment that homogenization of unlabeled
seedlings (pretreated with mevinolin for 44 h) in the presence of
[
H]mevalonate (50 µCi/plant) did not result
in labeled proteins being visible by autoradiography of the
lipid-extracted proteins. The only band visible on this autoradiograph
was that of free mevalonate at the electrophoretic front (not shown).
Figure 5: HPLC separation of lipids covalently bound to proteins of spinach seedlings. Spinach seedlings were labeled under standard conditions (see ``Experimental Procedures''). After extensive lipid extraction, proteins were subjected to methyl iodide hydrolysis (A) or alkaline hydrolysis (B). Released isoprenoids were extracted from the hydrolysis mixture and analyzed as described under ``Experimental Procedures.'' The elution times of unlabeled standards are shown: farnesol (F), geranylgeraniol (GG), phytol (P), and polyprenol-11 (PP-11).
Alkaline hydrolysis released a different set of isoprenoids from the labeled proteins (Fig. 5B). No farnesol was released under these conditions; however, some geranylgeraniol was liberated as well as long-chain polyprenols. Many products released by both methods of hydrolysis remain to be identified.
Figure 6:
Autoradiograph of an SDS-PAGE gel showing
proteins of various cellular fractions labeled in vivo by
[H]mevalonate. Seedlings were labeled and
fractionated as described under ``Experimental Procedures.''
Nuclear, chloroplastic, microsomal, cytosolic, and mitochondrial
fractions were prepared from the plant homogenate. Samples were
lipid-extracted and solubilized. The gel was loaded with 90 µg of
protein for all fractions except the mitochondrial fraction, for which
only 42 µg of protein was loaded, to allow detection of labeled
bands in all fractions on the same
autoradiograph.
The nuclear and mitochondrial fractions both contained many labeled polypeptides (Fig. 6), and the majority of labeled bands observed in the whole plant protein extract originate from these two fractions. The nuclear fraction shows major labeled bands at 47, 37, 33, and 5 kDa, as well as nine minor labeled components. Strongly labeled bands were detected in the mitochondrial fraction at 47, 33, and 6 kDa, with 10 weaker bands also being observed. There are some similarities in the patterns of bands detected in the nuclear and mitochondrial lanes, which are not due to significant levels of cross-contamination between the two fractions. Western blotting of these fractions has shown that the mitochondrial fraction contained undetectable levels of nuclear contamination, utilizing antibodies raised to proteins of the nuclear pore complex (data not shown). The nuclear fraction contained approximately 15% mitochondrial contamination as assessed by using two antisera raised to soluble mitochondrial proteins (not shown). The absence of significant levels of cross-contaminating membranes in all organelle fractions was seen by electron microscopy (data not shown).
To further study the nuclear location of labeled bands observed in
the initial fractionation experiments, a nuclear subfractionation
protocol was carried out. It can be seen in Fig. 7A that sequential treatment of the nuclear pellet with 0.2% Triton
X-100, DNase (250 µgml
) and RNase (150
µg
ml
), and 1.6 M NaCl resulted in
the release of different subsets of proteins. The autoradiograph of
this gel, shown in Fig. 7B, establishes that the
majority of the labeled polypeptides in the nucleus are located in the
Triton/salt-insoluble subfraction (nuclear matrix, N
), although two labeled bands (at 37 and 62 kDa)
were specifically removed by the Triton X-100 treatment (S
).
Figure 7:
Localization of
[H]mevalonate-labeled proteins in nuclear
subfractions. Seedlings were labeled with
[
H]mevalonate and fractionated as described under
``Experimental Procedures.'' Nuclei obtained from this
procedure were further subfractionated by sequential treatment with
0.2% Triton X-100, RNase (150 µg
ml
) and
DNase (250 µg
ml
), and 1.6 M NaCl.
Proteins present in the supernatant after each treatment are shown in lanes S1, S2, and S3 respectively. The lane Nm shows the proteins remaining in the pellet after the
above procedure. Proteins were detected by staining with Coomassie Blue (A). An autoradiograph of this gel is shown in B.
Molecular mass standards for both A and B are shown
on the left of the figure.
Isoprenylation of proteins has until now been studied in isolated cells (yeast and tissue culture), systems which take up sufficient labeled precursors and contain available protein acceptors. By using plants fed with radiolabel through the stem, our results show that the isoprenylation process can be studied in a complex organism in vivo. The labeling procedure developed for this study proved to be an effective tool for investigation of the mechanisms of the isoprenylation events. The level of isoprenylation found in these experiments with spinach seedlings is comparable with that reported previously (0.5%) in an optimized yeast system(36) .
Protein isoprenylation appears to be a frequently occurring event in the plant cell, and it is suggested from the time course of labeling, shown in Fig. 1, that protein-bound products of mevalonate are degraded simultaneously with the proteins to which they are associated, or they are specifically cleaved from them. Previous work (34, 37) indicates that in most cases the former hypothesis is most plausible.
The fact that mevinolin (present
during preincubation and labeling) results in stimulation of
[H]mevalonate metabolite incorporation into
proteins may be due to effects both on the endogenous pools of
precursors for isoprenylation (protein and isoprene) and on protein
synthesis itself. Mevinolin has been shown (Fig. 4B) to
stimulate [
S]methionine labeling of proteins.
The reason for this stimulation is, as yet, unknown, and it may
represent a genuine induction of protein synthesis. It has previously
been demonstrated that certain mevalonate pathway enzymes are
up-regulated after mevinolin treatment(38, 39) .
However, it is possible that the increased incorporation of
[
S]methionine into proteins is a consequence of
a higher intracellular concentration of the substrate resulting from a
mevinolin-induced alteration of cell membrane permeability, since
mevinolin may decrease the cellular concentrations of certain membrane
components. Thus, it is not yet possible to determine whether the
increase observed in [
H]mevalonate incorporation
into proteins in the presence of mevinolin (this paper and (34) ) is due to stimulation of protein synthesis or effects on
isoprene and protein acceptor pools. Neither of these possibilities can
be excluded until a method for estimating the intracellular
concentration of isoprenes is developed.
It is firmly established by
a number of studies that both farnesol and geranylgeraniol can be
covalently attached to cysteine residues in proteins by thioether
bonds. The results presented here demonstrate that spinach proteins
also contain thioether-linked farnesol and geranylgeraniol, as well as
additional longer-chain lipids. One of these has been identified as
phytol. The occurrence of farnesol and geranylgeraniol modification of
proteins is widely accepted and proved using physico-chemical methods.
However, several longer-chain isoprenes
(C-C
) have been reported as being
covalently bound to proteins(15, 17, 40) ,
although the linkage involved was not determined, and the isoprenoid
structures have yet to be confirmed. Thus, the posttranslational
modification of proteins with isoprenes other than farnesol and
geranylgeraniol remains largely unknown but represents an interesting
area for future research in all organisms.
It was routinely found that there was a significantly larger amount of geranylgeraniol than farnesol thioether-linked to spinach proteins as has also been shown in other systems, for example Chinese hamster ovary (41) and HeLa (42) cells. In our experiments, methyl iodide hydrolysis always resulted in a ratio of geranylgeraniol:farnesol greater than 2, and longer-chain isoprenes (such as phytol) were also seen, although at a significantly lower abundance. In a previous study(17) , the proteins of etiolated spinach seedlings were shown to covalently bind geranylgeraniol:farnesol in a ratio of 0.7. The variation in the levels of these thioether-linked isoprenes is probably a result of the very different developmental state of the seedlings used for the two studies. The methyl iodide hydrolysis results reported for green spinach tissue in this paper are more comparable with those of mammalian cells (41, 43) than etiolated spinach material in that the predominant protein-bound isoprene is geranylgeraniol. We have observed extensive modifications in the pattern of isoprenylated proteins in plants of different physiological condition, as well as during development and aging (this paper and (17) ). It appears that isoprenylated proteins may be important regulatory factors in the plant cell, and their amount and type of covalent association with isoprenoids vary depending on cellular requirements. It is also possible that the synthesis of certain isoprenylated proteins is induced by light. We are now analyzing the importance and extent of these alterations in isoprenylation pattern under a variety of conditions.
Upon alkaline hydrolysis, a set of isoprenoid products is released that are not related to those liberated by methyl iodine. The identity of most of these compounds remains to be determined; however, alkaline hydrolysis does appear to release long-chain polyprenols. Thus, it is clear that there are isoprenoid compounds associated with plant proteins via linkages other than thioether. We are presently also investigating whether any of the unidentified compounds detected after alkaline hydrolysis could be isoprenyl cysteines released or isoprenyl peroxides formed during the procedure.
The isoprenylated protein profile found in green spinach
tissue shows similarities to those detected in other material. It has
been reported previously that many isoprenylated proteins are
membrane-associated(7, 18, 44, 45, 46, 47) ,
and this is also observed in spinach seedlings. The identity of the
labeled bands shown in this paper is not known at the present time.
However, it is possible that the band of 68 kDa located in the nuclear
matrix represents a plant homologue of nuclear lamins (Fig. 7)(5, 6) , and the microsomal band at 24
kDa could be a small GTP-binding protein (Fig. 6)(44, 45, 48, 49) .
Isoprenylated proteins of 44-69 kDa have previously been reported
in the nuclear matrix of murine lymphoma cells(50) , and we
have demonstrated the occurrence of labeled nuclear matrix proteins in
this region. The isoprenylated -subunits of heterotrimeric
G-proteins have a molecular mass of 5-8
kDa(51, 52, 53) . As heterotrimeric
G-proteins have now been identified in plant
microsomes(54, 55) , it is most likely that the
radiolabeled band at 9 kDa observed in our experiments, unique to the
microsomal fraction, corresponds to a plant homologue of mammalian
G-protein
-subunits. The microsomal isoprenylated band of 52 kDa (Fig. 6) may be farnesylated ANJ1 (51 kDa), which was
demonstrated to be associated with Atriplex microsomal
membranes in vitro(19) . The identity of the
isoprenylated proteins in plant mitochondria remains to be established
as there are no previous reports of protein isoprenylation in this
organelle. However, it is possible that plant mitochondria contain an
isoprenylated homologue of DnaJ. A protein (SCJ1) displaying
significant homology to this molecular chaperone has recently been
identified in yeast mitochondria(56) . The deduced amino acid
sequence of SCJ1 does not include any recognizable isoprenylation
signal sequence, but it is possible that plant proteins contain
different motifs directing isoprenylation. Rat liver mitochondria are
known to produce large quantities of farnesyl
pyrophosphate(57) , which may be used for isoprenylation of
mitochondrial proteins. However, it cannot, at this point in the
investigation, be excluded that some of the bands recovered in the
mitochondrial fraction originate from peroxisomes.
The involvement of isoprenylated proteins in a range of cellular functions has previously been shown in animal and yeast systems. Identification of plant homologues to these proteins would be of great interest, and the methodology developed here will be applied to this end in the future. The occurrence of plant-specific isoprenylated proteins has been shown by their occurrence in chloroplasts. Isoprenylated proteins of other cellular compartments may prove to be unique to plants as we have observed many isoprenylated proteins that cannot be accounted for by comparison with labeled proteins identified in other organisms. As the mevalonate pathway is more diverse in plant than in animal cells(35) , it may be expected that there are differences in particular aspects of protein isoprenylation in the two systems.