(Received for publication, May 22, 1995)
From the
Members of the myristoylated alanine-rich protein kinase C
substrate (MARCKS) family are involved in several cellular processes
such as secretion, motility, mitosis, and transformation. In addition
to their ability to bind calmodulin and to cross-link actin filaments,
reversible binding to the plasma membrane is most certainly an
important component of the so far unknown functions of these proteins.
We have therefore investigated the binding of murine MARCKS-related
protein (MRP) to lipid vesicles. The partition coefficient, K, describing the affinity of myristoylated MRP
for acidic lipid vesicles (20% phosphatidylserine, 80%
phosphatidylcholine) is 5-8
10
M
, which is only 2-4 times
larger than the partition coefficient for the unmyristoylated protein.
Interestingly, the affinity of MRP for acidic lipid membranes is
20-30-fold smaller than reported for murine MARCKS (Kim, J.,
Shishido, T., Jiang, X., Aderem, A. A., and McLaughlin, S.(1994) J.
Biol. Chem. 269, 28214-28219). Since only a marginal binding
could be observed with neutral phosphatidylcholine vesicles, we propose
that electrostatic interactions are the major determinant of the
binding of MRP to pure lipid membranes. Although the myristoyl moiety
does not contribute drastically to the binding of MRP to vesicles,
photolabeling experiments with a photoreactive phospholipid probe show
that the fatty acid is embedded in the bilayer. The same membrane
topology was found for bovine brain MARCKS. Since the relatively low
affinity of MRP for vesicles is insufficient to account for a stable
anchoring of the protein to cellular membranes, insertion of the
myristoyl moiety into the bilayer might favor the interaction of MRP
with additional factors required for the binding of the protein to
intracellular membranes.
During the last decade, a growing number of myristoylated
proteins have been identified in eukaryotic cells. Myristoylation
refers to the attachment of a C14 saturated fatty acid (myristate) to
the N-terminal glycine residue of substrate proteins. The soluble
enzyme N-myristoyl transferase (NMT) ()cotranslationally transfers a myristoyl moiety from the
cofactor myristoyl-coenzyme A to the N terminus of nascent proteins
containing the consensus sequence
(Gly-X-X-X-Ser/Thr) (Johnson et
al., 1994). Although myristoylation is believed to be irreversible
(James and Olson, 1989), some evidence has been presented for a
demyristoylation activity in cytoplasmic fractions of brain
synaptosomes (da Silva and Klein, 1990; Manenti et al., 1994).
The importance of myristoylation has been highlighted by several
studies in which removal of the fatty acid leads to dramatic
alterations of cellular functions; myristoylation is necessary for the
transforming activity of v-Src (Kamps et al., 1985), for the
transcriptional suppressor activity of the HIV nef gene
product (Huang and Jolicoeur, 1994), and for signaling and transforming
functions of G protein
subunits (Gallego et al., 1992).
How myristoylation mediates the function of these proteins is, however,
unclear. One function of the fatty acid moiety is to promote
protein-protein interactions. In this respect myristoylation is
required for high affinity interaction of G protein
subunits with
the
subunits (Linder et al., 1991) and enhances the
binding of the HIV nef gene product to CD4 (Harris and Neil,
1994). Myristoylation also influences the conformation of proteins as
seen for the catalytic subunit of cAMP-dependent protein kinase
(Yonemoto et al., 1993) and for p56lck, a member of the Src
family, which are stabilized by the acylation (Nadler et al.,
1993). Myristoylation also promotes protein-ligand interactions as
indicated by the increased ability of myristoylated ADP-ribosylation
factor to exchange nucleotides (Franco et al., 1995). The most
obvious function of the myristoyl moieties is to mediate membrane
binding. Evidences for this role have been presented for several
proteins by analyzing the subcellular localization of unmyristoylated
mutants. This approach has successfully demonstrated that the myristoyl
moieties of nitric oxide synthase (Busconi and Michel, 1994), the
myristoylated alanine-rich protein kinase C substrate (MARCKS) (Graff et al., 1989), the product of the HIV nef gene (Yu
and Felsted, 1992), Src (David-Pfeuty et al., 1993), and the
subunit of Gz (Hallak et al., 1994) are necessary for
membrane binding. Myristoylation, however, is not sufficient to bind
proteins to membranes for several reasons. First, myristoylated
proteins are found in almost every subcellular compartment including
the cytosol (Magee and Courtneidge, 1985; Blenis and Resh, 1992).
Second, the apparent partition coefficient, K
,
describing the binding of acylated peptides to vesicles is about
10
M
and corresponds to a Gibbs
free energy of binding of 8 kcal, which is not sufficient to firmly
anchor proteins to membranes (Peitzsch and McLaughlin, 1993). These
observations indicate that proteins need some information, in addition
to myristoylation, in order to be targeted to their final subcellular
localization and to bind to membranes. A search for receptors for
proteins such as Src (Sigal and Resh, 1993), MARCKS (George and
Blackshear, 1992), and nitric oxide synthase (Busconi and Michel, 1994)
has, however, been unsuccessful so far. Recently, the emphasis has
shifted to dual binding motifs. Some myristoylated proteins, such as
several members of the G protein
subunit family (Casey, 1994) as
well as several members of the Src family (Resh, 1994), are
palmitoylated at a cysteine residue adjacent to the myristoylated
N-terminal glycine residue. This double acylation is sufficient to
anchor proteins firmly to the membrane (Blenis and Resh, 1992). Other
proteins such as MARCKS (Taniguchi and Manenti, 1993; Kim et
al., 1994a, 1994b) and Src (Sigal et al., 1994) contain a
basic domain that interacts electrostatically with membranes containing
negatively charged lipids. The affinity of the basic domains for acidic
phospholipids could provide the cell with a targeting mechanism to the
inner leaflet of the plasma membrane, which contains up to 30%
negatively charged lipids (Op den Kamp, 1981). In this respect,
McLaughlin and co-workers have proposed that the myristoyl moiety and
the basic domain of MARCKS act synergistically to firmly anchor the
protein to acidic lipid membranes (Kim et al., 1994b)
explaining the association of this protein with intracellular membranes
(Albert et al., 1986; Thelen et al., 1991) (for a
review see Vergères et al.(1995)).
The MARCKS family comprises two groups of proteins. The members of the first group, referred as to MARCKS, are 30-35-kDa proteins encoded by the macs genes. The second group, referred as to MARCKS-related protein (MRP), is composed of 20-kDa proteins encoded by the mrp genes and also called MacMARCKS and F52 in the literature (Aderem, 1992; Blackshear, 1993). Although macs and mrp are ubiquitously expressed, higher levels of mRNA are found in brain (MARCKS and MRP) and in reproductive tissues (MRP) (Lobach et al., 1993). The N terminus, which contains the consensus sequence for myristoylation, and the basic domain with the site of phosphorylation by protein kinase C, are conserved in MRP and MARCKS (Fig. 1). Receptor-mediated activation of protein kinase C alters the subcellular localization of MARCKS. Phosphorylation of the basic domain results in the translocation of MARCKS from the plasma membrane to the cytosol. This phenomenon is reversible, since dephosphorylation of MARCKS is concomitant with the relocation of the protein at the plasma membrane (Thelen et al., 1991). Recently, it was proposed that phosphorylation induces the cycling of MARCKS between the plasma membrane and lysosomes (Allen and Aderem, 1995). Although the molecular mechanisms underlying the association of MARCKS with membranes have been relatively well characterized (Taniguchi and Manenti, 1993; Kim et al., 1994a, 1994b; Allen and Aderem, 1995), nothing has been known about MRP so far. In analogy to MARCKS, one would expect that MRP binds to membranes. The slight differences in the sequences of the N terminus and of the basic domain might, however, modulate the binding and consequently give us some insights on how myristoylated proteins interact with membranes (Fig. 1). For these reasons we have purified the unmyristoylated form (unmyr MRP) as well as the myristoylated form (myr MRP) of recombinant murine MRP expressed in Escherichia coli and investigated the ability of the myristoyl moiety and of the basic domain to promote membrane binding. Although the myristoyl moiety is believed to contribute to the binding of proteins to membranes by inserting into the bilayer, no direct proof of this putative hydrophobic interaction has been presented until now. We have therefore also investigated the membrane topology of murine MRP and bovine MARCKS using a novel photoreactive phospholipid probe.
Figure 1: Structure of proteins of the MARCKS family. The amino acid sequence of the myristoylated N terminus, which contains the consensus sequence GXXXS recognized by NMT, as well as of the basic domain, which contains the serines phosphorylated by protein kinase C, are shown for murine MRP (uppersequence). Nonconserved residues (underlined in MRP sequence) are shown for murine and bovine MARCKS (lowersequences). The amino acid residues are numbered (top) according to the MRP sequence.
Two ml of LB containing the appropriate antibiotics were inoculated
with 20 µl of an overnight culture of either the
JM109(DE3)-pET3dF52M1 or the JM109(DE3)-pBB131NMT-pET3dF52M1 strain and
grown for 2 h at 37 °C. The culture was transferred to a tube, in
which 100 µCi of [H]myristate in ethanol had
previously been dried, and grown for an additional hour. The expression
of MRP and NMT was induced by adding 0.4 mM IPTG, and the
cells were grown for 5 additional hours before harvesting.
where f is the fraction of MRP bound to the
membrane. f
was calculated by estimating the
concentration of MRP in the supernatant following centrifugation of the
vesicles as described in the preceding paragraph. [L] (M) is the accessible lipid concentration and can be estimated
to 50% of the total lipids for 100-nm vesicles. K
(M
) relates the mole ratio of MRP
bound to the vesicles to the unbound MRP concentration and can be
regarded as an apparent association constant. K
was obtained by a least square fitting of the above equation. The
reciprocal of K
, 1/K
(M), can be regarded as an apparent dissociation
constant that is equal to the lipid concentration at which 50% of MRP
is bound to the vesicles (Peitzsch and McLaughlin, 1993).
Addition of IPTG to a log
phase culture of JM109(DE3) cells containing the plasmid pET3dF52M1
encoding MRP induces the expression of a heat-stable protein, which
appears as a double band on a 10% SDS-polyacrylamide gel and whose
apparent molecular weight is 37 kDa (Fig. 2, lane4). Since the double band is not present in the strain
containing the pBB131NMT plasmid only (lane2), the
protein results from transcription of the mrp gene.
Coexpression of NMT with MRP results in a slight decrease in the
apparent molecular weight of the double band (lane6). That this shift results from myristoylation of MRP is
shown in Fig. 3. When E. coli cells are grown in the
presence of [H]myristate a double band, whose
apparent molecular weight corresponds to MRP, is labeled radioactively
in the strain coexpressing MRP and NMT (lane1) but
not in the strain expressing MRP only (lane2).
Inspection of the Coomassie Blue-stained gel (Fig. 2) shows that
the myr MRP double band is apparently not significantly contaminated
with unmyr MRP (lane6). NMT can therefore
efficiently myristoylate MRP (>90%) in the E. coli strain
JM109(DE3). A comparison of the intensities of the Coomassie
Blue-stained MRP bands of the heated cell extracts with known amount of
the purified protein (see next paragraph) shows that approximately 20
mg of MRP is produced per 5 g of cell paste (about 1.5 liters of
culture). In the absence of IPTG, unmyr (lane3) and
myr (lane5) MRP are expressed to a lower but
measurable extent, showing that the DE3lacUV5 promoter is partially
leaky. Finally, we have observed that the starting pH of the media has
an effect on the expression of MRP. Since the expression gradually
decreases above pH 7.5 (data not shown) we have chosen a starting pH of
6.5 for the LB media.
Figure 2: Expression of MRP in E. coli. The E. coli strains JM109(DE3) containing the plasmids with the genes coding for NMT (lanes1 and 2), MRP (lanes3 and 4), and MRP + NMT (lanes5 and 6) were grown in the absence (lanes1, 3, and 5) or in the presence (lanes2, 4, and 6) of 0.4 mM IPTG. The cells were lysed for 10 min at 95 °C in the presence of 1% Triton X-100, and the equivalent of 150 µg, wet weight, of cell paste was loaded on a 10% polyacrylamide SDS gel. Proteins were visualized by Coomassie Blue staining. Molecular weight standards are shown to the right. The MRP double bands are indicated with arrowheads.
Figure 3:
Myristoylation of MRP in E. coli. The equivalent of 150 µg, wet weight, of cells grown in the
presence of [H]myristate was lysed as described
in Fig. 2and analyzed on a 10% SDS-polyacrylamide gel. The
radioactive label was detected by fluorography. Lane1, heated extract of cells containing the plasmids with
the genes coding for MRP and NMT. Lane2, cells
containing the plasmid coding for MRP only.
Figure 4: Purification of MRP. Coomassie Blue-stained SDS-polyacrylamide gel (10%) of 0.4 µg each of purified unmyr MRP (lane2) and myr MRP (lane3). Molecular mass standards (67, 43, and 30 kDa) are shown in lane1.
Since protein kinase C-dependent phosphorylation and binding to calmodulin are two hallmarks of proteins of the MARCKS family, we have characterized the interactions of purified MRP with these proteins. The binding of MRP to calmodulin was assessed using dansyl-calmodulin as a probe (Fig. 5). Incubation of 50 nM dansyl-calmodulin with 250 nM unmyr and myr MRP induces a shift in the fluorescence emission spectrum of dansyl from 500 to 490 nm as well as a 2.3-fold increase in the fluorescence intensity at 490 nm. These data show that both unmyr and myr MRP bind to calmodulin, in agreement with the observed retention of the proteins on a calmodulin affinity column. Finally, both unmyr and myr MRP are substrates for PKM, the catalytic fragment of protein kinase C, as judged by the incorporation of radioactive phosphate in the MRP double band (Fig. 6, lanes1 and 2, respectively).
Figure 5:
Binding of MRP to calmodulin. 250 nM unmyr (3) and myr (4) MRP were incubated with 50
nM dansyl-calmodulin in the presence of 1 mM CaCl. Fluorescence spectra were recorded between 390
and 600 nm with excitation at 340 nm. 1, buffer; 2,
calmodulin alone.
Figure 6:
Phosphorylation of MRP by PKM. 1
µM unmyr (lane1) and myr (lane2) MRP were incubated with 2.5 nM PKM in the
presence of 200 µM [P]ATP (0.25
µCi) and 10 mM MgCl
for 2 h at 37 °C. The
samples were analyzed on a 10% SDS-polyacrylamide gel. A,
Coomassie Blue staining; B,
autoradiography.
The appearance of MRP
as a double band raises the possibility that E. coli might
produce heterogeneous proteins. Several lines of evidence suggest,
however, that this is not the case. First, the double band could never
be separated on the various columns used for the purification. In
addition, myristoylation as well as phosphorylation do not alter the
double band pattern, indicating that the structure of the N terminus
and the phosphorylation state of the basic domain are identical for
each band. Finally, MRP is also recognized as a double band on Western
blots of macrophage extracts, ()suggesting that this pattern
might result from a conformational equilibrium between two forms of the
protein in electrophoresis buffer as a consequence of the weak affinity
of the negatively charged SDS for the acidic MRP.
Figure 7: Binding of MRP to phospholipid vesicles. 1 µM MRP was incubated with 2.5 mM PC/PS (4:1) (lane2) or 2.5 mM PC (lane3) sucrose-loaded vesicles. The vesicles were pelleted by centrifugation, and 25 µl of the supernatant, containing unbound MRP, was loaded on a 10% SDS-polyacrylamide gel. The gel was stained with silver. In a control experiment, the vesicles were omitted (lane1). Upperpanel, unmyr MRP; lowerpanel, myr MRP.
Figure 8:
Determination of apparent partition
coefficients for the binding of MRP to PC/PS (4:1) vesicles. 1
µM myr (A) and unmyr (B) MRP were
incubated with increasing amounts of PC/PS (4:1) vesicles and
centrifuged to separate free from membrane-bound protein. The
percentage of MRP bound to vesicles was calculated following scanning
densitometry of silver-stained SDS-polyacrylamide gels as described
under ``Experimental Procedures'' and is expressed as a
function of the accessible lipid concentration. The datapoints from five experiments are shown. The data is
fitted according to (see ``Experimental
Procedures'') using K values of 2
10
M
for unmyr MRP and 5
10
M
(brokencurve) and 8
10
M
(plaincurve) for
myr MRP.
Figure 9:
Structure of (I)-TID-PC/16.
Fig. 10shows the
results of the labeling experiments with MARCKS and MRP. In the
presence of PC/PS (4:1) vesicles, myr MARCKS and MRP are heavily
labeled. In the absence of acidic lipids, i.e. with PC
vesicles, the extent of labeling of myr MARCKS and MRP is decreased
more than 10-fold. When unmyr MARCKS and MRP are photolyzed in the
presence of PC/PS (4:1) vesicles (arrowheads), only a weak
labeling is observed. This labeling is, however significantly over
control values; we have estimated that unmyr MRP incorporates as much
as 10-15% of the radioactivity compared with the myristoylated
protein. In order to exclude the possibility that the radioactivity
associated with the proteins results from noncovalent binding of
unreacted reagent, vesicles containing (I)-TID-PC/16 were
photolyzed prior to incubation with unmyr MARCKS or MRP. These
controls, which are shown for the myristoylated proteins on the rightpanel, demonstrate that noncovalently bound
reagent is almost completely removed and consequently does not
contribute to the labeling observed when the solutions are photolized
subsequent to incubation of the proteins with the vesicles.
Figure 10:
Hydrophobic photolabeling of MRP and
MARCKS. unmyr as well as myr MRP and MARCKS were incubated with 100-nm
vesicles containing (I)-TID-PC/16. The vesicles consisted
of either PC or PC/PS (4:1). Following photolysis of the
protein-vesicle solutions, lipids were removed on DE52 gel, and the
protein-bound label was detected on a 10% SDS-polyacrylamide gel. The arrowheads indicate the position of the unmyristoylated
proteins. A, Coomassie Blue staining; B,
autoradiography.
The weak
labeling of the unmyristoylated proteins strongly suggests that the
major site of labeling with (I)-TID-PC/16 is the
myristoyl moiety. To confirm this hypothesis we have used the ability
of cell extracts to enzymatically demyristoylate MARCKS (Manenti et
al., 1994). myr MARCKS and MRP were labeled with (
I)-TID-PC/16, delipidated on DE52 gel, and finally
demyristoylated with a cytosolic fraction of brain synaptosomes (Fig. 11). Demyristoylation can easily be checked with MARCKS
since removal of the fatty acid shifts the apparent molecular mass of
the protein from 80 to 70 kDa (Fig. 11A) (Manenti et al., 1994). Since the 70-kDa band (arrowhead)
contains <5% of the radioactivity originally associated with the
intact protein, we conclude that (
I)-TID-PC/16 is mostly
bound to the myristoyl moiety of MARCKS (Fig. 11B). The
demyristoylation experiments with MRP follow essentially the same
pattern. Upon incubation with the synaptosomal extract, MRP is shifted
to an apparent higher molecular weight on SDS-polyacrylamide gel (panelA), suggesting that the protein is
demyristoylated (see Fig. 2and Fig. 4). Most of the
radioactivity originally associated with the protein is removed
following demyristoylation (panelB). The fraction of
label still associated with the protein after demyristoylation (about
30%) is, however, significantly higher than for MARCKS. Because of the
relative minor difference in apparent molecular weights between myr and
unmyr MRP (see Fig. 2and Fig. 4) it is unclear whether
the label remaining associated with the protein reflects an incomplete
removal of the myristoyl moiety or results from the labeling of other
parts of the protein. Finally, upon demyristoylation of MARCKS and MRP
photolyzed in the presence of PC vesicles, the low amount of label
associated with the proteins is removed (Fig. 11).
Figure 11:
Demyristoylation of photolabeled MRP and
MARCKS. Following photolysis of vesicles incubated with myr MRP and
MARCKS, the solutions were delipidated. Myr MARCKS and MRP were
demyristoylated enzymatically with cytosolic extracts of brain
synaptosomes and subsequently loaded on a 10% SDS-polyacrylamide gel.
Demyristoylation was assessed by analyzing the apparent molecular
weight of the proteins on a Coomassie Blue-stained 10%
SDS-polyacrylamide gel (A). (I)-TID-PC/16
remaining associated with MARCKS and MRP was detected by
autoradiography (B). The arrowheads indicate the
position of the unmyristoylated proteins.
Interestingly, the labeling of MARCKS with (I)-TID-PC/16 does not change the apparent molecular mass
of the protein, whereas addition of the myristoyl moiety increases the
apparent molecular mass from 70 to 80 kDa (Fig. 11). These
observations indicate that, in contrast to the phospholipid analogue (
I)-TID-PC/16, the myristoyl moiety might specifically
alter the conformation of MARCKS and consequently its affinity for SDS.
Another intriguing observation is that the myristoyl moiety of MARCKS
can still be removed following labeling with (
I)-TID-PC/16. The putative ``demyristoylase''
therefore most likely does not specifically recognize the myristoyl
moiety of MARCKS. The information contained at the N terminus of MARCKS
is thus likely to be important for enzymatic demyristoylation.
In order to investigate how MRP interacts with membranes we have compared the binding of unmyr and myr MRP to vesicles of different lipid compositions. As a source of proteins we have used an E. coli system in which myr MRP can be produced by coexpressing yeast NMT (Blackshear et al., 1992). Both unmyr and myr MRP were expressed, purified to homogeneity under mild conditions, and characterized. We have shown that both unmyr and myr MRP bind dansyl-calmodulin and are phosphorylated by the catalytic fragment of protein kinase C. Whether myristoylation modulates the interactions of MRP with calmodulin and protein kinase C is currently under investigation and will be reported elsewhere.
The role of the basic
domain was analyzed by changing the lipid composition of the vesicles
and introducing acidic lipids to mimic the composition of the plasma
membrane (Kim et al., 1994a, 1994b). Both unmyr and myr MRP
require acidic lipids for their binding, in agreement with the model
proposed for MARCKS that predicts that the basic domain interacts
electrostatically with acidic lipid membranes (Taniguchi and Manenti,
1993; Kim et al., 1994a, 1994b). The partition coefficient of
unmyr MRP in the presence of PC/PS (4:1) vesicles is, however, 3 orders
of magnitude smaller than for the peptide corresponding to the basic
domain of MARCKS (K = 4
10
M
) (Kim et al., 1994a). This
large difference might result from a steric hindering of the basic
domain in the protein. Alternatively, negative charges, close to the
basic domain, could inhibit the binding of MRP to acidic lipid
membranes via electrostatic repulsion, as suggested for MARCKS (Kim et al., 1994b). A third explanation might also be found in the
structure of the basic domain; MRP contains a proline at position 96
instead of a serine in other MARCKS proteins (see Fig. 1). Since
the basic domain has been hypothesized to be an amphipathic
-helix
(Graff et al., 1989), this proline might alter the structure
of the helix by introducing either a
30-degree kink or a turn
(Richardson and Richardson, 1989) and potentially decrease the
partition coefficient of MRP. This hypothesis is supported by the
observation that unmyr MARCKS has a 50-fold higher affinity for PC/PS
(4:1) vesicles (K
= 1.3
10
M
) (Kim et al., 1994b).
Expression of myristoylation-deficient mutant proteins shows that
the myristoyl moiety is necessary to promote the binding of otherwise
acylated proteins to membranes (see Introduction). Our results show
that myristoylation is clearly not sufficient for membrane binding
since we have estimated that the apparent partition coefficient of myr
MRP in the presence of PC vesicles is smaller than 10M
. The affinity of myr MRP for
neutral membranes is therefore at least 2 orders of magnitude smaller
than for myristoylated model peptides, which have partition
coefficients of 10
M
(Peitzsch
and McLaughlin, 1993). This observation suggests that the myristoyl
moiety of MRP is in a conformation that inhibits its insertion into
neutral membranes. Furthermore, in the presence of acidic lipid
membranes, the myristoyl moiety only increases the binding of MRP to
PC/PS (4:1) vesicles by a factor of 2-4. In contrast to MARCKS
(Kim et al., 1994b), a strong cooperative effect between the
basic domain and the myristoyl moiety is not observed, and we therefore
propose that the major determinant of the binding of MRP to pure lipid
membranes is the basic domain rather than the myristoyl moiety.
Interestingly, the conclusion that the myristoyl moiety of a protein is
not required for its binding to phospholipid vesicles has also been
proposed for the ADP-ribosylation factor ARF1 (Franco et al.,
1993) and is likely to be extended to other proteins.
Fluorescent
fatty acid labels incorporated into the palmitoylation sites of
rhodopsin have demonstrated that the palmitoyl moiety of this protein
is situated in the membrane (Moench et al., 1994). In spite of
the considerable recent interest in myristoylated proteins, a direct
demonstration of such a topology for myristoyl moieties has not been
presented so far. In order to investigate this aspect, we have
photolabeled MRP as well as MARCKS with (I)-TID-PC/16.
The observations that the myristoyl moiety is required to obtain a
strong labeling and that demyristoylation results in a loss of the
label, demonstrate that the myristoyl moiety of MRP and MARCKS is
incorporated in the bilayer. This represents to our knowledge the first
direct evidence that the fatty acid moiety of myristoylated proteins
can insert into phospholipid bilayers. In the presence of PC vesicles,
the labeling of myr MRP and MARCKS is drastically reduced,
demonstrating that electrostatic interactions are required to promote
the insertion of the myristoyl moiety into the membrane.
A low but
nonetheless significant labeling can be observed when unmyr MRP and
MARCKS are photolized in the presence of PC/PS (4:1) vesicles. This
observation indicates that secondary hydrophobic interactions might
also be involved in membrane binding. The basic domain, which can be
modeled as an amphipathic -helix, contains 6 hydrophobic residues,
which are clustered in two domains of the putative
-helix (Graff et al., 1989). These residues could potentially insert in the
hydrophobic bilayer and participate in the binding. In this respect,
replacement of Phe
and Phe
(Phe
and Phe
in the MRP sequence) by alanines results in
a twofold decrease in the binding of the basic domain of MARCKS to
membranes (Kim et al., 1994a). In analogy to MARCKS proteins,
the N-terminal domain of the ADP-ribosylation factor ARF1 also contains
a repeat of hydrophobic residues, IFXXLFXXF, whose
structure is compatible with that of an amphipathic helix and which
might be critical to the membrane attachment of that protein (Kahn et al., 1992).
myr MRP binds to PC/PS (4:1) vesicles with a
partition coefficient of 5-8 10
M
which is 20-30-fold smaller
than for MARCKS (Kim et al., 1994b). Whereas the affinity of
MARCKS for PC/PS (4:1) vesicles is high enough to account for a stable
binding of the protein at the plasma membrane (Kim et al.,
1994b), the partition coefficient of MRP is clearly insufficient. Since
myristoylation does not drastically increase the binding of MRP to
membranes, the cooperative model proposed for MARCKS (Kim et
al., 1994b) cannot be applied to MRP. Based on our finding, as
well as on the conclusions obtained for MARCKS by McLaughlin and
co-workers (Peitzsch and McLaughlin, 1993; Kim et al., 1994a,
1994b), we propose a model for the binding of MRP to membranes (Fig. 12). In solution, the myristoyl moiety of MRP is in a
close conformation. The basic domain targets MRP to a negatively
charged membrane, such as the plasma membrane. This interaction is too
weak to allow a stable anchoring of the protein but allows an efficient
insertion of the myristoyl moiety into the bilayer. This particular
conformation is then recognized by a membrane protein, which in turn
provides the additional binding energy to firmly anchor MRP at the
membrane.
Figure 12: Putative model for the binding of MRP to acidic lipid membranes.