(Received for publication, June 14, 1994; and in revised form, October 17, 1994)
From the
The histidine-rich protein hisactophilin is known to be associated with the inner surface of the plasma membrane and to be present as a soluble protein in the cytoplasm of Dictyostelium discoideum cells. Mass spectrometry of hisactophilin from the cytosol or extracted from a membrane fraction showed that none of the hisactophilin purified from D. discoideum cells had the mass predicted from the known cDNA-derived amino acid sequence of the protein. Electrospray mass spectrometry and liquid secondary ion mass spectrometry of tryptic fragments separated by reversed-phase high performance liquid chromatography (HPLC) identified the most hydrophobic peptide as a myristoylated fragment from the N terminus of hisactophilin. Taken together the analytical data, it is concluded that all hisactophilin in D. discoideum cells is N terminally modified by myristoylation. By reversed-phase HPLC, two isoforms of hisactophilin, HsI and HsII, were recovered from the cytosolic as well as the membrane fraction of D. discoideum cells. Whereas the masses of HsI fragments produced by trypsin fit into the previously published sequence of hisactophilin (myristoylation considered), HsII is another protein distinguished from HsI by several amino acid exchanges. HsI and HsII can form homo- and heterodimers by disulfide bridges. Hisactophilin is phosphorylated in vivo. Both isoforms proved to be substrates of membrane-associated threonine/serine kinase from D. discoideum, which may regulate the interaction of hisactophilin with the plasma membrane.
Highly motile amoeboid cells as those of Dictyostelium need sophisticated mechanisms for coupling of their actin skeleton
to the plasma membrane. In order for a cell to move, it is essential to
regulate this coupling locally at the leading edge and also at the
sites of contact with a surface to which the cell adheres. In Dictyostelium cells, two proteins have been described that are
capable of coupling actin filaments to membranes. One of them is
ponticulin, a transmembrane glycoprotein that attaches laterally to
actin filaments and binds them to the cytoplasmic phase of the cell
envelope(1, 2) . The other protein is hisactophilin,
which exists in a plasma-membrane bound and a free cytoplasmic state
and binds in vitro to actin in a strictly pH-dependent
manner(3) . The high content of 26% histidine residues is
thought to be responsible for binding of the protein to actin at pH
values below the pK of histidine and its dissociation from
actin filaments beyond pH 7.0. NMR has shown that hisactophilin
consists of a barrel, which forms a backbone around a 3-fold axis
of symmetry, and of three major loops exposed on the surface of the
molecule which contain nearly all of the histidine
residues(4) .
In this paper modifications of hisactophilin
are studied which might be implicated in reversible attachment to the
plasma membrane. Earlier results have indicated that during metabolic
labeling of D. discoideum cells with
[H]palmitic acid radioactive label is
incorporated into hisactophilin(5) . Here we demonstrate that
all hisactophilin is myristoylated in D. discoideum cells. In
addition, evidence is provided for the presence of two isoforms of
hisactophilin and their phosphorylation by a membrane-associated
kinase.
HPLC was performed using Kontron Instruments
(München, Germany) pump 420 and detector 430 at a
wavelength of 206 nm and a RP18 Lichrospher column (125 4 mm;
Merck, Darmstadt, Germany). A gradient system of solvent A (0.1%
trifluoroacetic acid (Sigma, Deisenhofen, Germany) in water) and
solvent B (0.1% trifluoroacetic acid in acetonitrile; Lichrosolv,
Merck) at a flow rate of 1 ml/min was used throughout. Linear gradients
of 0-80% B in A in 80 min were used for final purification of the
protein, and isoforms HsI and HsII were separated by a linear gradient
of 0-35% B in A in 10 min, followed by an isocratic elution at
35% B. Fractions were collected manually and concentrated by
evaporation in a Speed Vac centrifuge.
For proteolytic cleavage 1 mg/ml of HPLC-purified, denatured, and reduced hisactophilin was digested with either trypsin or endoprotease Lys-C at 37 °C. Trypsin, sequencing grade (Boehringer Mannheim, Germany), was added at a concentration of 0.1 mg/ml in 100 mM Tris-HCl, pH 8.5, with 10% acetonitrile. For peptide sequencing endoprotease Lys-C, sequencing grade, was used for cleavage at a concentration of 0.01 mg/ml in buffer containing 50 mM Tris-HCl, 50 mM Tricine, pH 8.5, 1 mM EDTA, and 10% acetonitrile. The peptides were fractionated by HPLC using the same column and solvents as for uncleaved hisactophilin. A linear gradient of 0-80% B in A in 80 min was employed to separate tryptic fragments, and a gradient of 0-50% solvent B in A in 100 min for Lys-C cleavage products.
For LSI-MS analysis in a mass spectrometer MAT900 (Finnigan MAT, Bremen, Germany), the peptides were dissolved in acetonitrile/acetic acid/water (4:1:5) and mixed with the matrix (glycerol/monothioglycerol 1:1). The energy of the ionizing cesium ions was 20 keV.
For gas chromatography-mass spectrometry analysis the mass spectrometer CH7A (Finnigan MAT) was coupled with a gas chromatograph HRGC 5160 (Carlo Erba, Milano, Italy). The all-glass system consisted of a silica capillary coated with Durabond 1 (S & W. Scientific, Folsan, CA), which was introduced directly into the ion source (70 eV). The temperature program was 5 min at 100 °C, following 5 °C/min up to 250 °C with a helium flow of about 1 ml/min. The inlet temperature was 260 °C. For derivatization, the peptides were hydrolyzed as for amino acid analysis, evaporated, dried, and treated with hexamethyldisilazan/trimethyl-chlorosilan/pyridin (2:1:7) (Pierce) according to Sweeley et al.(8) .
Peptides of HsII were sequenced using a pulsed liquid-phase sequencer 477 equipped with an on-line phenylthiohydantoin-analyzer 120A (Applied Biosystems).
Figure 1: Comparison of hisactophilin purified from Dictyostelium cells (D.d.) with hisactophilin expressed in transformed E. coli cells (E.c.). The proteins were chromatographed under identical conditions on a reversed-phase column with a linear acetonitrile gradient. The bar below the curves indicates the fractions of Dictyostelium hisactophilin pooled for preparing fragments as shown in Fig. 2.
Figure 2: Separation of tryptic fragments of hisactophilin from Dictyostelium cells by reversed-phase HPLC. The most hydrophobic fragment eluting at 56-60% acetonitrile (bar) was analyzed by LSI-MS. The inset shows the presence of a single mass corresponding to that of the 3 N-terminal amino acids G-N-R plus one myristoyl moiety.
To answer the first
question, modification of the hisactophilin by fatty acid was examined.
Labeling by [H]fatty acids in vivo revealed that the label from palmitic acid as well as myristic
acid was incorporated into hisactophilin. To determine the type of
bond, the entire HPLC fraction of cytosolic Dictyostelium hisactophilin was treated with hydroxylamine at pH 7 or 10 and
rechromatographed by HPLC. Presence of thioester or ester bonds should
result in a shift in retention time toward the unmodified bacterially
expressed hisactophilin. No shift was found and the two peaks did not
merge, suggesting modification of hisactophilin from Dictyostelium cells by hydroxylamine stable amide bonds. Metabolic labeling with
[
H]myristic acid or
[
H]palmitic acid gave a similar result: the
tritium label was not removed from hisactophilin by treatment with
hydroxylamine at pH 10.
In order to determine the type of fatty acid
linked to hisactophilin and to localize the bond, the protein was
cleaved with trypsin, and the fragment with the highest retention time
on a reversed-phase HPLC column was analyzed by LSI-MS (Fig. 2).
The peak of the MH-ion was found at m/z = 556.1 (Fig. 2, inset). Amino acid
analysis of this fragment resulted in Gly, Asx, and Arg in equimolar
ratios, consistent with the sequence G-N-R of the N-terminal tryptic
cleavage product predicted from the known hisactophilin
sequence(3) . The fragment differed in mass from the tripeptide
by 210 Da, which is exactly the mass of a myristoyl residue. In order
to confirm that the N terminally bound fatty acid is myristic acid, an
aliquot of the tryptic fragment was hydrolyzed and trimethylsilylated.
Analysis by gas chromatography-mass spectrometry revealed only one
major peak in the gas chromatogram, which coincided in retention time
and mass spectrum with authentic trimethylsilylester of myristic acid.
Figure 3: Separation of two isoforms of hisactophilin by isocratic HPLC at 35% acetonitrile. Peak A represents hisactophilin II, and peak B represents hisactophilin I.
Both the proteins of peak A and B reacted with antibodies mAb 54-11-10 and 54-165-12 previously used to label hisactophilin(3, 14) . In the following we refer to the protein of peak B as hisactophilin I (HsI), and to the protein of peak A as hisactophilin II (HsII). The same peptide of high retention time with the mass of myristoyl-G-N-R was found by LC-MS after trypsin treatment of both proteins, indicating that myristoylation is common to hisactophilins I and II.
Under non-reducing conditions, three different masses were detected in mixtures of purified HsI and HsII, which coincided with the predicted masses of homo- and heterodimers. Covalent modification of cysteine residues by 4-vinylpyridine resulted in masses of 13,641 and 13,820 Da, which correspond to the masses of HsI and HsII plus 105 Da, the mass of one ethylpyridine moiety/hisactophilin monomer. These results indicate 1 free cysteine residue to be present in each hisactophilin isoform.
A portion of
hisactophilin is known to be attached in Dictyostelium cells
to the inner face of the plasma membrane. To examine whether the same
isoforms as from the cytosol can be isolated from a membrane fraction,
hisactophilin was purified from a 10,000 g pellet of a Dictyostelium cell homogenate. The hisactophilin was extracted
with non-ionic detergent from the 10,000
g pellet and
purified essentially as the cytosolic one. HPLC of the extracted
hisactophilin revealed two peaks with retention times coincident with
those of HsI and HsII from the cytosol. ES-MS established that the
masses of the two isoforms from the membrane fraction corresponded to
the masses of HsI (13, 536 Da) and HsII (13, 715 Da) as purified from
the cytosol (Fig. 4). However, the ratios of the two isoforms
might differ in the soluble and detergent-extracted fraction: in the
material purified from the cytosol an excess of HsI over HsII was
found, in the membrane extract more HsII than HsI was recovered.
Figure 4: Comparison of hisactophilin isoforms from the cytosol and membrane fraction. Reconstructed masses of hisactophilins from the cytosolic fraction (A) and membrane extract (B) determined by ES-MS are shown.
The
isoforms purified by HPLC (Fig. 3) could also be separated by
isoelectric focusing in a shallow pH gradient. The less basic isoform
proved to be HsI, and the slightly more basic form HsII (Fig. 5). Bacterially expressed hisactophilin focussed
essentially as HsI. As judged from the
[H]myristic acid label incorporated into the
isoforms and from immunoblotting, the ratio of HsI to HsII was >1 in
the cytosolic fraction and about 1 in an extract from membranes.
Figure 5:
Separation of hisactophilins I and II from
cytosolic and membrane fractions by two-dimensional-electrophoresis. Horizontal direction, isoelectric focussing; vertical
direction, SDS-PAGE. Top panels, cells were metabolically
labeled with [H]myristic acid during starvation
until harvest at 8 h of development. Hisactophilins were
immunoprecipitated and, after electrophoresis, fluorographed for
H incorporation. Bottom panels, proteins were
separated under the same conditions and subjected to immunoblotting
with hisactophilin-specific antibody.
In summary, the two hisactophilin isoforms separated by HPLC have different but uniform masses, they give rise to the same N-terminal peptide corresponding to the mass of myristoyl-G-N-R and, finally, hisactophilins purified from cytosolic and membrane fractions have the same masses. From these results we conclude that all hisactophilin in D. discoideum cells is myristoylated.
Figure 6:
Comparison of the masses of tryptic
fragments from hisactophilin I and II as detected by LC-MS with the
known hisactophilin I sequence. Cleavage sites for trypsin as inferred
from the HsI sequence are indicated and the corresponding masses of
fragments shown on top of the sequence. All predicted masses of HsI
fragments were detected by the electrospray MS ( = Cys
blocked with ethylpyridine). The HsII fragments are provisionally
aligned with HsI fragments according to similar mass and retention time
in a reversed-phase column.
Figure 7: Reversed-phase HPLC of fragments obtained by digestion with endoprotease Lys-C for mass determination and amino acid sequencing. The peaks analyzed are indicated by bars, and masses are indicated below the curve. N-terminal sequences of the two larger peptides were determined, and 2 amino acid replacements as compared to HsI were detected (underlined). The mass of the small fragment with the largest retention time corresponded to the N-terminal myristoyl-hexapeptide predicted from the HsI sequence as a Lys-C cleavage product.
Two
sharply separated fragments with masses of 3,706 and 2,766 Da,
respectively, did not coincide with any of the fragments predicted from
the HsI sequence. These fragments were N terminally sequenced. In the
3,706-Da fragment, 2 amino acid exchanges were discovered among the
first 17 amino acid residues: instead of Ala and
His
of HsI, Val and Gly were found in HsII. In the
2,766-Da fragment, the first 14 amino acid residues fit to the HsI
sequence, confirming that the two proteins designated as HsI and HsII
are closely related to each other. In addition, the mass of the HsII
fragment with the largest retention time, eluting from the HPLC column
behind the uncleaved protein (Fig. 7), was determined to be 902
Da. This fits exactly with the myristoyl-GNRAFK fragment predicted from
the HsI sequence. From the mass spectrometry and sequencing data, it
can be inferred that HsI and HsII have the same N-terminal region, but
that more than eight differences in sequence are distributed downstream
along their polypeptide chains.
The membrane fraction proved to be an appropriate source
of hisactophilin kinase. As a substrate, a mixture of HsI and HsII from
the cytosol was employed. Kinase extracted with detergent
cofractionated on a DE52-cellulose column with the hisactophilins, but
could be separated on a Sephacryl S-300 column. The kinase eluted
behind the excluded volume but in front of the hisactophilins, which
can be easily separated due to their atypical elution behind the salt
peak(3) . The kinase preparation was free of endogenous
substrate and did efficiently phosphorylate both hisactophilins with a
pH optimum of 6.4 and a temperature optimum at 30 °C. Activity of
the kinase required Mg but not Mn
ions, which suggested that the enzyme is a threonine/serine
kinase. This was confirmed by total hydrolysis of hisactophilins
phosphorylated in vitro with kinase from Dictyostelium cells and [
-
P]ATP (Fig. 8). 95%
of the label was recovered in phosphothreonine and 5% in phosphoserine.
No phosphotyrosine was detected.
Figure 8:
Phosphoamino acids of hisactophilin after
phosphorylation in vitro. A mixture of HsI and HsII as
purified from the cytosol was incubated with solubilized and partially
purified kinase from a crude D. discoideum membrane fraction
together with [-
P]ATP. After total
hydrolysis and electrophoretic separation of phosphoamino acids, an
autoradiograph was scanned for
P incorporation at the
positions of phosphotyrosine (Y), phosphothreonine (T), and phosphoserine (S).
In order to explore whether
hisactophilin is also phosphorylated in vivo, cells of D.
discoideum were metabolically labeled during growth with
[P]phosphate. Fig. 9shows that label is
incorporated into hisactophilin, indicating that within the cells
hisactophilin is not only modified by myristoylation but also subject
to phosphorylation.
Figure 9:
Phosphorylation of hisactophilin in
vivo. After growth in the presence of
[P]phosphate, cells were lysed with SDS. An
aliquot of the cell lysate, hisactophilin immunoprecipitates, and
controls were separated by SDS-PAGE and either stained with Coomassie
Blue (lanes 1-4), or blotted, followed by indirect
labeling with an anti-hisactophilin antibody, mAb 54-165-12, and
anti-mouse IgG conjugated to alkaline-phosphatase (lanes
6-8). Lane 1, crude cell lysate. Lanes 2 and 6, hisactophilin precipitated from the lysate with mAb
54-11-10. For comparison purified hisactophilin (lanes 3 and 7) and protein-A precipitated mAb 54-11-10 (lanes 4 and 8) were applied. Lane 5,
autoradiograph of lane 6 showing the
P-label on
hisactophilin. Although Coomassie Blue shows a single band of
precipitated protein which coincides with the antibody-labeled
hisactophilin band (lanes 2 and 6), the
autoradiograph visualizes multiple phosphorylated bands in addition to
the one of hisactophilin (lane 5). These traces of proteins
coprecipitating with hisactophilin might represent phosphorylated
kinases.
Figure 10: Scheme of hisactophilin insertion into the inner lipid layer of the plasma membrane. For constructing the diagram, the structure of bacterially expressed, non-acylated hisactophilin I as determined by NMR (4) was used. To the N terminus a myristoyl moiety was attached by modeling. Dark areas represent histidine residues exposed on the protein surface, which are thought to be involved in pH-sensitive actin binding. Membrane phospholipids were assembled by computer modeling. Part of an actin filament representing four monomers is shown according to scale. The nucleating effect on actin polymerization (3) suggests that hisactophilin binds to F-actin at a site matched by two monomers.
Hisactophilin efficiently binds at pH 6.5 to actin filaments(3) . Bundling of actin filaments by hisactophilin has been observed by Urban (5) . This activity is possibly a function of hisactophilin dimers that are produced under non-reducing conditions by disulfide bridge formation. The bundling activity indicates that hisactophilin binds laterally to F-actin. Hisactophilin also nucleates actin polymerization (3) , which suggests that it stabilizes actin oligomers by binding to a cleft between two monomers. In Fig. 10four monomers of an actin filament, as constructed from refined x-ray fiber diffraction data (17) , are shown in scale with hisactophilin I to demonstrate that sizes and shapes would fit.
Three mechanisms by which an equilibrium between
cytoplasmic and membrane-bound hisactophilin can be maintained in Dictyostelium cells will be discussed here. 1) As in the
subunits of heterotrimeric G proteins, membrane binding might be
supported by reversible, covalent linkage of palmitic acid to a
cysteine residue. In vivo radiolabel from myristic acid as
well as palmitic acid is incorporated into hisactophilin. However,
since hisactophilin I contains only 1 cysteine residue, which is free
in the purified protein, it is more likely that palmitic acid is
converted by the cells into myristic acid which is then linked to the N
terminus of hisactophilin. 2) Binding to negatively charged membrane
lipids might be enhanced by positively charged amino acids as in the
adhesion plaque protein MARCKS (20) and in proteins of the Src
family (21) , where lysine residues are clustered in the
N-terminal region. Phosphorylation by protein kinase C relieves MARCKS
from its membrane-bound state(15) . Since hisactophilin is
phosphorylatable by membrane-associated threonine/serine kinase, and is
also phosphorylated in vivo, this modification might be
responsible for dissociation of the protein from the membrane. 3) In
contrast to other myristoylated proteins(22) , the
hisactophilin I sequence does not show any clustering of lysine
residues in the N-terminal region. In HsI only 1 lysine but several
histidine residues are located in the polypeptide chain close to the N
terminus(3) . The pK of histidine of 6.8 makes the net
charge of hisactophilin sensitive to small changes in the cytoplasmic
pH. The presence of proton pumps in the membrane of endosomes (23) might be responsible for a rise of pH at the cytoplasmic
surface of these vesicles, which may cause hisactophilin to detach from
the endosomal membrane.
Note Added in Proof-The cDNA sequence of hisactophilin II (EMBL/Genbank accession number U13671) and its genomic sequence (A. A. Noegel, personal communication) confirm the mass-spectrometric data presented in this paper.