(Received for publication, April 13, 1995; and in revised form, July 25, 1995)
From the
N-Myristoyltransferase (NMT) catalyzes the
co-translational addition of myristic acid to the N-terminal glycine of
many cellular, viral, and fungal proteins which are essential to normal
cell functioning and/or are potential therapeutic targets. We have
found that bovine brain NMT exists as a heterogeneous mixture of
interconvertible high molecular mass multimers involving 60-kDa NMT
subunit(s). Gel filtration chromatography of partially purified NMT at
low to moderate ionic strength yields NMT activity eluting as 391
± 52 and 126 ± 17 kDa peaks as well as activity which
profiles the protein fractions and likely results from NMT
nonspecifically associating with background proteins and/or column
matrix. Chromatography in 1 M NaCl causes 100% of this
activity to elute as a single peak of
391 kDa. Subsequent treatment
of the
391 kDa activity peak with an NMT peptide reaction product (i.e.N-myristoyl-peptide) results in
75% of the
activity re-eluting as a
126-kDa peak in 1 M NaCl.
Rechromatography also yields small amounts of a
50-kDa NMT monomer
which increases with prior storage at 4 °C. Up to 5 NMT subunits
were identified by SDS-polyacrylamide gel electrophoresis and specific
immunoblotting with a human NMT peptide antibody and by
cofactor-dependent chemical cross-linking with an
I-peptide substrate of NMT. The prominent 60 kDa and
minor 57-, 53-, 49-, and 47-kDa NMT immunoblotted subunits co-migrate
with five of nine silver-stained proteins in an enzyme preparation
purified >7,000-fold with
50% yield by selective elution from
octyl-agarose with the myristoyl-CoA analog, S-(2-ketopentadecyl)-CoA. Storage at 4 °C also leads to
conversion of the larger NMT subunit(s) into 49 and 47 kDa forms with
no loss of NMT activity. These results identify two interconvertible
forms of NMT in bovine brain that result from NMT subunit
multimerization and/or complex formation with other cellular proteins.
The data also identify a fully active NMT monomer which arises from
subunit proteolysis. This study thus reveals a previously unappreciated
level of NMT complexity which may have important mechanistic and/or
regulatory significance for N-myristoylation in mammalian
cells.
N-Myristoylation is the co-translational covalent
attachment of myristic acid in amide linkage to the N-terminal glycine
residue of a number of mammalian, viral, and fungal
proteins(1, 2, 3, 4) . For many
proteins synthesized on free polyribosomes, co-translational N-myristoylation is required for their proper subcellular
compartmentalization and subsequent biological function. For example,
N-terminal myristate, in conjunction with N-terminal basic residues, is
instrumental in effecting interactions of the protein tyrosine kinase,
p60, with the plasma membrane (5) which
in turn is essential for
p60
-mediated cellular
transformation(6) . N-Myristoylation is also required
for the plasma membrane association of the polyprotein precursor of the
human immunodeficiency virus internal structural polyprotein, p55, from
which site it directs the assembly of virus capsids and their budding
from infected cells(7, 8) . N-Myristoylation
of certain proteins of Candida albicans and Cryptococcus
neoformans, pathogenic fungi which affect immunocompromised
patients, is essential for their vegetative
growth(9, 10) . The central role for myristate in
these processes has made N-myristoylation a chemotherapeutic
target for anti-viral (11, 12, 13, 14) and anti-fungal (9, 15, 16) therapies.
N-Myristoylation results from the catalytic transfer of
myristic acid from myristoyl-CoA to appropriate protein substrates by
myristoyl-CoA:protein N-myristoyltransferase (NMT) ()(EC 2.3.1.97). NMT has been purified to homogeneity from
the yeast Saccharomyces cerevisiae(17) , extensively
characterized(1, 17, 18, 19, 20, 21, 22, 23, 24, 25) ,
and shown to be a
53-kDa monomer. The hNMT gene has been isolated
by functional complementation of a yNMT temperature-deficient mutant
and predicts a
48-kDa protein having 44% homology with the yeast
enzyme(26) .
In contrast to yNMT, NMTs purified from several mammalian sources exhibit varying molecular masses, charge heterogeneity, and/or peptide substrate specificities. For example, while native NMT partially purified from human erythroleukemia cells (27) or purified to near homogeneity from bovine spleen (28) exhibits apparent molecular masses by gel filtration consistent with a monomeric 48-58-kDa enzyme, the enzyme from murine leukemia L1210 cells (29) and bovine brain(30, 31, 32, 33, 34, 52) display apparent molecular masses up to 390 kDa. Furthermore, at least two distinct forms of NMT have been isolated from L1210 cells (29) while chromatofocusing (30) and ion-exchange chromatography (32, 34) have resolved as many as four separate forms of the enzyme from bovine brain. Assuming that there is a single mammalian gene(26) , these data suggest the existence of homo- and/or heteromultimeric NMT complexes. The functional significance and molecular basis for multiple high molecular mass NMTs and NMT subunits ranging in size from 48 to 67 kDa remains unexplained(27, 28, 33, 35) .
To begin to address these questions, we have examined the hydrodynamic properties and subunit structure of bovine brain NMT (bNMT). We have determined that the bNMT exists in vitro as two multisubunit complexes which can be interconverted by manipulation of ionic strength and/or by treatment with an N-myristoyl-peptide product of the NMT reaction. We have also identified a subunit heterogeneity for bNMT which arises in part from proteolysis of larger subunit precursor(s) and which apparently accounts for the accumulation of a fully active monomeric form of the enzyme. Our data thus provide in one system an explanation for the disparity of data concerning the native NMT structure(s) including physiologically relevant processes which may account for the enzyme quaternary structure and activity. We also provide information for circumventing problems encountered during the manipulation and purification of NMT from complex tissues such as bovine brain.
Figure 1:
Gel filtration of bNMT. bNMT was
partially purified by (NH)
SO
fractionation and DEAE-Sepharose ion-exchange chromatography and
applied to a Sephacryl S-200 HR column equilibrated in gel filtration
buffer (50 mM potassium phosphate (pH 7.4) buffer and
proteolytic enzyme inhibitors) (part A) or gel filtration
buffer containing 1 M NaCl (part B). Fractions 34-37 (peak I) from part B containing NMT activity were concentrated, desalted, treated with
10 mMN-myristoyl-GSSKSKPKD and applied to a
Sephacryl S-100 HR column in gel filtration buffer containing 1 M NaCl (part C). Fractions were monitored for
absorbance at 280 nm (-) and for NMT activity(- - - -). Fractions
36-38 (peak II) from part C were pooled and
concentrated for subsequent purification and analysis. The apparent
molecular masses of the activity peaks are indicated by the arrows (see Table 1).
To test this hypothesis, the NMT activity (peak
I) from the Sephacryl S-200 HR column shown in Fig. 1B was pooled, concentrated, re-equilibrated in 50
mM potassium phosphate (pH 7.4) buffer without NaCl, and
treated with 10 mM of a synthetic N-myristoylated
peptide corresponding to amino acids 2-10 of p60 (i.e.N-myristoyl-GSSKSKPKD). In addition to
being amphipathic, the N-myristoyl-peptide is an enzymatic
product of the NMT reaction. Upon subsequent rechromatography on a
Sephacryl S-100 HR column equilibrated in 50 mM potassium
phosphate (pH 7.4) buffer containing 1 M NaCl, only
25% of
the applied activity eluted close to the original peak I while
the majority of the applied activity (
75%) eluted with an apparent
molecular mass identical with peak II and 1-2% of the
activity eluted as a third peak of
50 kDa (peak III) (Fig. 1C). Comparable results were obtained when the
experiment was carried out using Sephacryl S-200 HR, Superdex 200, or
Superose 12 columns (Table 1). When the activity in peak I (Fig. 1C) was again treated with 10
mMN-myristoyl-GSSKSKPKD and rechromatographed on the
same Sephacryl S-100 HR column as described above, the applied activity
eluted with a distribution similar to that shown in Fig. 1C except that the amount of activity in the
50 kDa peak
increased with lengthened time of storage at 4 °C prior to
rechromatography (data not shown). Furthermore, when the enzyme in peak
II was rechromatographed on a Superdex 200 column equilibrated
in 100 mM Tris-CAPS (pH 9.4) buffer containing 1 M NaCl, the NMT activity once again eluted as the original
391-,
126-, and
50-kDa peaks (data not shown). Together these
experiments provide evidence for two major forms of native bNMT of
391 and
126 kDa as well as a minor
50 kDa form which
accumulates during storage. The experiments also reveal an equilibrium
between the
391 and
126 kDa forms which is readily manipulated
by changes in ionic strength and/or by the presence of an N-myristoyl-peptide. Finally, our ability to manipulate this
multimerization phenomenon proved useful in the eventual purification
of the enzyme.
Figure 3: Silver staining and immunoblotting of bNMT. Aliquots from the Sephacryl S-100 HR NMT (1.5 µg of protein) and octyl-agarose NMT fractions (0.16 µg of protein) were analyzed by SDS-PAGE and either silver stained for protein or immunoblotted for bNMT subunits. Part A, silver stain: lane 1, Sephacryl S-100 HR NMT fraction; lane 2, octyl-agarose NMT fraction. Part B, immunoblot: lane 1, Sephacryl S-100 HR NMT fraction blotted with an affinity purified hNMT peptide antibody which had been preincubated with 20 µM peptide antigen (+); lane 2, Sephacryl S-100 HR NMT fraction blotted with untreated(-) antibody; lane 5, octyl-agarose NMT fraction blotted with an untreated(-) antibody.
Figure 2:
Cofactor-dependent radioiodine chemical
cross-linking of bNMT subunits. Part A, the autoradiography of
SDS-PAGE analysis of the radioiodine chemical cross-linking of bNMT
subunits in the Sephacryl S-100 HR NMT fraction (i.e. peak II from Fig. 1C). Equal aliquots of protein
(40-50 µg) were cross-linked with
GSSKSKPKDPSQRRR-I-Y and 1 mM
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in the presence of
increasing concentrations of the N-myristoyl-CoA analog, S-(2-ketopentadecyl)-CoA (lanes 1-4).
Competitive blocking of radioiodine cross-linking was demonstrated by
the addition to reaction mixtures (identical to that shown in lanes
3 and 5) of increasing concentrations of the
non-radioactive peptide (lanes 6-8). Lane 1,
control without(-) myristoyl-CoA analog; lane 2, 4
µM myristoyl-CoA analog; lane 3, 10 µM myristoyl-CoA analog; lane 4, 20 µM myristoyl-CoA analog; lane 5, 10 µM myristoyl-CoA analog; lane 6, 10 µM myristoyl-CoA analog plus 5 µM peptide; lane
7, 10 µM myristoyl-CoA analog plus 20 µM peptide; lane 8, 10 µM myristoyl-CoA analog
plus 50 µM peptide. Part B, radioactive
cross-linking shown in part A was quantitated by densitometry.
Radioactivity corresponding to the 64-66 kDa bands (p66+p64) or the 47 kDa band (p47) was
normalized to the level of radioactivity in the nonspecifically labeled
75 kDa band (p75) in each respective lane and plotted as a
percentage of the maximum radioactivity of the same band in lane 4 from part A. The percentage radiolabeling in lanes
1-4, and 6-8 of part A are presented
as bar graphs and explained in the legend on the
right.
Multiple NMT subunits were also identified in the purified enzyme preparations by immunoblotting with an affinity purified hNMT antibody. For this analysis, a polyclonal antibody was raised against a synthetic peptide encoded by the hNMT gene(26) . Antibody specificity was confirmed by immunoblotting of bacterially expressed hNMT and by total competition of that immunostaining by preincubation of the antibody with 20 µM of the peptide antigen (data not shown). Immunoblotting of the Sephacryl S-100 HR NMT fraction with this affinity purified antibody revealed a major stained band of 60 kDa and four lesser stained bands corresponding to polypeptides of 57, 53, 49, and 47 kDa (Fig. 3B, lane 2). Again, the immunostaining of all five bands was blocked by preincubation of the antibody with 20 µM of the peptide antigen (Fig. 3B, lane 1). A similar analysis of the octyl-agarose NMT fraction revealed the same immunostaining pattern (Fig. 3B, lane 3) except for the 53 kDa band which is not seen in Fig. 3B but which was evident in other blots from the same preparation, although at a reduced intensity compared to the blot from the Sephacryl S-100 HR NMT fraction (data not shown). These experiments thus identify five polypeptides in our most purified enzyme fractions with immuno-cross-reactivity to hNMT. Analysis of duplicate aliquots of the octyl-agarose NMT fraction by SDS-PAGE and either immunoblotting or silver staining revealed that the five immunostained bands had electrophoretic mobilities identical with five of the nine silver-stained bands (indicated by triangle markers in Fig. 3, A and B). Also, the pattern of immunoblotted bands (Fig. 3B, lane 2) was notably similar to the pattern of radioiodine cross-linked bands (see Fig. 2A) except for the difference in apparent molecular weights of the upper specifically cross-linked bands and the reduced resolution of the broad lower cross-linked band, most likely resulting from the addition of covalently linked peptide. These data thus reveal an exceptional coincidence of cross-linking, immunoblotting, and silver staining, which is consistent with the identification of up to 5 NMT subunits in the enzyme fractions purified from bovine brain.
The effect of storage on NMT
subunit activity and heterogeneity was also examined. Immunoblotting of
the octyl-agarose NMT fraction at different times during storage at 4
°C revealed a gradual conversion of the higher molecular mass NMT
subunits (i.e. 60, 57, and 53 kDa) into the lower molecular
mass forms (i.e. 49 and 47 kDa) over several months (Fig. 4A). During the same time period the
corresponding NMT activity was not significantly affected (Fig. 4C). However, when a fresh octyl-agarose NMT
sample was frozen at -15 °C for 10 days, the typical
immunostained pattern of five bands (Fig. 4A, lane
1) was converted to a doublet corresponding to the 47 kDa band and
a new slightly faster migrating species of 46 kDa (Fig. 4A, lane 5) while NMT activity was
concomitantly reduced by 30% (Fig. 4C, frozen). Subunit heterogeneity was also examined in a freshly
prepared tissue homogenate; the 60 and 47 kDa bands were in
approximately the same proportions as seen in the fresh octyl-agarose
NMT sample (see Fig. 4A, lane
1), while the other intermediate forms were not observed (data not
shown).
Figure 4: Effect of storage on bNMT heterogeneity. Aliquots of freshly purified octyl-agarose NMT fraction were stored refrigerated or frozen and analyzed by immunoblotting and silver staining and assayed for enzyme activity. Part A, immunoblotting of bNMT in a fresh octyl-agarose control (lane 1) or octyl-agarose stored at 4 °C for 3 days (lane 2), for 48 days (lane 3), for 70 days (lane 4), or for 10 days at -15 °C (lane 5). Part B, silver stain of fresh octyl-agarose control: fresh (lane 1) or frozen for 10 days (lane 2). Part C, bNMT activity for fresh octyl-agarose control, stored for 10, 38, 48, or 85 days at 4 °C, or frozen at -15 °C for 10 days. Each analysis represents the average ± range (n = 2) except for the control (mean ± standard error (n = 4)) and the 85 day (n = 1).
The effect of freezing on the NMT subunit pattern was also followed by silver staining (Fig. 4B). This analysis confirmed the loss of the 60 and 49 kDa bands. However, the retention of a 57 kDa silver-stained band after freezing suggested the presence of still another non-NMT protein which co-migrates with the 57-kDa NMT subunit in the octyl-agarose NMT fraction. Together, these data indicate that some or all of the 49- and 47-kDa subunits result from proteolytic processing of larger precursors. Since the same proportion of 60- and 47-kDa subunits were observed in freshly prepared brain extracts as were in the highly purified octyl-agarose NMT preparations and that the conversion of the 60-kDa subunit to the 49- and 47-kDa subunits required several months, it appears that this processing is very slow. It is likely, therefore, that NMT subunit heterogeneity is not totally the result of post-extraction proteolysis but reflects a similar in vivo process, possibly with regulatory significance.
We have found that bNMT activity exists as two
interconvertible oligomeric complexes which we ascribe to a reversible
multimerization of 60 kDa NMT subunit(s). Evidence for this
conclusion includes (i) the conversion of a mixture of
391 and
126 kDa forms of NMT (see Fig. 1A) into a single
391-kDa species by treatment with 1 M NaCl (see Fig. 1B), (ii) the subsequent partial dissociation of
this
391-kDa multimer back into the
126 kDa form upon treatment
with an N-myristoyl-peptide product of NMT (see Fig. 1C), and (iii) the reappearance of both forms of
the enzyme when the isolated
391- and
126-kDa enzymes are
rechromatographed. The data also reveal the accumulation during storage
of a fully active monomeric form of the enzyme (i.e.
50
kDa) presumably resulting from proteolysis of the
60-kDa subunit.
Considering the co-purification of several unidentified proteins with
our >7,000-fold purified enzyme preparation, the high molecular
weight forms of NMT could also involve other cellular proteins.
Nevertheless, assuming that the
391- and
126-kDa enzymes
represent hexamers and dimers, respectively, of a
60-kDa NMT
subunit, we propose the following model to explain the
interrelationships described in this study (Fig. 5). We
speculate that dimers made up of two
60-kDa NMT subunits are formed
independent of ionic strength but are disassociated by SDS sample
buffer. At low to moderate ionic strength, NMT exists primarly in the
form of dimers and hexamers as illustrated by our finding of two
prominent high molecular mass forms (i.e.
126 and
391
kDa) by gel filtration in the absence of 1 M NaCl (see Fig. 1A). Significant amounts of activity are also
found in the later eluting fractions presumably as a result of
nonspecific ionic interactions between NMT and background proteins
and/or column matrix. Upon treatment with 1 M NaCl, 100% of
the NMT activity is converted into a hexamer by blocking nonspecific
interactions and by enhancing hydrophobic surface contacts between the
dimers (see Fig. 1B). Subsequent treatment with N-myristoyl-peptide and 1 M NaCl dissociates most of
the hexamers into dimers due to the binding of the myristoyl moiety to
hydrophobic contact sites and/or pockets (i.e. myristoyl-CoA-binding sites) of the dimers thus interfering with
dimer-dimer associations. We further suggest that the accumulation of
the
50-kDa monomer during storage reflects the removal of
end-to-end subunit contact sites by proteolysis.
Figure 5:
Model accounting for multiple forms of
bNMT. Under low to moderate ionic strength conditions (see Fig. 1A), NMT exists as a mixture of 391-kDa
hexamers (
25%),
126-kDa dimers (
50%), and enzyme activity
nonspecifically interacting with background protein and/or column
matrix (
25%). Dimer formation is designated by end-to-end contacts
between two
60-kDa NMT subunits and is presumably in a reversible
equilibrium with the NMT hexamer proposed to result from side-to-side
contacts between dimers. Treatment with 1 M NaCl (see Fig. 1B) blocks the nonspecific enzyme interactions and
shifts the equilibrium in favor of formation of the
391-kDa hexamer
(100%) due to the enhancement of hydrophobic side-to-side contacts
between the NMT dimers. The addition of an N-myristoyl-peptide
followed by 1 M NaCl (see Fig. 1C) partially
reverses formation of the hexamer due to the binding of the N-myristoyl moiety to the hydrophobic surfaces and/or pockets (i.e. myristoyl-CoA-binding sites) of the dimers with the
resulting formation of a mixture of
391-kDa hexamers (
25%),
126-kDa dimers (
75%), and
50-kDa monomers (1-2%). The
accumulation of the
50-kDa monomer increases with longer storage
and presumably reflects the proteolytic cleavage of N-terminal contact
ends from the
60-kDa NMT subunit.
In addition to our work reported here and previously(30, 31) , this model is consistent with the report that 200 µM myristoyl-CoA promotes the dissociation of a large broad bNMT activity peak (i.e. 150-60 kDa by gel filtration) into 66 and 43 kDa forms and that relative amounts of the smaller form increased with prolonged storage(33) . This latter study showed that the myristoyl-CoA substrate promotes a dissociation similar to that observed with our N-myristoyl-peptide product. Since N-myristoyl-peptides have been shown to competitively inhibit the binding of myristoyl-CoA to the acyl-CoA-binding site of yNMT(21) , it is possible that the dissociation of NMT multimers induced by N-myristoyl-peptide (or myristoyl-CoA) envisioned in our model may reflect a process for regulating bNMT activity.
We have also identified a number of bNMT polypeptides
which are immuno-cross-reactive with hNMT but which exhibit apparent
molecular masses up to 12 kDa larger than that predicted from the
1248-nucleotide open reading frame assigned to the human
enzyme(26) . It is unlikely that this difference is due to
abnormal migration of the bNMT subunits during SDS-PAGE because of the
close homology between bNMT and hNMT as revealed by comparable
immunoblotting with an hNMT peptide antibody and partial amino acid
sequencing (33) and because hNMT (and yNMT) expressed in
bacteria exhibit apparent molecular masses on SDS-PAGE within 2% of
their predicted values(19, 43) . ()This
assumption is also consistent with the apparent absence in bNMT of
covalently linked carbohydrate or other obvious structural features
which might account for anomalous electrophoretic
mobility(44, 45) . Therefore, assuming that the bNMT
gene is similar to the gene described for hNMT, then one possible
explanation for the apparent size discrepancy is the presence in the
hNMT gene of an in-frame methionine start codon 183 nucleotides
upstream of the assigned start site which defines an open reading frame
encoding a protein 6.7 kDa larger than that predicted for the expected
human gene product. We have found that a recombinant hNMT translated
from that upstream start site co-migrates with our 57-kDa bNMT
subunit.
This indicates that either the major 60-kDa
species could result from initiation at yet another start site even
further upstream or that the 5` sequences of the bNMT and hNMT genes
are different(16, 23) .
Our experiments indicate
that the subunit heterogeneity of bNMT may in part reflect proteolysis
of larger subunit precursor(s). It is unlikely that this cleavage
occurs from the C terminus because of our finding that proteolysis did
not affect bNMT activity and the fact that the five C-terminal residues
of yNMT and at least Leu through Lys
(of
416 residues) of hNMT are essential for yeast and human NMT catalytic
activities(23, 26, 27) . Furthermore, since
our antibody recognizes amino acids 27-38 of hNMT and must be
present in order for the fragments to be immunostained, we conclude
that NMT subunit heterogeneity most likely results from cleavage of
N-terminal sequences preceding the antibody epitope. The
removal of up to 12 kDa of the bNMT N terminus with no apparent effect
on enzyme activity is also consistent with the suggestion that the
N-terminal domains of NMTs may have in vivo regulatory
functions reflecting species-specific
requirements(16, 23) . The possibility that these
N-terminal residues could be responsible for subunit multimerization
and/or complex formation with other proteins is consistent with the
presumed removal of this 12-kDa N-terminal domain during storage as
shown by SDS-PAGE (see Fig. 4) and the parallel accumulation of
a monomeric (i.e.
50 kDa) form of NMT as detected by gel
filtration (see Fig. 1C). The N terminus of NMT could
also mediate physiologically important protein-protein interactions
which (i) determine targeting to specific intracellular
compartments(38, 46, 47) , (ii) facilitate
association with ribosomal N-terminal processing
complexes(48) , and/or (iii) regulate co-substrate availability
through associations with intracellular proteins such as acyl-CoA
synthetase or acyl-CoA binding
proteins(47, 49, 50, 51) . It is
also possible that the N-terminal proteolysis noted in this study
reflects in vivo processes designed to release a fully active
enzyme from regulatory constraints. The extent to which this latter
mechanism may be operational could account for reports of different
sized NMT subunits from different mammalian
sources(27, 28, 33, 35) .
The purification of NMT from mammalian sources has been difficult due to its tendency to lose activity and/or to separate into multiple molecular forms during a variety of fractionation procedures(30, 32, 33, 34) . These difficulties result in part from nonspecific ionic interactions between NMT and other macromolecules and/or chromatographic supports. They also reflect a dynamic equilibrium between several multimeric and monomeric forms as determined by ionic strength, proteolysis, and/or protein concentration(34) . A key to the eventual purification of NMT and its subsequent characterization was the application of conditions (i.e. 1 M NaCl) to block nonspecific ionic interactions while preserving enzyme activity. The purification also depended upon our ability to manipulate the oligomeric structure of NMT and by its selective elution from octyl-agarose with a high affinity cofactor analog. Furthermore, our successful isolation of bNMT in both high purity and high yield provide assurance that the properties described in this study are representative of the majority of NMT activity in bovine brain rather than that of a minor form of the enzyme. We have thus provided answers to questions regarding the size differences and heterogeneity of the enzyme within this species and in comparison with yNMT and hNMT. We have also clarified previously unappreciated structural and hydrodynamic properties which could have important implications for understanding in vivoN-myristoylation.