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INTRODUCTION |
Aminoacyl-tRNA synthetases are the family of enzymes responsible
for correctly linking tRNA molecules with amino acids so that the
latter may be subsequently incorporated into growing polypeptide
chains. In addition to this fundamental role in protein biosynthesis,
these enzymes have additional cellular roles, such as synthesis of
signaling dinucleotides. The aminoacyl-tRNA synthetase enzyme family is
also widely used in the course of study of such processes as evolution
of modular proteins and mechanisms of RNA-protein recognition (reviewed
in Refs. 1 and 2). Recently, attention has turned to the aminoacyl-tRNA
synthetases as potential targets for new antibiotics (reviewed in Ref.
3). Additional medical interest is due to the observation that the most
widely recognized myositis-specific autoantibodies are directed against
aminoacyl-tRNA synthetases (reviewed in Ref. 4)
A unique feature of aminoacyl-tRNA synthetases from higher eukaryotes
is the existence of a high molecular mass multisynthetase complex. As
isolated from a variety of cell types ranging from mammals to insects,
this approximately 1 × 106-Da particle contains nine
aminoacyl-tRNA synthetase activities: the arginyl-tRNA synthetase
dimer, the aspartyl-tRNA synthetase dimer, glutaminyl-tRNA synthetase,
isoleucyl-tRNA synthetase, leucyl-tRNA synthetase, the lysyl-tRNA
synthetase dimer, methionyl-tRNA synthetase, and the
glutamyl-/prolyl-tRNA synthetase
(GluProRS),1 which is a
bifunctional polypeptide. The multisynthetase complex also contains
three additional proteins that are identified by their apparent masses:
p43, which contains the complete sequence of the endothelial
monocyte-activating polypeptide II cytokine (5); p38, which is of as
yet unknown function; and p18, which may mediate association of the
multisynthetase complex with elongation factor 1 (6).
In order to fully understand the biological function(s) of this
intriguing particle, structural details of the multisynthetase complex
are necessary. Thus far, electron microscopy studies have shown that
the particle measures approximately 27 nm in diameter and appears to be
a cup or elongated "U" shape (7) as suggested by the distinct
triangular, rectangular, and square-shaped orientations visible in
negatively stained electron micrographs. A general idea of the
distribution of polypeptides within the complex has been gained through
dissociation of the native particle into subcomplexes (8, 9).
Additional evidence in support of interactions between certain of the
synthetase polypeptides has been provided through use of the yeast
two-hybrid system (10), binding experiments monitored by surface
plasmon resonance (11), and chemical cross-linking (12, 13). Within the
constraints of these data, a working model has been presented (13),
which is composed of three domains arranged into a "Y"-shape made
up of two "arms" and a "base." The composition of each domain
as well as likely positions of individual polypeptides within the
domains have been suggested, but prior to this study there have been no
precise localizations of specific components or sites within the
multisynthetase complex.
This article describes the first localization of a particular
polypeptide within the particle. Specifically, the bifunctional glutamyl-/prolyl-tRNA synthetase has been visualized by immunoelectron microscopy using an antibody directed against prolyl-tRNA synthetase.
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EXPERIMENTAL PROCEDURES |
Protein Purification--
The multisynthetase complex was
isolated from rabbit reticulocyte lysate (Green Hectares) as described
previously (13). Using antisera from two separate rabbits that were
immunized with rat liver prolyl-tRNA synthetase (14), immunoglobulin
was partially purified by precipitation with 45% ammonium sulfate,
desalting with buffer exchange into 10 mM Tris-HCl, pH 7.5, over 10-DG columns (Bio-Rad), then chromatography over DEAE-Trisacryl M
(IBF) eluting with a 0-300 mM KCl gradient. As assessed by
immunoblot analysis (15), the immunoglobulins specifically reactive
with GluProRS were in a peak eluting with approximately 100 mM KCl.
Immunoelectron Microscopy--
Samples of approximately 12 µg
of multisynthetase complex in storage buffer (50 mM
Hepes-NaOH, pH 7.4, 1 mM dithiothreitol, 0.5 mM
EDTA, 50% glycerol) were incubated with 5 or 10 µl of one of the two
immunoglobulin preparations plus 50 µl of 25 mM
Hepes-NaOH, pH 7.2, 25 mM NaCl for 2 h at room
temperature, then immunocomplexes were isolated by HPLC gel filtration
(GPC 300 column, Alltech) in the same buffer. Three different
preparations of multisynthetase complex were used. In order to
stabilize the multisynthetase complex for electron microscopy
glutaraldehyde was added to 0.3% (7) and then the immunocomplexes
negatively stained with 1% aqueous uranyl acetate. Electron
micrographs were obtained with a LEO 912AB microscope operated at 80 kV
at absolute magnification of 31,500 or 40,000 using the spectrometer
slit to increase contrast by removing inelastically scattered electrons.
Image Analysis--
Immunocomplexes were identified on
micrographs and the orientation of the multisynthetase complex and
antibody binding site were recorded. Of 413 immunocomplexes analyzed,
only 14 were inconsistent with the primary localization. For
preparation of figures, electron micrograph negatives were digitized at
an optical resolution of 1000 dpi using an Agfa Duoscan flatbed
scanner. Composite figures were prepared using standard graphics
programs (Imgworks, Showcase and Snapshot) on a Silicon Graphics
Indigo2 workstation, then converted to tif format.
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RESULTS |
Specificity of Antibody Binding--
The original description and
characterization of the antibodies directed against rat liver
prolyl-tRNA synthetase (14) demonstrated specific reaction with the
GluProRS polypeptide within the multisynthetase complex. That the
immunoglobulin fractions prepared for this study retained that
specificity was checked by immunoblot analysis (data not shown). Thus,
immunoelectron microscopic visualization of the site of antibody
binding is a reliable indicator of the position of this polypeptide
within the particle.
Localization of GluProRS within the Multisynthetase
Complex--
Panel A of Fig.
1 shows a gallery of individual particles
of negatively stained multisynthetase complex with one bound antibody. Examples of each type of characteristic orientation (7) of the particle
are given. In column A, triangular views of the particle are
presented. Panels 1-3 show a "Y"-like orientation in
which two arms and a base are visible. These likely correspond to the three domains of the working model of the complex (13). The site of
antibody binding is clearly seen to be near the division between one
arm and the base. In panels 4-6 of column A, the
particle appears slightly rotated along the long axis so that the cleft between the arms is no longer visible. These can be termed
"intermediate" views. The site of antibody binding is again at the
division between the large end of the triangle and the smaller base.
This is consistent with the placement based on the first three panels.
Column B shows rectangular views of the multisynthetase
complex. These have been interpreted to correspond to an approximately
90° rotation around the long axis of the particle from the Y-like
view (7). The symmetry of this projection precludes assigning one end
or the other as an arm or the base. However, the site of antibody
binding is consistently located at or near the midpoint of the long
axis. This would correspond to the position seen in the triangular
orientations, that is, near the division of the two portions of the
particle. Column C shows square-like views of the
multisynthetase complex. The images in panels 1-3 have a
central "hole" into which stain has penetrated, while those in
panels 3-6 do not. These are considered to be views from
the "top" and "bottom" of the particle, respectively, given the
interpretation that the triangular and rectangular orientations are
"front" and "side" views of the "cup". The site of antibody binding is on the periphery of the multisynthetase particle as suggested by the clear view of bound antibodies. It is also near the
midpoint of the particle axis that is orthogonal to the rotational axis
relating the triangular and rectangular orientations.

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Fig. 1.
Galleries of negatively stained
aminoacyl-tRNA synthetase complex with bound antibodies directed
against prolyl-tRNA synthetase. Scale marker equals 25 nm.
Panel A, individual multisynthetase particles with one bound
antibody. Triangular views (column A) show the site of
antibody binding near the division between the arms and base of the
particle. Rectangular views (column B) place the site of
antibody binding near the center of the long axis of the particle.
Square views (column C) indicate the site of antibody
binding at the periphery of the particle. Panel B, images
with one antibody linking two multisynthetase particles.
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Panel B of Fig. 1 is a gallery of images where two particles
of the multisynthetase complex are linked by a single antibody. The
dimers shown in column A all contain one particle in which the central
cleft between the arms is visible, that is, front views. In each, the
site of antibody binding is again at or near the division between an
arm and the base. In panels 3 and 5, the other
antigen combining site of the antibody is linked to a multisynthetase particle in the rectangular orientation. Again, the binding site is
near the center of the long axis of the complex. Panels 1 and 2 of column B clearly show antibodies linking
the midpoints of two rectangular views and a rectangular with an
intermediate orientation, respectively. The remaining images in
column B show linked particles in the top or bottom
orientations, which also emphasize the location of the antibody binding
site near the midpoint of the complex.
Approximately 3% of immunocomplexes analyzed cannot be interpreted in
a manner consistent with the existence of only one antibody binding
site near the intersection of two domains of the multisynthetase particle. Examples of these are shown in Fig.
2. In column A are immunocomplexes that appear to have two antibody molecules bound to one
multisynthetase complex. In each case, one site is consistent with that
described above, while the other is on the opposite side of the
particle. These views suggest the presence of another binding site, but
their rare occurrence indicates that it is likely nonspecific binding.
In column B, two antibodies are also bound, but their
binding sites are very close together. However, both are consistent
with localization of the GluProRS polypeptide near the midpoint of the
particle. Column C shows dimers that are linked by two
antibodies. Panels 1 and 2 each have one
multisynthetase particle in the "front" view, which again clearly
show that the primary antibody binding site is near the intersection of
an arm and the base. The rectangular view of one of the particles in panel 3 emphasizes the location of the epitopes near the
midpoint of the long axis.

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Fig. 2.
Examples of multisynthetase particles with
two bound antibody molecules. Scale marker equals 25 nm.
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The above data are summarized in a schematic (Fig.
3) showing the relationship of the
different particle orientations. When viewed from the front where the
central cleft is visible, the major antibody binding site is near the
division between one arm and the base of the particle. Partial rotation
of the particle around the long axis gives an intermediate view, while
the rectangular orientation can be considered the side view. In both,
the primary antibody binding site is still clearly visible near the
midpoint of the particle. The top and bottom orientations are obtained by orthogonal rotation, shown here from the intermediate view. Antibody
binding is near the midpoint of one side of each square view. The area
in the drawings representing the primary antibody binding site
encompasses sections of both an arm and the base. This is to account
for the variability of antibody location along the long axis as seen in
both triangular (Fig. 1, panel A, column A) and
rectangular (Fig. 1, panel A, column B)
orientations, as well as the examples of closely spaced binding of two
antibodies (Fig. 2, columns B and C). As
suggested by the examples shown in column A of Fig. 2, a
secondary minor binding site is depicted on the opposite side of the
particle.

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Fig. 3.
Schematic indicating the relationship of
characteristic orientations of electron microscopic images of the
multisynthetase complex with patterned boxes indicating the primary and
minor binding sites of the antibody directed against prolyl-tRNA
synthetase.
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DISCUSSION |
The immunoelectron microscopic data presented here provide the
first visualization of a specific site within the multisynthetase complex. The GluProRS polypeptide has been localized within a defined
area of the particle by mapping the binding of antibody directed
against ProRS. The primary area of antibody binding observed in this
study overlaps significantly with the placement of GluProRS in the base
of the three domain working model, which is based largely on
cross-linking data (13).
Although the primary area of antibody binding observed here is centered
around the midpoint of the particle axes, it encompasses sections of
both the domain termed the base as well as one arm domain. Not only is
this range of locations noticeable in immunocomplexes containing one
antibody (Fig. 1, panels A and B), but there are also images where two antibodies are bound in close proximity on the
multisynthetase complex (Fig. 2, columns B and
C). One explanation of these data is that the ProRS domain
of GluProRS is very elongated and bridges the two particle domains. The
range of binding site placements would thus be due to antibodies within the polyclonal immunoglobulin preparations that recognize multiple epitopes within the polypeptide. A second possibility is that the two
closely bound antibodies are detecting two copies of GluProRS within
the complex. A dimer can be envisioned to more easily cover the
approximately 10-nm spread of the binding site. A stoichiometry of two
copies of the bifunctional synthetase has been reported in complex from
sheep liver (16), while others have observed variability from 1.0 to
1.5 in preparations from rat liver, rabbit liver, and rabbit
reticulocytes (17). In addition, when mammalian prolyl-tRNA synthetase
is isolated in its low molecular mass form, it occurs as a dimer (14,
18). It would thus seem likely that the bifunctional polypeptide form
is also a dimer. However, in preparations from this laboratory, the
estimated molar ratio of the GluProRS polypeptide has always been close
to one (7, 9), but this is a relative value based on densitometric
analysis of bands on polyacrylamide gels. The presence of small numbers
of complexes with an additional GluProRS polypeptide bound at a
separate site within the complex would explain the observation here of the second, but rare, antibody binding site. This would also be consistent with the specificity of binding demonstrated by immunoblot analyses.
If a monoclonal antibody to the GluProRS were available, the
appropriate explanation could be readily determined and the binding site likely restricted considerably. As an alternative,
tRNAPro and/or tRNAGlu can be used as
structural probes for the two active sites within this bifunctional
enzyme. Such experiments are currently under way.