Abstract
Multicellular eukaryotes contain a macromolecular assembly of nine aminoacyl-tRNA synthetase activities and three auxiliary proteins. One of these, p43, is the precursor of endothelial monocyte-activating polypeptide II (EMAP II), an inflammatory cytokine involved in apoptotic processes. As a step toward understanding this paradoxical association, the EMAP II portion of p43 has been localized within the rabbit reticulocyte multisynthetase complex. Immunoblot analysis demonstrates strong reaction of anti-EMAP II antiserum with p43, as well as cross-reactivity with isoleucyl-tRNA synthetase. Electron microscopic images of immunocomplexes show two antibody binding sites. The primary site is near the midpoint of the multisynthetase complex at the intersection of the arms with the base. This site near the lower edge of the central cleft is assigned to the C-terminal cytokine portion of p43. The secondary site of antibody binding is in the base of the particle and maps the location of isoleucyl-tRNA synthetase. These data allow refinement of the three-domain model of polypeptide distribution within the multisynthetase complex. Moreover, the central location of p43/EMAP II suggests a role for this polypeptide in optimizing normal function and in rapid disruption of essential cellular machinery when apoptosis is signaled.
Highlights
Multicellular eukaryotes contain a macromolecular assembly of nine aminoacyl-tRNA synthetase activities and three auxiliary proteins
Assignment of the Primary Antibody Binding Site to p43 and the Secondary Site to Isoleucyl-tRNA Synthetase—Immunoblot analysis shows that the strongest reaction of antibodies within the anti-endothelial monocyte-activating polypeptide II (EMAP II) antiserum is with p43
Regardless of whether this is because of higher titer of this population, stronger affinity, or both, this result suggests that the majority of bound antibodies observed in electron microscopic images of immunocomplexes will react with this polypeptide
Summary
The secondary site of antibody binding is in the base of the particle and maps the location of isoleucyl-tRNA synthetase. These data allow refinement of the three-domain model of polypeptide distribution within the multisynthetase complex. One role for p43 may be in tRNA trafficking (see Ref. 9 and references therein), but a variety of other biological functions are possible, because it is a precursor form [10] of endothelial monocyte-activating polypeptide II (EMAP II).. As an initial step toward understanding this intriguing association, this study localizes the cytokine portion of p43 within the multisynthetase complex using antibodies directed against EMAP II. The information obtained is used to refine the threedomain model of the particle and to propose a role for p43 within the multisynthetase complex
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