Role of the N- and C-terminal extensions on the activity of mammalian mitochondrial translational initiation factor 3

Abstract

Mammalian mitochondrial translational initiation factor 3 (IF3(mt)) promotes initiation complex formation on mitochondrial 55S ribosomes in the presence of IF2(mt), fMet-tRNA and poly(A,U,G). The mature form of IF3(mt) is predicted to be 247 residues. Alignment of IF3(mt) with bacterial IF3 indicates that it has a central region with 20-30% identity to the bacterial factors. Both the N- and C-termini of IF3(mt) have extensions of approximately 30 residues compared with bacterial IF3. To examine the role of the extensions on IF3(mt), deletion constructs were prepared in which the N-terminal extension, the C-terminal extension or both extensions were deleted. These truncated derivatives were slightly more active in promoting initiation complex formation than the mature form of IF3(mt). Mitochondrial 28S subunits have the ability to bind fMet-tRNA in the absence of mRNA. IF3(mt) promotes the dissociation of the fMet-tRNA bound in the absence of mRNA. This activity of IF3(mt) requires the C-terminal extension of this factor. Mitochondrial 28S subunits also bind mRNA independently of fMet-tRNA or added initiation factors. IF3(mt) has no effect on the formation of these complexes and cannot dissociate them once formed. These observations have lead to a new model for the function of IF3(mt) in mitochondrial translational initiation.

Figure 1

Domain organization of prokaryotic IF3 and mammalian IF3_mt and deletion constructs for IF3_mt. (A) Schematic diagram of the organization of E.coli IF3 and IF3_mt and its deletion derivatives. IF3_mt begins with an N-terminal import sequence that is not shown. The full-length version of the mature protein (IF3_mtFL) encompasses residues 32–278. The region with homology to E.coli IF3 begins at residue 61 and goes through residue 245. Deletion of the N-terminal extension (IF3_mtΔN) gives a derivative that includes amino acids 61–278. Deletion of the C-terminal extension (IF3_mtΔC) includes amino acids 32 through and including residue 245. The deletion of both extensions (IF3_mtΔNC) gives a construct that includes residues 61 through 245. (B) Analysis of the purity of the full-length and deletion derivatives by SDS–PAGE. Samples (1–2 µg) of IF3_mtFL and its deletion derivatives were applied to a 12% SDS–PAGE gel and stained with Coosmassie blue.

Figure 2

Activity of IF3_mt and its deletion derivatives in initiation complex formation. (A) The activity of IF3_mtFL (closed squares), IF3_mtΔN (closed circles), IF3_mtΔC (open triangles) and IF3_mtΔNC (open squares) was tested in initiation complex formation on mitochondrial 55S ribosomes using poly(A,U,G) as the mRNA. A blank containing no IF3_mt has been subtracted from each value (0.29 pmol). (B) Activities of IF3_mtFL (closed squares) and IF3_mtΔNC (open squares) were tested on mitochondrial 55S ribosomes using a transcript of the cytochrome oxidase subunit 2 gene as the mRNA. A blank containing no IF3_mt (0.11 pmol) has been subtracted from each value. (C) The activities of IF3_mtFL (closed squares), IF3_mtΔN (closed circles), IF3_mtΔC (open triangles) and IF3_mtΔNC (open squares) were tested on E.coli 70S tight couples using poly(A,U,G) as mRNA. A blank containing no IF3_mt (0.34 pmol) has been subtracted from each value. (D) Model for the N- and C-domains of Bacillus stearothermophilus IF3 created from the PDB coordinates (1TIF and 1TIG) using MolMol (55) indicating the location of the N- and C-terminal extensions. Homology modeling suggests that the N-domain of IF3_mt has a similar fold to that observed with the B.stearothermophilus factor (23). The C-domain of IF3_mt is not as highly conserved and cannot be modeled accurately. However, it is probable to have a similar overall fold.

Figure 3

Activity of E.coli IF3 and IF3_mt in proofreading the initiation complex. The abilities of E.coli IF3 (circles) and IF3_mt (squares) to promote the dissociation of a pre-formed complex [E.coli 30S:poly(U):[¹⁴C]AcPhe-tRNA] (open symbols) were measured in the presence of various concentrations of IF3 as described in Materials and Methods. The value for 100% complex remaining is 2.1 pmol. The effects of E.coli IF3 and IF3_mt on a pre-formed complex [30S:poly(A,U,G):[³⁵S]fMet-tRNA] (closed symbols) were tested under similar conditions. The 100% value for these experiments was 0.14 pmol.

Figure 4

Effect of E.coli IF3, IF3_mt and its derivatives on the binding of [³⁵S]fMet-tRNA to mitochondrial 28S subunits in the presence and absence of mRNA. Initiation complexes were prepared containing [³⁵S]fMet-tRNA, IF2_mt and 28S subunits in the presence and absence of poly(A,U,G) as mRNA. After incubation with the indicated amounts of IF3_mt or its derivatives, the amount of complex remaining was determined using a nitrocellulose filter-binding assay. The data are reported as the relative amount of binding obtained since data from different experiments were combined and the absolute numbers obtained depend on the percentage of active 28S subunits in different preparations. For these experiments, the value normalized to 1 for binding in the presence of mRNA represents 0.71 pmol while the value bound in the absence of mRNA was 0.35 pmol. For the experiments testing the effects of deletion of the N- and C-terminal extension, the level of binding obtained in the absence of mRNA and IF3_mt was 0.12 pmol. IF3_mtFL in the presence of mRNA (closed circles), IF3_mtFL in the absence of mRNA (closed squares), IF3_mtΔN (open circles) or IF3_mtΔC (open triangles) or E.coli IF3 (open squares) were added as indicated. The amount of fMet-tRNA retained on the small subunit was measured as described in Materials and Methods.

Figure 5

Effect of the addition of 39S subunits on the mRNA-independent binding of [³⁵S]fMet-tRNA formed in the presence and absence of IF3_mt. (A) The 28S subunits were incubated with [³⁵S]fMet-tRNA and IF2_mt in the absence (closed circles) or presence (open circles) of IF3_mt. Reaction mixtures were analyzed by sucrose density gradient centrifugation and the position of the [³⁵S]fMet-tRNA was located by filtering appropriate fractions as described in Materials and Methods. (B) [³⁵S]fMet-tRNA binding was initially carried out with 28S subunits in the presence of IF2_mt but in the absence of mRNA. Reactions mixtures were prepared in the absence (closed circles) or presence (open circles) of IF3_mt. Following assembly of these complexes, 39S subunits (0.066 µM) were added and the incubation was continued for an additional 5 min at 27°C. The resulting complexes were then analyzed on sucrose gradients.

Figure 6

Proposed model for the role of IF3_mt in initiation complex formation in mammalian mitochondria. For a description of the steps see text.