The effect of spermine on the initiation of mitochondrial protein synthesis

Abstract

Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2(mt)) and mitochondrial initiation factor 3 (IF3(mt)). This observation indicates that IF2(mt) and IF3(mt), while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3(mt) dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2(mt). The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.

Figure 1. Effect of spermine on initiation complex formation on 55S ribosomes in the presence and absence of various mRNAs

A. [³⁵S]fMet-tRNA binding to mitochondrial 55S particles (6 pmol, 60 nM) was tested in the presence and absence of 10 μg poly(A,U,G) mRNA and the presence of IF2_mt (8 pmol, 80 nM) and IF3_mt (8 pmol, 80 nM) at different concentrations of spermine. B. [³⁵S]fMet-tRNA binding to mitochondrial 55S ribosomes was tested in the presence of saturating amounts of poly(A,U,G) (5 μg), the 29-mer cytochrome oxidase start site (2.24 μM), or the AUG triplet (6 μg, 53 μM) at different concentrations of spermine as described in Materials and Methods.

Figure 2. Effect of spermine on IF3_mt-induced 55S dissociation

A. [³⁵S]fMet-tRNA binding to mitochondrial 55S particles (8 pmol, 80 nM) was tested at different concentrations of IF3_mt with and without spermine (0.1 mM) using 10 μg poly(A,U,G) as the mRNA at a fixed concentration of IF2_mt (8 pmol, 80 nM). B. Fractionation profiles of mitochondrial 55S ribosomes after centrifugation on 10–30% sucrose gradients in 5 mM Mg²⁺. Mitochondrial 55S ribosomes (8 pmol, 80 nM) were incubated at 5 mM MgCl₂ with or without spermine (0.1 mM) in the absence or presence of IF3_mt (41 pmol, 410 nM) at 37 °C for 15 min. The samples were then placed on ice for 10 min and subsequently layered on a 10–30% sucrose density gradient (gradients were prepared with and without spermine, respectively, corresponding to the reaction conditions used) and analyzed as described in Materials and Methods.

Figure 3. Effect of IF2_mt on initiation complex formation in the presence and absence of spermine

[³⁵S]fMet-tRNA binding to mitochondrial 55S particles (6 pmol, 60 nM) was tested with and without spermine (0.1 mM) the indicated concentrations of IF2_mt.

Figure 4. Effect of spermine on initiation complex formation on 28S subunits in the presence and absence of initiation factors

[³⁵S]fMet-tRNA binding to mitochondrial 28S subunits (4 pmol, 40 nM) was tested using 10 μg poly(A,U,G) as the mRNA in the presence and absence of IF2_mt (14 pmol, 140 nM) at different concentrations of spermine.