Abstract
The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.
Highlights
Synthesized proteins are targeted to, and translocated across, membranes either post-translationally or co-translationally
As these studies have shown, stable ribosome-nascent chain complexes (RNCs) represent the initial targeting stages of proteins that are targeted to, and imported across, membranes co-translationally (26, 30 –32). matrix protein malate dehydrogenase (mMDH) was used to make RNCs because we had previously shown that the mMDH nascent chain is sufficient to stabilize ribosome binding to mitochondria and others have suggested that mMDH may be imported co-translationally [15, 17, 19]
Most of the studies investigating mitochondrial protein import have been performed in yeast and may not represent how proteins are targeted to mammalian mitochondria because of the differences in the transmembrane protein transfer that exist between yeast and mammalian cells [47]
Summary
Preparation of High Salt-washed Mitochondria—Crude mitochondria were isolated from Sprague-Dawley rat livers as previously described [22]. The disintegrations/min of RNC-mitochondria complexes was measured and the number of moles of RNCs bound was calculated using the following assumptions: 1) each RNC was loaded with a complete nascent chain; 2) there are 7 methionines in the mMDH-Met nascent chain and 4 methionines in the Luc nascent chain; 3) the specific activity of the [35S]methionine was 1,000 Ci/mmol; 4) the radiolabeled methionine concentration in the translation reaction was 0.61 M and the unlabeled methionine concentration was 3.79 M; and 5) 85% the radioactivity in RNC preparations was contained within a ribosome-associated nascent chain (see Fig. 1C). To determine the contribution of cations on RNC binding, RNCmitochondria complexes were prepared under the conditions described above, except that the reaction was scaled up to a final volume of 280 l. The counts/min measurements between experiments were normalized by calculating the relative counts/min in each fraction compared with the total within each experiment
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