In organello labelling and SDS-PAGE have been utilized to examine polypeptide synthesis in isolated mitochondria from normal and S-sterile maize (B37) seedlings under various temperature conditions. This was partially based on a concern that the temperature shifts involved in transferring the mitochondria from the 4 C isolation conditions to the 30-37 C labelling conditions used in in organello studies might be generating a "heat shock-like" condition and giving rise to synthesis of HSPs, which might then be interpreted as normal mitochondrial protein synthetic products.
Mitochondria were isolated from five day old etiolated coleoptiles (R.J. Kemble et al., 1980; Genetics 95:451-458), treated with DNAase and/or RNAase to eliminate cytoplasmic nucleic acids and purified over sucrose, all at 4 C. The mitochondria were gently resuspended in 150 ul of a buffer containing 50 mM Tris-HCl (pH 7.2), 20 mM MgCl2, 40 mM KCl, 2 mM dithiothreitol, 2 mM ATP, 100 mg/ml BSA, 25 umol of 19 amino acids (minus methionine), 50 UCi of 35S-methionine and sterile water to volume. The reaction mixtures were incubated for 60 minutes at 30, 37 or 42 C and then put on ice. After centrifugation, pelleted mitochondria were solubilized and 25,000 cpm of acid-precipitable lysates were subjected to SDS-PAGE as described elsewhere (C.L. Baszczynski et al., 1982; Can. J. Biochem. 60:569-579).
Incorporation of labelled precursor into polypeptides was 30-35% less at 42 C than at 30 C. While a broad spectrum of newly synthesized polypeptides was observed at 30 C in mitochondria from both N and S cytoplasms, incubation at 37 or 42 C resulted in a depression of synthesis of many of these polypeptides and a marked enhancement in synthesis of a prominent polypeptide of approximately 57 kD. Sterile mitochondrial isolations and nuclease pretreatments of intact mitochondria preclude the possibility of this polypeptide being a product of translation of non-organellar mRNA. In addition, it does not correspond in size to any of the previously described maize cellular HSPs (C.L. Baszczynski et al., 1982).
Since the 57 kD polypeptide is not present in samples labelled at 30 C, it would appear that the transfer from 4 C to 30 C does not lead to an HS response. However, it is clear that a similar shift to 37 C does give rise to a heat shock response, such that the experimental temperature conditions must be taken into consideration when evaluating protein products. Earlier work on maize cellular HSPs revealed that the precise temperature regime of an experiment influences the spectrum of newly synthesized polypeptides (Baszczynski et al., 1984; Ph.D. Thesis, UW.O., London, Canada).
The present results substantiate an earlier MNL report of HSP synthesis in normal maize mitochondria (C.M. Nebiolo and E.M. White, 1984; 58:144-145) as well as reports in a recent abstract (R.M. Sinibaldi and T.H. Turpen, 1985, 1st Int. Congr. Pl. Mol. Biol., PO-2-229); both suggest that a 50-60 kD polypeptide synthesized following a heat shock is encoded within the mitochondria. In addition the present report indicates that cms-S mitochondria do not differ from the normal cytoplasm in response to heat shock conditions.
C.L. Baszczynski and R.J. Kemble
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