Pet54 interacts with COX1 mRNA, and this interaction is independent of Mss51.
A, mitochondria were solubilized with dodecyl maltoside, and Pet54–3xMyc or untagged Pet54 (−) was subjected to immunoprecipitation with antibody anti-Myc. One-fourth of the immunoprecipitate (IP) was resolved by SDS-PAGE and transferred to a PVDF membrane for Western blotting. The membrane was probed with anti-Myc antibody and with anti-citrate synthase antibody (CS) as a negative control for interaction. The total fraction represents 5% of the mitochondrial extract used for immunoprecipitation. *, nonspecific bands from the immunoglobulin heavy chain used for immunoprecipitation. ♦, nonspecific bands when the anti-Myc antibody was used. B, RNA was extracted from the total (T) and immunoprecipitate fractions. Each fraction was divided in two, and cDNA was prepared in the presence (+) or absence (−) of reverse transcriptase (RT) using primers for the COX1 and VAR1 5′-UTRs. The (−) RT lanes represent a negative control for DNA contamination. The PCR products were run on an agarose gel. ●, bands due to primer dimers. C and D, mitochondria from cells carrying Pet54–3xMyc or the untagged Pet54 and either the wild type MSS51 or the Δmss51 deletion were treated and analyzed as in A and B.