FIGURE 4:

MRX8 is essential for COX1 translation initiation and elongation. (A) Tenfold serial dilutions of Δmrx8 cells in either XPM78a or XPM171a expressing either wild-type MRX8 or vector were spotted on YPD, SD-Arg. and SGly. (B) Top: Newly synthesized mitochondrial protein products were measured in either XPM78a or XPM171a with the ∆mrx8 allele in their nuclear genome expressing either wild-type MRX8 or vector at 16°C by incorporation of [³⁵S]methionine and cysteine in the presence of cycloheximide to inhibit cytosolic translation. Mitochondria from labeled cells were isolated, and proteins were separated on 17.5% SDS–PAGE. Radiolabeled proteins were transferred onto a nitrocellulose membrane and visualized by phosphoimaging. The positions of mtDNA-encoded proteins are indicated. As control a Coomassie-stained gel is shown. Representative images of multiple trials are shown. Bottom: Mitochondria was isolated from Δmrx8 cells in either XPM78a or XPM171a expressing either wild-type MRX8 allele or vector cultured at 16°C. Equivalent amounts of mitochondrial proteins were separated via SDS–PAGE and subjected to immunoblot analysis. Samples were analyzed using antibodies to Arg8 and F1β.