Mkt1 and Pbp1 are both required for proper translation of nuclear-encoded mitochondrial proteins regulated by Puf3. (A) Western blot showing levels of the direct Puf3 target Mrp51, the indirect Puf3 target Cox2, and the non-Puf3 target Por1 in indicated strains grown to log phase in YPD and after switch to YPL medium for the indicated times. Numerical values represent relative densities of Mrp51 and Cox2 bands compared with corresponding G6PDH bands. pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells exhibited reduced levels of Mrp51 and Cox2 but not Por1. (B) Growth curves of indicated strains cultured in YPD medium. Cells were monitored using an automated plate reader during incubation at 30°C, and measurements were collected every 30 min. WT, pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells grew at similar rates. Representative traces from a single experiment are depicted (n = 6 per group). (C) Growth curves of indicated strains grown in YPL medium using the method described in B. pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells grew more slowly than WT. Representative traces from a single experiment are depicted (n = 6 per group). (D) Relative abundances of proteins detected by TMT-MS in pbp1∆ cells compared with WT during log phase growth in YPD medium (n = 3 per group). Statistical significance (P < 0.05) was calculated using unpaired, two-sided t test. The set of nuclear-encoded mitochondrial proteins encoded by mRNAs previously shown to be directly regulated by Puf3 (cis Puf3 targets) was significantly reduced based on Fisher’s exact test (Lapointe et al., 2018). Accompanying plot depicts representative mitochondrial protein GO terms enriched among proteins found to be significantly decreased (fold-change < log₂(0.75)). (E) Relative abundances of proteins detected by TMT-MS in mkt1∆ cells compared with WT during growth in YPD medium (n = 3 per group). Statistics were calculated using tests described in D. Accompanying plot depicts representative mitochondrial protein GO terms enriched among proteins found to be significantly decreased (fold-change < log₂(0.75)). (F) Relative abundances of proteins detected by TMT-MS in pbp1∆ cells compared with WT 3 h following switch from YPD to YPL medium (n = 3 per group). Statistics were calculated using tests described in D. Accompanying plot depicts representative mitochondrial protein GO terms enriched among proteins found to be significantly decreased (fold-change < log₂(0.75)). (G) Relative abundances of proteins detected by TMT-MS in mkt1∆ cells compared with WT 3 h following switch from YPD to YPL medium (n = 3 per group). Statistics were calculated using tests described in D. Accompanying plot depicts representative mitochondrial protein GO terms enriched among proteins found to be significantly decreased (fold-change < log₂(0.75)). (H) Western blot depicting levels of Cox2, Pbp1-Flag, and Por1 in cells grown in YPD and after switch to YPL medium. Pbp1Δ500-503 and Pbp1-4A strains had reduced levels of Cox2 and Pbp1-Flag. (I) Growth curves of indicated strains cultured in YPL medium. Growth was monitored using the method described in B. Pbp1Δ500-503 and Pbp1-4A strains had slower growth compared with WT. Representative traces from a single experiment are depicted (n = 3 per group). Source data are available for this figure: SourceData F3.