Figure 4. The Mkt1/Pbp1 complex... | Rockefeller University Press

$The Mkt1/Pbp1 complex genetically and physically interacts with Puf3. (A) Western blot assessing Cox2 and Por1 levels from cells grown in YPD to log phase and following switch to YPL medium. puf3∆, pbp1∆puf3∆, and mkt1∆puf3 cells appear to exhibit increased Cox2 protein levels compared with pbp1∆ and mkt1∆ cells. (B) Growth curves of indicated strains collected using an automated plate reader during incubation in YPL medium. Cells were incubated at 30°C, and measurements were obtained every 30 min. KO strains (pbp1∆, mkt1∆, puf3∆, pbp1∆puf3∆, and mkt1∆puf3) exhibited reduced growth compared with WT cells (n = 4 per group). (C) RT-qPCR analysis of Puf3-target mRNA levels from cells collected during log phase growth in YPD and then 3 h after switching to YPL medium. P values were calculated using unpaired, two-sided t test (mean ± SD, n = 5, except n = 4 for mkt1∆ group). *P < 0.05; **P < 0.01; ***P < 0.001. (D) RT-qPCR analysis of non–Puf3-target mRNA levels from cells in C collected during log phase growth in YPD and then 3 h after switching to YPL medium. P values were calculated using unpaired, two-sided t test (mean ± SD, n = 5, except n = 4 for mkt1∆ group). *P < 0.05. (E) Western blot assessing Puf3-HA protein levels from cells grown in YPD to log phase and then 1, 3, and 5 h after switching to YPL medium. Puf3 protein levels were decreased in KO cells, while Puf3 phosphorylation persisted. (F) co-IP assessing interactions between Flag-tagged Mkt1 and Pbp1 with Puf3-HA in cells cultured to log phase in YPD and 3 h following switch to YPL medium. Mkt1-Flag and Pbp1-Flag interacted with Puf3-HA preferentially in YPL medium. Note: Mkt1-Flag did not interact with Puf3-HA in pbp1∆ cells, while Pbp1-Flag and Puf3-HA continued to interact in mkt1∆ cells despite diminished Pbp1-Flag levels. (G) Polysome profile of WT cells collected 3 h following switch from YPD to YPL medium and associated western blot for Mkt1-Flag, Pab1, and Puf3-HA in collected fractions. Numerical values represent relative densities of Mkt1-Flag and Puf3-HA bands compared with corresponding Pab1 bands. Mkt1-Flag and Puf3-HA localized to polysome fractions. (H) Polysome profile of pbp1∆ cells collected 3 h following switch from YPD to YPL medium and associated western blot for Mkt1-Flag, Pab1, and Puf3-HA in collected fractions. Mkt1-Flag was less present in polysome fractions. Source data are available for this figure: SourceData F4.$

Figure 4.

The Mkt1/Pbp1 complex genetically and physically interacts with Puf3. (A) Western blot assessing Cox2 and Por1 levels from cells grown in YPD to log phase and following switch to YPL medium. puf3∆, pbp1∆puf3∆, and mkt1∆puf3 cells appear to exhibit increased Cox2 protein levels compared with pbp1∆ and mkt1∆ cells. (B) Growth curves of indicated strains collected using an automated plate reader during incubation in YPL medium. Cells were incubated at 30°C, and measurements were obtained every 30 min. KO strains (pbp1∆, mkt1∆, puf3∆, pbp1∆puf3∆, and mkt1∆puf3) exhibited reduced growth compared with WT cells (n = 4 per group). (C) RT-qPCR analysis of Puf3-target mRNA levels from cells collected during log phase growth in YPD and then 3 h after switching to YPL medium. P values were calculated using unpaired, two-sided t test (mean ± SD, n = 5, except n = 4 for mkt1∆ group). *P < 0.05; **P < 0.01; ***P < 0.001. (D) RT-qPCR analysis of non–Puf3-target mRNA levels from cells in C collected during log phase growth in YPD and then 3 h after switching to YPL medium. P values were calculated using unpaired, two-sided t test (mean ± SD, n = 5, except n = 4 for mkt1∆ group). *P < 0.05. (E) Western blot assessing Puf3-HA protein levels from cells grown in YPD to log phase and then 1, 3, and 5 h after switching to YPL medium. Puf3 protein levels were decreased in KO cells, while Puf3 phosphorylation persisted. (F) co-IP assessing interactions between Flag-tagged Mkt1 and Pbp1 with Puf3-HA in cells cultured to log phase in YPD and 3 h following switch to YPL medium. Mkt1-Flag and Pbp1-Flag interacted with Puf3-HA preferentially in YPL medium. Note: Mkt1-Flag did not interact with Puf3-HA in pbp1∆ cells, while Pbp1-Flag and Puf3-HA continued to interact in mkt1∆ cells despite diminished Pbp1-Flag levels. (G) Polysome profile of WT cells collected 3 h following switch from YPD to YPL medium and associated western blot for Mkt1-Flag, Pab1, and Puf3-HA in collected fractions. Numerical values represent relative densities of Mkt1-Flag and Puf3-HA bands compared with corresponding Pab1 bands. Mkt1-Flag and Puf3-HA localized to polysome fractions. (H) Polysome profile of pbp1∆ cells collected 3 h following switch from YPD to YPL medium and associated western blot for Mkt1-Flag, Pab1, and Puf3-HA in collected fractions. Mkt1-Flag was less present in polysome fractions. Source data are available for this figure: SourceData F4.

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