Fig. 3.

Eso1-dependent Psm3^K106 acetylation during the cell cycle. (A) The anti-Psm3^K106Ac antibody reacts with Psm3 in a K106-dependent manner in total extracts (left) and GFP immunoprecipitates (middle). (Right) Psm3 detection by anti-Psm3^K106Ac antibodies is dependent on Eso1, but not on Wpl1. (B and C) Psm3^K106 acetylation during the cell cycle. (B) Cells bearing the thermosensitive mutation cdc10-129 were shifted to the restrictive temperature to induce G₁ arrest. Samples were taken at regular time intervals after the temperature shift to monitor Psm3^K106Ac on total protein extracts as the cells progressed from an asynchronous population (time zero) to a homogeneous G₁ arrest. Flow cytometry analysis of the DNA content (right) showed that Psm3^K106Ac started to decrease as the G₁ peak appeared. The drift in the G₁ peak to the right at later time points was due to an increase in the mitochondrial DNA content as the cells elongated (47). (C) Cells were arrested in G₁ by nitrogen deprivation and released synchronously into the cell cycle. Psm3^K106Ac probed from total protein extracts was weakest in cells with a G₁ DNA content and began to rise at the time of DNA replication. Anti-Psm3 antibodies were used to adjust protein loading so that similar amounts of Psm3 were present in all samples.