Fig. 4.
The Jjj1 C-terminus is important for the role of Jjj1 in ribosome biogenesis. A) Analysis of wild-type cells and Δjjj1 cells containing empty vector (—), or expressing Jjj1 or Jjj11-339 from the native promoter, was as follows: Left: strains were serially diluted, spotted on minimal medium plates, then incubated at 23°C for 3 days or 30°C for 2 days. Right: Cell extracts were prepared from Δjjj1+JJJ1 and Δjjj1+jjj11-339 cultures used for serial dilutions and subjected to immunoblot analysis using antibody specific to the Jjj1 N-terminus and antibody specific to Ssc1 as a loading control. B) Δjjj1 cells containing empty vector and Δjjj1 cells expressing wild-type Jjj1 or Jjj11-339 from the native promoter were grown at 23°C, lysed. The lysate was then centrifuged through a sucrose gradient. The migration of ribosomal subunits, monosomes and polysomes was monitored by absorbance at 254 nm and plotted versus the time course of fraction collection. Arrows denote half-mer polysome peaks. C) Δjjj1 cells expressing either Jjj1-GFP or Jjj11-339 -GFP from the native promoter, and the nucleus-specific protein RFP-Pus1 as a nuclear marker, were grown at 30°C prior to imaging by fluorescence and differential interference contrast (DIC) microscopy. Representative images show DIC, localization of Jjj1-GFP or Jjj11-339 -GFP as indicated, localization of RFP-Pus1, a nuclear marker, and an image overlay (merge).