Figure 4
From: Distinct functional relevance of dynamic GTPase cysteine methylation in fission yeast
Cysteine methylation is important to regulate sexual differentiation mediated by palmitoylated Ras1. (a) In fission yeast cellular morphogenesis is regulated by unpalmitoylated Ras1 from endomembranes. (b) Pheromone signaling is modulated by plasma membrane-tethered and palmitoylated Ras1 to activate MAPK Spk1. (c) Deconvolved images of cells from control and mam4∆ strains expressing a GFP-CRIB fusion (GTP-bound Cdc42) were grown in YES medium and observed by fluorescence microscopy. (d) Serial dilutions of suspensions of control, mam4∆, Cdc42-L160S, and mam4∆ Cdc42-L160S strains were spotted on YES plates and incubated at either, 25, 30, and 34 °C for 3 days. Results representative of three independent experiments are shown. (e) Control, mam4∆, erf2∆, and mam4∆ erf2∆ strains of the h+ mating type were mixed with wild type h− cells, poured on SPA plates, and incubated at 25 °C. The percentage of conjugation efficiency (as mean ± SD) was determined after 24 and 48 h of incubation by microscopic counting of number of vegetative cells, zygotes, and asci. Biological triplicate samples (≥300 cells) were counted for each cross. (f) Control and mam4∆ strains were grown in EMM2 medium and transferred to the same medium lacking nitrogen source. TCA extracts were obtained from samples taken at different times, and activated Spk1 was detected with anti-phospho-p44/42 antibody. Relative units as mean ± SD for Spk1 activation (biological triplicates) were determined with respect to the internal control (anti-Cdc2 blot) at each time point.*P < 0.05. (G) Spk1 activation in cultures from control, erf2∆, and mam4∆ erf2∆ strains was detected and quantified as described in (f). *P < 0.05.
