Figure 2
From: Distinct functional relevance of dynamic GTPase cysteine methylation in fission yeast
Mam4 function differentially affects GTPase localization at the plasma membrane. (a) Deconvolved images of cells from control and mam4∆ strains expressing GFP-Rho1 fusions grown in YES medium and observed by fluorescence microscopy. Representative fluorescence intensity plots (as arbitrary fluorescence units) were generated from line scans across the cell width (dotted white lines). (b) Images of cells from control and mam4∆ strains expressing Cdc42-GFPSW genomic fusions observed by fluorescence microscopy. Representative fluorescence intensity plots were generated from line scans across the cell length (dotted white lines). (c) Images of cells from control and mam4∆ strains expressing GFP-Rho2-HA-CCIIS genomic fusions observed by fluorescence microscopy. Representative fluorescence intensity plots were obtained as described in (a). (d) Deconvolved images of mixed control (GFP-Rho2-HA-CCIIS, mCherry-Atb2) and mam4∆ (GFP-Rho2-HA-CCIIS) cells were observed by fluorescence microscopy. (e) Images of cells from control and mam4∆ strains expressing GFP-Ras1 genomic fusions observed by fluorescence microscopy. Representative fluorescence intensity plots were obtained as described in (a). (f) Deconvolved images of mixed control (GFP-Ras1, mCherry-Atb2) and mam4∆ (GFP-Ras1) cells were observed by fluorescence microscopy. (g) Rho2 palmitoylation assayed by the acyl-biotinyl switch assay in cell lysates from control and mam4∆ strains expressing a GFP-Rho2-HA genomic fusion. Biotinylation is specific for proteins containing a free sulfhydryl generated after hydroxylamine cleavage (+HX). Total extracts from the strains were included as loading controls. GFP-Rho2-HA fusion was detected employing anti-HA antibody. Percentage of palmitoylation (as mean ± SD) in control and mam4∆ cells was determined from biological duplicates. (h) Ras1 palmitoylation by the acyl-biotinyl switch assay in control and mam4∆ strains expressing a GFP-Ras1 genomic fusion was determined as above. GFP-Ras1 fusion was detected employing anti-GFP antibody. Percentage of palmitoylation (as mean ± SD) in control and mam4∆ cells was determined from biological duplicates.
