Subcellular fractionation of PfRab5B and GAP45 GFP chimeras.

When fused to GFP, the first 28 amino acids of PfRab5B confer a partial membrane association to the fusion protein. PfRab5B28-GFP is found to varying degrees in the four fractions studied. Substitution of glycine at position two with alanine (PfRab5B28 G2A) results in loss of all membrane association, as does substitution of cysteine at position three (PfRab5B28 C3A). A known myristoylated and palmitoylated protein, GAP4529-GFP is found exclusively in membrane fractions and a G2A substitution results in an entirely soluble protein (GAP4529 G2A). A substitution of cysteine at position five causes a reduction in the proportion of protein associated with membranes (GAP4529 C5A). Double substitutions of glycine and cysteine result in a cytosolic protein for both PfRab5B28 and GAP4529 GFP fusion proteins. HL: hypotonic lysis buffer supernatant, HS: high salt buffer supernatant, CS: carbonate buffer supernatant, CI: carbonate buffer insoluble.