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.将PfRab5B的前28个氨基酸与绿色荧光蛋白（GFP）融合后，可赋予该融合蛋白部分膜结合能力。研究发现，PfRab5B28-GFP在四种受试组分中均有不同程度的分布。将第2位甘氨酸替换为丙氨酸（PfRab5B28 G2A）会使融合蛋白完全丧失膜结合活性，第3位半胱氨酸的丙氨酸替换（PfRab5B28 C3A）也可产生相同效果。已知的肉豆蔻酰化与棕榈酰化蛋白GAP4529-GFP仅存在于膜组分中，其G2A突变体（GAP4529 G2A）则完全转变为可溶性蛋白。将第5位半胱氨酸替换为丙氨酸会降低膜结合蛋白的占比（GAP4529 C5A）。对于PfRab5B28与GAP4529的GFP融合蛋白而言，甘氨酸与半胱氨酸的双位点突变均可使蛋白变为胞质可溶性形式。组分缩写说明：HL为低渗裂解缓冲液上清液，HS为高盐缓冲液上清液，CS为碳酸盐缓冲液上清液，CI为碳酸盐缓冲液不溶组分。