COMPARISON OF THE BIOCHEMICAL-PROPERTIES OF UNPROCESSED AND PROCESSEDFORMS OF THE SMALL GTP-BINDING PROTEIN, RAB6P

The rab6 protein (rab6p) belongs to a large family of ras-like low-mol ecular-mass GTP-binding proteins thought to be involved in the regulat ion of intracellular transport in mammalian cells. When expressed in t he baculovirus/insect cell system, two major forms of rab6p are obtain ed; a 24-kDa cytosolic unprocessed form and a 23-kDa membrane-bound fo rm which represents the processed lipid-modified protein. Here, we hav e purified both forms to homogeneity and we have studied and compared their biochemical properties. Unprocessed and processed rab6p display similar binding-rate constants (k(on)) for GDP and GTP (1 - 1.9 muM-1 min-1). However, significant differences exist in the dissociation con stants of bound guanine nucleotides. Processed rab6p in low and high m agnesium solutions displays similar k(off) values for GTP and GDP. How ever, unprocessed rab6p has a k(off) value higher for GDP than for GTP in both low and high magnesium solutions. Their intrinsic GTPase acti vities also differ; unprocessed rab6p has an almost undetectable GTPas e activity, whereas that of processed rab6p is in the same range as th at reported for other ras and ras-like GTP-binding proteins (0.01 2 +/ - 0.002 min-1). These results suggest that post-translational modifica tions of rab6p might induce subtle changes in the three-dimensional st ructure of the protein which affect the guanine-nucleotide-binding/hyd rolysis activity.