Comparison of the biochemical properties of unprocessed and processed forms of the small GTP-binding protein, rab6p.
The rab6 protein (rab6p) belongs to a large family of ras-like low-molecular-mass GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. When expressed in the baculovirus/insect cell system, two major forms of rab6p are obtained; a 24-kDa cytosolic unprocessed form and a 23-kDa membrane-bound form which represents the processed lipid-modified protein. Here, we have purified both forms to homogeneity and we have studied and compared their biochemical properties. Unprocessed and processed rab6p display similar binding-rate constants (kon) for GDP and GTP (1-1.9 microM-1 min-1). However, significant differences exist in the dissociation constants of bound guanine nucleotides. Processed rab6p in low and high magnesium solutions displays similar koff values for GTP and GDP. However, unprocessed rab6p has a koff value higher for GDP than for GTP in both low and high magnesium solutions. Their intrinsic GTPase activities also differ; unprocessed rab6p has an almost undetectable GTPase activity, whereas that of processed rab6p is in the same range as that reported for other ras and ras-like GTP-binding proteins (0.012 +/- 0.002 min-1). These results suggest that post-translational modifications of rab6p might induce subtle changes in the three-dimensional structure of the protein which affect the guanine-nucleotide-binding/hydrolysis activity.
Yang
C
C
Unité de Génétique Somatique, URA CNRS 361, Institut Pasteur, Paris, France.
Mollat
P
P
Chaffotte
A
A
McCaffrey
M
M
Cabanié
L
L
Goud
B
B
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D009036:Moths; D011499:Protein Processing, Post-Translational
D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins