Selection and application of recombinant antibodies as sensors of rab protein conformation.
The existence of a conformational switch of Rabs and other small GTPases involved in intracellular transport regulation has been known for many years. This switch is superimposed on the membrane association/dissociation cycle for most of these GTPases. While these processes are key features of the dynamics of intracellular transport events, surprisingly very few previous studies have focused on the dynamics of the GDP/GTP cycle of Rab proteins in time and space. The main reason for this is the lack of tools available to dynamically probe for Rab GTPases conformation switches and membrane association/dissociation, in particular in vivo. We recently reported the in vitro selection of conformation-specific recombinant antibodies specific to the GTP-bound conformation of Rab6 proteins. These antibodies were obtained in vitro by phage display, a rather simple, rapid, and cheap technique. We additionally showed that these conformation-specific antibodies can be expressed in living cells to follow endogenous Rab6 in its activated conformation in vivo. The same strategy could be used to study other conformation switching mechanisms and, in general, to study the switching between states that antibodies can distinguish (e.g., phosphorylation, ubiquitination).
Nizak
Clément
C
Moutel
Sandrine
S
Goud
Bruno
B
Perez
Franck
F
D000906:Antibodies / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D020691:rab GTP-Binding Proteins / Q000737:chemistry*
D000906:Antibodies; D011994:Recombinant Proteins; D020691:rab GTP-Binding Proteins