Detection of activated Rab7 GTPase with an immobilized RILP probe.
The dynamic and coordinated exchange of multiple GTPases between the cytosol and the phagosome membrane represents a critical process during phagosome biogenesis. In particular, acquisition of Rab7 is crucial for progression to the stage where formation of phagolysosomes is observed. Optimal Rab7 effector function requires its conversion to the GTP-bound form where it becomes activated. In light of this regulatory node, the GDP/GTP switch on the Rab7 molecule represents a tractable event to dissect the control of phagosome maturation by intracellular pathogen or their products. Direct measurement of Rab7 activation requires 32P-GTP binding to renatured Rab7 recovered by pull downs and resolved by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and autoradiography. Here, we describe a novel, alternative, nonradioactive assay to measure Rab7 activity which takes advantage of the specific binding of activated (GTP bound) Rab7 to its effector RILP (Rab7 interacting lysosomal protein). Active Rab7 bound to immobilized recombinant RILP on latex beads can be detected quantitatively by either classical Western blotting or flow cytometry.
Division of Infectious Diseases, Department of Medicine, University of British Columbia and Vancouver Coastal Health Institute, Vancouver, BC, Canada.
Jim
J
Sun
Ala-Eddine
AE
Deghmane
Cecilia
C
Bucci
Zakaria
Z
Hmama
D048868:Adaptor Proteins, Signal Transducing / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D005434:Flow Cytometry; D055767:Immobilized Proteins / Q000378:metabolism*; D015151:Immunoblotting; D007840:Latex; D051379:Mice; D008863:Microspheres; D008967:Molecular Biology / Q000379:methods*; D015335:Molecular Probes / Q000378:metabolism*; D009163:Mycobacterium bovis / Q000502:physiology; D011485:Protein Binding; D015203:Reproducibility of Results; D020691:rab GTP-Binding Proteins / Q000032:analysis*; D000090802:rab7 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D055767:Immobilized Proteins; D007840:Latex; D015335:Molecular Probes; D000090802:rab7 GTP-Binding Proteins; C000716465:rab7 GTP-binding proteins, mouse; D020691:rab GTP-Binding Proteins