Recombinant antibodies to the small GTPase Rab6 as conformation sensors.
Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.
CNRS UMR144, Institut Curie, 26 rue d'Ulm, F75248 Paris Cedex 05, France.
Clement
C
Nizak
Solange
S
Monier
Elaine
E
del Nery
Sandrine
S
Moutel
Bruno
B
Goud
Franck
F
Perez
D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007135:Immunoglobulin Variable Region; D008164:Luminescent Proteins; D009154:Mutation; D019151:Peptide Library; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D022161:Transport Vesicles / Q000737:chemistry; D020691:rab GTP-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D001426:Bacterial Proteins; D007135:Immunoglobulin Variable Region; D008164:Luminescent Proteins; D019151:Peptide Library; C094135:Rab6 protein; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D006153:Guanosine Diphosphate; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020691:rab GTP-Binding Proteins