Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway.
The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.
Evans
Timothy M
TM
Institute for Molecular Bioscience, Department of Biochemistry, The University of Queensland, Brisbane QLD 4072, Australia.
Ferguson
Charles
C
Wainwright
Brandon J
BJ
Parton
Robert G
RG
Wicking
Carol
C
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008647:Mesocricetus; D051379:Mice; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D009154:Mutation; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*
D018076:DNA, Complementary; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D049452:Green Fluorescent Proteins; D001710:Biotin; D004247:DNA; C486614:RAB23 protein, human; C431746:Rab23 protein, mouse; D020691:rab GTP-Binding Proteins