A role for the small GTPase Rab21 in the early endocytic pathway.
Rab proteins comprise a family of monomeric GTPases that control cellular membrane traffic. Rab21 is a poorly characterised member with no known function. Human Rab21 cDNA from K562 cells was subcloned into GFP expression vectors to generate Rab21 and Rab21 mutants defective in either GTP hydrolysis (Rab21 Q78L) or binding (Rab21 T33N) for transfection studies in HeLa cells. Confocal fluorescence microscopy and ultrastructural studies revealed Rab21 to be predominantly localised to the early endocytic pathway, on vesicles containing earlyendosomal antigen 1 EEA1, transferrin receptor and internalised ligands. EEA1 was localised to enlarged endosomes in Rab21 wild-type expressing cells but the GTP hydrolysis and GDP binding mutants had unique phenotypes labelling tubular reticular structures and the trans-Golgi network, respectively. Early endosome localisation for Rab21 was confirmed in a hepatoma cell line that allowed analysis of the subcellular distribution of the endogenous protein. Comparison of the localisation of Rab21 with other Rabs revealed extensive colocalisation with early endocytic variants Rab4, Rab5, Rab17 and Rab22 but much less overlap with those associated with late endosomes, recycling endosomes and the early secretory pathway. Cells expressing Rab21 T33N had defects in endocytosis of transferrin and epidermal growth factor and failed to effectively deliver the latter ligand to late endosomes and lysosomes for degradation. Collectively, our data provide the first characterisation of Rab21 function in early endosome dynamics.
Simpson
Jeremy C
JC
Cell Biology and Biophysics Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Griffiths
Gareth
G
Wessling-Resnick
Marianne
M
Fransen
Jack A M
JA
Bennett
Holly
H
Jones
Arwyn T
AT
D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004705:Endocytosis; D011992:Endosomes / Q000648:ultrastructure; D004815:Epidermal Growth Factor / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism / Q000648:ultrastructure; D018613:Microscopy, Confocal; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins; D014168:Transferrin; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D020691:rab GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins