Abstract
Several Rab GTPases have been localized to distinct compartments of the endocytic pathway. Rab4 is associated with early endosomes and recycling vesicles and regulates membrane recycling from early endosomes. Rab7 is localized to late endosomes and is involved in the regulation of membrane transport between late endosomes and lysosomes. Although Rab4 and Rab7 appear to regulate distinct transport events in endocytosis, it is not clear whether they perform their activities in related or entirely distinct intracellular compartments. To address this question, we generated stable cell lines expressing Rab4 tagged with a novel X31 influenza hemagglutinin (NH) epitope tag. These antibodies are characterized in this paper and were used to immunoisolate endocytic vesicles with cytoplasmically exposed NHRab4. Immunoisolated membranes contain internalized 125I-transferrin, but are devoid of Rab7. Confocal immunofluorescence microscopy showed that the early endosomal GTPases Rab4 and Rab5 both do not codistribute with Rab7 within the same cell. These observations suggest that each of the three Rab GTPases operationally defines a distinct station of the endocytic pathway.
Highlights
Receptor-mediated endocytosis is a process in which eukaryotic cells internalize proteins and solutes
Distribution of Endosomal Rab Proteins rescence data employing the novel novel X31 influenza hemagglutinin (NH) epitope tag show that Rab4 and Rab7 are essentially associated with distinct nonoverlapping endocytic compartments
To define the epitopes recognized by the novel NH antibodies, we performed a deletion analysis of constructs consisting of Rab4 with 5Јextensions of oligonucleotides encoding truncated versions of the NH epitope tag
Summary
TfR, transferrin receptor; hTfR, human TfR; Tf, transferrin; CHO, Chinese hamster ovary; GST, glutathione S-transferase; PBS, phosphate-buffered saline. Distribution of Endosomal Rab Proteins rescence data employing the novel NH epitope tag show that Rab and Rab are essentially associated with distinct nonoverlapping endocytic compartments. In NHRab4/GRab (Rab with N-terminal vesicular stomatitis virus G protein epitope tag), we observed that Rab and Rab are associated with distinct endocytic organelles. These results suggest that the early endocytic compartments characterized by Rab or Rab and the late endosomes associated with Rab are separate structures
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