Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes.
Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.
Vonderheit
Andreas
A
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland.
Helenius
Ari
A
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002522:Chlorocebus aethiops; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D012672:Semliki forest virus / Q000378:metabolism*; D014162:Transfection; D014709:Vero Cells; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
C494127:E1 glycoprotein, Semliki forest virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins; C093846:early endosome antigen 1; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D020823:ADP-Ribosylation Factor 1; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D015739:Nocodazole