Endosome fusion induced by diphtheria toxin translocation domain.
Endosomal cargo travels through a dynamic vesicle network en route to degradation by lysosomes or recycling through the Golgi apparatus back to the cell surface. Rab5 is a key determinant of the early endosomes by organizing effector proteins in specific subdomains and mediating early endosome fusion. We find that early endosome morphogenesis and maturation is disrupted by diphtheria toxin (DT). Rab5 bound endosomes increase in size and in Rab5 content due to luminal toxin exposure, whereas Rab7 positive endosomes are not detectably altered. These changes appear to be caused by an activity of the toxin entry domain (T domain) as mutations inactivating either the receptor binding (CRM107) or ADP-ribosyl transferase (CRM197) activities do not inhibit the effect of DT on endosome morphogenesis. In contrast, mutations in the T domain or diminishing the endosomal pH gradient, which prevents T domain membrane insertion, inhibit these endosome changes. The change in size appears to be due to changing the early endosome fission-fusion equilibrium. The Rab5 membrane exchange rate, assessed with photoactivatable GFP-Rab5, decreases in the presence of DT. These changes to endosomes may reflect activities of the T domain that mediate toxin entry to the cytosol. The nontoxic mutant DT, CRM197, yields a new tool to manipulate endosome dynamics in living cells.
Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive MSC 3704, Bethesda, MD 20892, USA.
Antonella
A
Antignani
Richard J
RJ
Youle
D002470:Cell Survival / Q000187:drug effects; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000494:pharmacology*; D011992:Endosomes / Q000187:drug effects* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D014162:Transfection; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D004167:Diphtheria Toxin; D020696:rab5 GTP-Binding Proteins