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Vol. 16, Issue 4, 2077-2090, April 2005

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Role of Ubiquitin and Proteasomes in Phagosome Maturation

Warren L. Lee ^*  , Moo-Kyung Kim , Alan D. Schreiber , and Sergio Grinstein ^*

^* Programme in Cell Biology, Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; Interdepartmental Division of Critical Care Medicine, Department of Medicine, University of Toronto, Toronto M5S 3J3, Ontario, Canada; Division of Respirology, Department of Medicine, University of Toronto, Toronto M5S 3J3, Ontario, Canada; and Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Submitted June 8, 2004; Revised December 27, 2004; Accepted January 26, 2005
Monitoring Editor: Keith Mostov

Phagosomes undergo multiple rounds of fusion with compartments of the endocytic pathway during the course of maturation. Despite the insertion of vast amounts of additional membrane, the phagosomal surface area remains approximately constant, implying active ongoing fission. To investigate the mechanisms underlying phagosomal fission we monitored the fate of Fc receptors (FcR), which are known to be cleared from the phagosome during maturation. FcR, which show a continuous distribution throughout the membrane of nascent phagosomes were found at later times to cluster into punctate, vesicular structures, before disappearing. In situ photoactivation of receptors tagged with a light-sensitive fluorescent protein revealed that some of these vesicles detach, whereas others remain associated with the phagosome. By fusing FcR to pH-sensitive fluorescent proteins, we observed that the cytoplasmic domain of the receptors enters an acidic compartment, indicative of inward budding and formation of multivesicular structures. The topology of the receptor was confirmed by flow cytometry of purified phagosomes. Phagosomal proteins are ubiquitylated, and ubiquitylation was found to be required for formation of acidic multivesicular structures. Remarkably, proteasomal function is also involved in the vesiculation process. Preventing the generation of multivesicular structures did not impair the acquisition of late endosomal and lysosomal markers, indicating that phagosomal fusion and fission are controlled separately.

Address correspondence to: Sergio Grinstein (sga{at}sickkids.ca).

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