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MBC in Press, published online ahead of print February 9, 2005
Mol. Biol. Cell 10.1091/mbc.E04-06-0464

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Submitted on June 8, 2004
Revised on December 27, 2004
Accepted on January 26, 2005

Role of Ubiquitin and Proteasomes in Phagosome Maturation

Warren L. Lee,*{dagger}{ddagger} Moo-Kyung Kim,{sect} Alan D. Schreiber,{sect} and Sergio Grinstein*

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

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{gamma} receptors (Fc{gamma}R), which are known to be cleared from the phagosome during maturation. Fc{gamma}R, 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, while others remain associated with the phagosome. By fusing Fc{gamma}R 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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