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Molecular and Cellular Biology, April 2003, p. 2501-2514, Vol. 23, No. 7
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.7.2501-2514.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Modulation of Rab5 and Rab7 Recruitment to Phagosomes by Phosphatidylinositol 3-Kinase

Otilia V. Vieira,1 Cecilia Bucci,2 Rene E. Harrison,1 William S. Trimble,1 Letizia Lanzetti,3 Jean Gruenberg,4 Alan D. Schreiber,5 Philip D. Stahl,6 and Sergio Grinstein1*

Cell Biology Program, Hospital for Sick Children and Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada,1 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita degli Studi di Lecce, 73100 Lecce,2 Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy,3 Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland,4 Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283,5 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 631106

Received 4 September 2002/ Returned for modification 29 November 2002/ Accepted 30 December 2002

Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.

* Corresponding author. Mailing address: Division of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Phone: (416) 813-5727. Fax: (416) 813-5028. E-mail: sga{at}sickkids.on.ca.

Molecular and Cellular Biology, April 2003, p. 2501-2514, Vol. 23, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.7.2501-2514.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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