Reconstitution of recycling from the phagosomal compartment in streptolysin O-permeabilized macrophages: role of Rab11.
By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells in vesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasma membrane following phagosome biogenesis. Here, we report a new approach to the study of recycling from the phagosomal compartment: streptolysin O- (SLO) permeabilized macrophages. In this semi-intact cell system, energy and cytosol are required to efficiently reconstitute recycling transport. Addition of GDPbetaS strongly inhibits this transport step, suggesting that a GTP-binding protein modulates the dynamics of cargo exit from the phagosomal compartment. GTPases of the Rab family control vesicular trafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role of Rab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilized macrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycled proteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, we confirm that overexpression of Rab11:S25N substantially decreases recycling from phagosomes in intact cells. These findings show the requirement of a functional Rab11 for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilized macrophages likely constitute a useful tool to identify new molecules involved in regulating transport along the phagocytic pathway.
Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza 5500, Argentina.
Natalia
N
Leiva
Martín
M
Pavarotti
María I
MI
Colombo
María T
MT
Damiani
D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D002463:Cell Membrane Permeability; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010588:Phagosomes / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013301:Streptolysins / Q000494:pharmacology*; D013873:Thionucleotides / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D001426:Bacterial Proteins; D020558:GTP Phosphohydrolases; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate; D011993:Recombinant Fusion Proteins; D013301:Streptolysins; D013873:Thionucleotides; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C025964:streptolysin O; C023427:guanosine 5'-O-(2-thiodiphosphate)