Isoform-specific membrane targeting mechanism of Rac during Fc gamma R-mediated phagocytosis: positive charge-dependent and independent targeting mechanism of Rac to the phagosome.
Rac1 and Rac2 are capable of stimulating superoxide production in vitro, but their targeting and functional mechanisms are still unknown. In the present study, we found that Rac1, 2, and 3 all accumulate at the phagosome during Fc gammaR-mediated phagocytosis, and that the order of accumulation (Rac1 > Rac3 > Rac2) depends on the net positive charge in their polybasic (PB) regions (183-188 aa). Although all GFP-tagged prenylated PB regions of Rac isoforms (GFP-Rac(PB)) and GFP-tagged prenylated 6 Ala (GFP-6A) accumulated during phagocytosis, GFP-Rac2(PB) and GFP-6A showed weak accumulation at the phagosome through a linear structure connecting the phagosome and endomembranes. The PB region of Rac1 showed strong phospholipid interaction with PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and phosphatidic acid, however, that of Rac2 did not. Constitutively active Rac2, GFP-Rac2(Q61L), was predominantly localized at the endomembranes; these endomembranes fused to the phagosome through the linear structure during phagocytosis, and this accumulation mechanism did not depend on positive charge in the PB region. Our conclusion is that Rac1 directly targets to the phagosome using the positively charged PB region and this accumulation mechanism is likely enhanced by the phospholipids. In addition to this mechanism, Rac2 has a positive charge-independent mechanism in which Rac2 initially targets to endomembranes and then these endomembranes fuse to the phagosome.
Ueyama
Takehiko
T
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan.
Eto
Mika
M
Kami
Keiichiro
K
Tatsuno
Toshihiko
T
Kobayashi
Toshihiro
T
Shirai
Yasuhito
Y
Lennartz
Michelle R
MR
Takeya
Ryu
R
Sumimoto
Hideki
H
Saito
Naoaki
N
D024361:Amino Acids, Basic / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D002474:Cell-Free System / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D050506:Mutant Chimeric Proteins / Q000502:physiology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010587:Phagocytosis / Q000276:immunology*; D010588:Phagosomes / Q000201:enzymology* / Q000276:immunology; D010743:Phospholipids / Q000378:metabolism; D017368:Protein Prenylation; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000502:physiology*; D055672:Static Electricity; D013481:Superoxides / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000097766:RAC2 GTP-Binding Protein
D024361:Amino Acids, Basic; D007527:Isoenzymes; D050506:Mutant Chimeric Proteins; D010446:Peptide Fragments; D010743:Phospholipids; D017452:Receptors, IgG; D013481:Superoxides; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C498771:Rac3 protein, mouse