DOPC/DOPS liposomes enriched in PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ cause only minimal dissociation of Rac1(mant-GDP)·RhoGDI complexes. 1 nmol of Rac1(mant-GDP)·RhoGDI complex was mixed with liposomes (90 nmol of total phospholipids) comprising 0.74 mm (82.22 mol %) DOPC and 0.16 mm (17.77 mol %) DOPS (A); 0.74 mm (81.30 mol %) DOPC, 0.16 mm (17.58 mol %) DOPS, and 10 μm (1.10 mol %) PtdIns (3,4)P₂ (B); or 0.74 mm (81.30 mol %) DOPC, 0.16 mm (17.58 mol %) DOPS, and 10 μm (1.10 mol %) (PtdIns(3,4,5)P₃ (C). In all experiments the Rac1(mant-GDP)·RhoGDI complex was preincubated with phospholipid liposomes for 12 min at room temperature. The mixtures were subjected to gel filtration on a Superose 12 FPLC column, and the eluates were monitored in line for the fluorescent signal of Rac1(mant-GDP). In each panel, peak 1 represents Rac1(mant-GDP) dissociated from the Rac1(mant-GDP)·RhoGDI complex and bound to the liposomes eluting in the exclusion volume, and peak 2 represents the non-dissociated Rac1(mant-GDP)·RhoGDI complex. The numbers next to peaks 1 and 2 express the amount of Rac1(mant-GDP) dissociated from the complex and left in the complex, respectively, expressed as a percentage of the total amount of Rac1(mant-GDP) present in the complex injected into the column. Calculations were based on the integration of the relevant peaks. The panels illustrate representative individual experiments of at least two to three experiments performed for each combination of components.

All GEF constructs display nucleotide exchange activity on prenylated Rac1 bound to DOPC/DOPS liposomes. DOPC/DOPS liposomes (180 nmol of total phospholipids) were mixed with 2 nmol of prenylated Rac1(GDP), 10 nmol of mant-GDP, and one of the following components: buffer (A), 3 nmol of TrioN (B), 3 nmol of Tiam1(C1199) (C), 3 nmol of Tiam1(C1199) KK/AA, RR/AA (D), or 3 nmol of Tiam1(C374) (E). Reaction mixtures were incubated for 12 min at room temperature. The mixtures were subjected to gel filtration on a Superose 12 FPLC column, and the eluates were monitored in line for the fluorescent signal of Rac1(mant-GDP). In each panel, peak 1 represents liposome-bound prenylated Rac1 labeled with mant-GDP eluting in the exclusion volume, and peak 2 represents free prenylated Rac1 labeled with mant-GDP eluting in a volume corresponding to its molecular mass. The numbers next to peaks 1 and 2 express the percentage of the total amount of prenylated Rac1 labeled with mant-GDP associated with the respective peaks. Calculations were based on the integration of the areas of peaks 1 and 2 in the five experimental situations (A, B, C, D, and E), which are listed in the table at the bottom of the figure. The panels illustrate representative individual experiments of three experiments performed for each combination of components. The numerical results in the table are means ± S.E. of three experiments.

Replacement of mant-GMPPNP by mant-GDP eliminates the dissociation of Rac1·RhoGDI complexes by anionic liposomes and TrioN, but nucleotide exchange on Rac1 in complex with RhoGDI is conserved. 2 nmol of Rac1(GDP)·RhoGDI complex and 10 nmol of mant-GDP were mixed with DOPC/DOPS liposomes (90 nmol of total phospholipids) and 3 nmol of TrioN (A), DOPC/DOPS/PtdIns(3,4,5)P₃ liposomes (90 nmol of total phospholipids) and 3 nmol of TrioN (B), 3 nmol of TrioN without liposomes (C), and DOPC/DOPS/PtdIns(3,4,5)P₃ liposomes (90 nmol of total phospholipids) without TrioN (D). In all experiments, the reaction mixtures were incubated for 12 min at room temperature. The mixtures were subjected to gel filtration on a Superose 12 FPLC column, and the eluates were monitored in line for the fluorescent signal of Rac1(mant-GDP). In each panel, peak 1 represents Rac1 dissociated from the Rac1(GDP)·RhoGDI complex, labeled with mant-GDP, and bound to the liposomes eluting in the exclusion volume, and peak 2 represents the non-dissociated Rac1(mant-GDP)·RhoGDI complex. The areas of fluorescent peak 2 appear in the tables below the upper and lower panels and represent means ± S.E. of three experiments. The panels illustrate representative individual experiments of three experiments performed for each combination of components. w/o, without.

Amphiphile-independent oxidase activation in native versus PtdIns(3,4,5)P₃-enriched phagocyte membranes by Rac1(GDP)·RhoGDI complexes, guanine nucleotides, and GEFs. In these experiments, native phagocyte membranes or membranes supplemented with 10 μm PtdIns(3,4,5)P₃ were prepared as described under “Experimental Procedures.” Oxidase activation was assayed in a cell-free system consisting of either native or supplemented membranes (equivalent to 5 nm cytochrome b₅₅₉ heme) mixed with prenylated Rac1(GDP) (100 nm), p67^phox (300 nm), one of the GEF constructs (200 nm), and GTP (1 μm) (A) or with Rac1(GDP)·RhoGDI complex (100 nm), p67^phox (300 nm), one of the GEF constructs (400 nm), and either GDP or GTP (1 μm) (B). Assay mixtures were incubated for 5 min at room temperature in the absence of an anionic amphiphile, and production was initiated by the addition of NADPH (240 μm). The results are means ± S.E. of three experiments. Error bars represent S.E. w/o, without.

Schematic representation of GEF constructs used in this study. A, domain architecture of full-length Trio and His₆-tagged TrioN. Numbers indicate the position of amino acids in the full-length protein. DHn and DHc stand for the N- and C-terminal Dbl homology domains, respectively; SH3 stands for Src homology 3 domain; Ig stands for Ig-like domain, and STK stands for serine-threonine kinase domain. B, domain architecture of full-length Tiam1 and its His₆-tagged derivatives Tiam1(C1199) (residues 394-1591) and Tiam1(C374) (residues 1033-1406). Numbers indicate the position of amino acids in the full-length protein. RBD stands for Ras-binding domain, and PDZ stands for PSD-95 Discs large/ZO-1 homology domain.

Exposure of Rac1(GDP)·RhoGDI complexes to anionic liposomes, mant-GMMPNP, and TrioN causes dissociation of complexes, GDP to mant-GMPPNP exchange on Rac1, and binding of Rac1-GMPPNP to liposomes. 2 nmol of Rac1(GDP)·RhoGDI complex were mixed with 10 nmol of mant-GMPPNP and DOPC/DOPS liposomes (90 nmol of total phospholipids) and 3 nmol of TrioN (A), DOPC/DOPS/PtdIns(3,4,5)P₃ liposomes (90 nmol of total phospholipids) and 3 nmol of TrioN (B), or DOPC/DOPS/PtdIns(3,4,5)P₃ liposomes (90 nmol of total phospholipids) and buffer (C). In all experiments, the reaction mixtures were incubated for 12 min at room temperature. The mixtures were subjected to gel filtration on a Superose 12 FPLC column, and the eluates were monitored in line for the fluorescent signal of Rac1(mant-GMPPNP) (upper panels). In each panel, peak 1 represents Rac1 dissociated from the Rac1(GDP)·RhoGDI complex, labeled with mant-GMPPNP, and bound to the liposomes eluting in the exclusion volume, and peak 2 represents the nondissociated Rac1(GDP)·RhoGDI complex. The areas of the fluorescent peak 1 in the three experimental situations (A, B, and C) are listed in the table associated with the figure and represent means ± S.E. of three experiments. The panels illustrate representative individual experiments of at least three experiments performed for each combination of components. The dissociation and translocation of Rac1 to liposomes was confirmed by Western blot analysis (lower panels). 35 μl of each fraction (0.6 ml), corresponding to peak 1 (fractions 11 to 14), were subjected to SDS-PAGE followed by immunoblotting with anti-Rac1 antibody as described under “Experimental Procedures.” Rac1·RhoGDI complex, applied to the first lane at the left, served as a control for the ability of anti-Rac1 antibody to detect Rac1. The figure illustrates representative individual experiments of two performed with Rac1(GDP)·RhoGDI complexes mixed with liposomes in the three experimental conditions (A, B, and C). D, detection of liposome-bound His₆-tagged TrioN by ELISA. 25-μl aliquots of fractions 13 and 14, corresponding to peak 1, were used to coat the wells of 96-well microplates. His₆-tagged TrioN was detected using anti-polyhistidine antibody as described under “Experimental Procedures”; results are means ± S.E. of three experiments. Error bars represent S.E. E, SDS-PAGE analysis of peak 2. 35 μl of each fraction corresponding to peak 2 (fractions 20-24) were analyzed by SDS-PAGE. D and E illustrate representative individual experiments of at least two to three experiments performed. w/o, without.

The N-terminal PH domain of Tiam1 is essential for promoting the dissociation of the Rac1(GDP)·RhoGDI complex in the presence of PtdIns(3,4,5)P₃-enriched liposomes and mant-GMPPNP. 2 nmol of Rac1(GDP)·RhoGDI complex and 10 nmol of mant-GMPPNP were mixed with DOPC/DOPS/PtdIns(3,4,5)P₃ liposomes (90 nmol of total phospholipids) and 3 nmol of Tiam1(C1199) (A), DOPC/DOPS/PtdIns(3,4,5)P₃ liposomes (90 nmol of total phospholipids) and 3 nmol of Tiam1(C1199) KK/AA, RR/AA (B), DOPC/DOPS/PtdIns(3,4,5)P₃ liposomes (90 nmol of total phospholipids) and 3 nmol of Tiam1(C374) (C), DOPC/DOPS liposomes (90 nmol of total phospholipids) and 3 nmol of Tiam1(C1199) (D), DOPC/DOPS liposomes (90 nmol of total phospholipids) and 3 nmol of Tiam1(C1199) KK/AA, RR/AA (E), and DOPC/DOPS liposomes (90 nmol of total phospholipids) and 3 nmol of Tiam1(C374) (F). In all experiments, the reaction mixtures were incubated for 12 min at room temperature. The mixtures were subjected to gel filtration on a Superose 12 FPLC column, and the eluates were monitored in line for the fluorescent signal of Rac1(mant-GMPPNP). In each panel, peak 1 represents Rac1 dissociated from the Rac1(GDP)·RhoGDI complex, labeled with mant-GMPPNP, and bound to the liposomes eluting in the exclusion volume, and peak 2 represents the non-dissociated Rac1(GDP)·RhoGDI complex. The areas of fluorescent peak 1 in the six experimental situations are presented in the tables beneath the panels illustrating the in-line recording of fluorescence (A, B, C, D, E, and F) and represent means ± S.E. of three experiments. The panels illustrate representative individual experiments of at least three experiments performed for each combination of components. G, H, and I, detection of liposome-bound His₆-tagged Tiam1 constructs by ELISA. 25-μl aliquots of fractions 13 and 14, corresponding to peak 1, derived from the experiments illustrated in A-F were used to coat the wells of 96-well microplates. The His₆-tagged Tiam1 proteins were detected using anti-polyhistidine antibody as described under “Experimental Procedures.” Results are means ± S.E. of three experiments. Error bars represent S.E.

Extrapolation of the in vitro mechanism of Rac·RhoGDI dissociation by the cooperative action of PtdIns(3,4,5)P₃-containing liposomes, GTP, and GEF to events hypothesized to occur in the course of oxidase activation in the intact phagocyte. The proposed sequence of events is as follows. 1, in the PM of the resting cell, represented by a phospholipid composition of less than 20% anionic lipids (phosphatidylcholine (PC)/phosphatidylserine (PS)), the Rac·RhoGDI complexes are in the cytosol. 2 and 3, upon phagocyte stimulation, PtdIns(3,4,5)P₃ is generated on the cytosolic aspect of the PM resulting in a marked increase in negative charge. A small proportion of the Rac·RhoGDI complexes dissociates spontaneously, and Rac(GDP) translocates to the PtdIns(3,4,5)P₃-enriched PM. 4, a marked enhancement of the dissociation of Rac·RhoGDI complexes takes place upon the translocation of a Rac-specific GEF to the PM by virtue of the affinity of the PH domain of GEF for PtdIns(3,4,5)P₃. This leads to GDP to GTP exchange on Rac, also bound to the PM, preventing reassociation with RhoGDI due to the lower affinity of the latter for Rac(GTP). 5, membrane-associated Rac(GTP) interacts with downstream effectors, exemplified by p67^phox. Intrinsic or GTPase-activating protein-enhanced GTPase activity leads to the conversion of Rac(GTP) to Rac(GDP), which reassociates with RhoGDI and is returned to the cytosol. “Minus” symbols represent the negative charge of the phospholipids on the cytosolic aspect of the PM, “plus” symbols represent the positive charge of the polybasic C terminus of Rac, and “ip” stands for isoprenyl. PI3K, phosphoinositide 3-kinase. Some elements in this scheme are inspired by a model proposed by Bokoch et al. (see Fig. 7 in Ref. 67).