Further manipulations were performed at 4C

Further manipulations were performed at 4C. promote its release from the plasma membrane. Our data support a different conclusion: membrane association of persists whether the protein WAY-100635 Maleate is activated and/or depalmitoylated. MATERIALS AND METHODS Nucleotides were purchased from Boehringer Mannheim. Guanosine 5-(3-harboring the plasmid pQE60C6HAPT1 (19). Cells were harvested by centrifugation, and the pellet was frozen with liquid nitrogen. Further manipulations were performed at 4C. The cell pellet was thawed in 250 ml of 50 mM Tris?HCl, pH 8.0, supplemented with 32 g/ml WAY-100635 Maleate each of l-1-tosylamido-2-phenylethyl chloromethyl ketone and 1-chloro-3-tosylamido-7-amino-2-heptanone. Lysozyme (50 mg) and DNase (50 g) were added sequentially to the stirring suspension. After 30 min, the suspension was centrifuged at 100,000 for 30 min. APT1 was purified from the supernatant fraction by nickel nitrilotriacetic acid affinity chromatography (Qiagen, Santa Clarita, CA) on a 10-ml column. The resin was washed with 100 ml of 50 mM Tris?HCl, pH 8/10 mM imidazole/100 mM NaCl and eluted by addition of 75 ml of 50 mM Tris?HCl, pH 8/100 mM imidazole. The eluted protein was concentrated to 20 ml in a Centriprep30 (Amicon) and dialyzed twice against 2 liters of 20 mM Na Hepes, pH 8/2 mM MgCl2/1 mM EDTA. The WAY-100635 Maleate yield of essentially homogeneous APT1 was 600 mg. The activity of the recombinant enzyme was not distinguishable from that of APT1 purified from rat liver (19). WAY-100635 Maleate Membranes were prepared from HEK 293 cells that had been transfected to express HA-tagged s and incubated for 1 h in [3H]palmitic acid (1 mCi/ml) to radiolabel palmitoylated proteins. Aliquots of membrane protein (100 g suspended in a solution of 20 mM Na Hepes, pH 8/2 mM MgCl2/1 mM EDTA/1 mg/ml BSA) were incubated with the indicated guanine nucleotide (100 M) and purified APT1 for 30 min at 30C (1 mg/ml membrane protein). To quantify depalmitoylation of subunit substrates, a portion of Rabbit Polyclonal to BCLAF1 each reaction mixture (65%) was solubilized and immunoprecipitated with the antibody to the HA epitope tag of s. The amount of tritium associated with HA-tagged s was quantified by phosphorimager analysis. To separate membrane-bound and soluble subunits, the remaining portion of each WAY-100635 Maleate reaction mixture was centrifuged at 200,000 in and and and (Table ?(Table1).1). On average, there were about twice as many gold particles per group on membranes incubated with GTPS as there were on those incubated with GDPS, suggesting that i concentrated into fewer sites (Table ?(Table1).1). These differences in the number of gold particles per group and per m2 (GDPS vs. GTPS) were significant, as analyzed by Tukeys test ( 0.05). The increases in the amount and concentration of immunogold labeling of GTPS-treated membranes are consistent with the altered pattern of immunofluorescence (Fig. ?(Fig.1).1). Table 1 Quantification of immunogold-labeling of on plasma?membranes and and and (44), Hepler (51), Edgerton (52), and we (17) found little, if any, difference in the subcellular distribution of palmitoylation site mutants of s or q compared with wild-type proteins when they were expressed in HEK 293, COS, or Sf9 cells. Moreover, when we depalmitoylated HA-tagged s with APT1, the protein was not released from membranes, even when GTPS was also present (Fig. ?(Fig.3).3). Although removal of palmitate may decrease the avidity of subunits for the membrane somewhat, the bulk of the available data does not support the notion of substantial release of unpalmitoylated s or q from the membrane. Immunofluorescence studies indicate that nonpalmitoylated cysteine (Cys-3) mutants of i subfamily members (ref. 53 and unpublished data) and the Src family tyrosine kinase p59fyn (54) are found predominantly on intracellular membranes. It is not known whether the lack of palmitate or alteration of the cysteine residue prevents these proteins from attaining or maintaining their proper association with the plasma membrane. The capacity of q to stimulate.