February 18, 2025

2002;277:41268C41273

2002;277:41268C41273. incorporated with different efficiencies into Golgi complex to plasma membrane vesicular carriers, and 2) the different deacylation rates of single-acylated H-Ras influence differentially its overall exchange between different compartments by nonvesicular transport. Taken together, our results show that individual S-acylation sites provide singular information about H-Ras subcellular distribution that is required for GTPase signaling. INTRODUCTION Ras family proteins are monomeric guanosine triphosphatases (GTPases) that couple extracellular signals to the intracellular effector pathways that control cell proliferation, differentiation, and survival (Wennerberg 0.05; *** 0.001. We then explored the possibility that the differences observed in subcellular distribution could be a consequence of alterations in membrane association and/or a posttranslational modification of H-Ras. To test this, we performed biochemical experiments to analyze the membrane binding and extent of lipidation of H-Ras when expressed in CHO-K1 cells. Our results demonstrated that H-Ras(WT), H-Ras(C181S), H-Ras(C184S), ddATP and H-Ras (C181S,C184S) were preferentially bound to cellular membranes, as these partitioned mostly to the pellet fraction after ultracentrifugation at 400,000 (Figure 1C). In addition, Triton X-114 partition assays of membrane and cytosolic fractions revealed that all of the H-Ras proteins were enriched in the detergent phase, which indicates the highly hydrophobic character of the protein ddATP conferred by farnesylation and/or S-acylation (Figure 1D; Gomez and Daniotti, 2005 ). Because live-cell imaging experiments suggested that H-Ras(C181S) and H-Ras(C184S) behave differently from the nonacylatable H-Ras(C181,184S), these mutants might be posttranslationally S-acylated. To try to confirm this, we used two independent methods. First, Rabbit Polyclonal to POU4F3 we directly evaluated the S-acylation of these mutants (and H-RasWT as control) by acyl biotinyl exchange (ABE) assays (Wan 0.05; ** 0.01; *** 0.001; **** 0.0001. One possible explanation for the different deacylation kinetics of H-Ras at the plasma membrane compared with endomembrane could be differential interaction between APTs and their substrates at these two subcellular locations in CHO-K1 cells. Although both APT1 and APT2 are able to deacylate Ras ddATP (Dekker and resuspended in 400 l of 5 mM Tris-HCl (pH 7.0) in the presence of protease inhibitors. Pellets were dispersed by repetitive pipetting and vortexing. After 30 min of incubation, pellets were passed 60 times through a 25-gauge needle. Nuclear fractions and unbroken cells were removed by centrifuging twice at 4C for 5 min at 600 using a TLA 100.3 rotor (Beckman Coulter). The supernatant (S1 fraction) was removed, and the pellet (P1 fraction) was resuspended in 400 l of 5 mM Tris-HCl (pH 7.0). Both fractions were further ultracentrifuged at 400,000 (2007) with some modifications. Briefly, transfected CHO-K1 cells grown in 100-mm dishes were washed with cold PBS, harvested, lysed, and centrifuged as described ddATP in the preceding subsection. Supernatant was removed, and Triton X-100 was added to a final concentration of 1 1.7% and incubated with end-over-end rotation at 4C for 1 h. The proteins were precipitated with chloroform/methanol (1:4 vol/vol) and resuspended in SB (4% SDS, 50 mM Tris HCl, pH 7.4, 5 mM EDTA) with 10 mM (2006) . Confocal microscopy and image acquisition Confocal images were collected using an Olympus FluoView FV1000 confocal microscope (Olympus Latin America, Miami, FL) equipped with a multiline argon laser (458, 488, and 514 nm) and two heliumCneon lasers (543 and 633 nm, respectively). CFP was detected by using laser excitation at 458 nm, a 458/514-nm excitation dichroic mirror, and a 470- to 500-nm band-pass emission filter. YFP was acquired by using laser excitation at 514 nm, a 458/514-nm excitation dichroic mirror, and a 530/570-nm band-pass emission filter. Cherry protein was acquired with a laser excitation at 543 nm, a 458/543/633-nm excitation dichroic mirror, and a 560-nm long-pass emission filter. Alexa Fluor 647 was acquired with a laser excitation at 633 nm, a 488/543/633-nm excitation dichroic mirror, and a 650-nm long-pass emission filter. For CFP/YFP/Cherry acquisition, images were sequentially acquired in line ddATP mode. This minimizes the bleedthrough between channels mainly due to overlapping emission spectra of these fluorochromes. Live-cell experiments were performed at 37C.