Tài liệu Báo cáo khoa học: Electrical properties of plasma membrane modulate subcellular distribution of K-Ras ppt

Electrical properties of plasma membrane modulate subcellular distribution of K-Ras Guillermo A Gomez and Jose L Daniotti ´ ´ ´ ´ ´ Centro de Investigaciones en Quımica Biologica de Cordoba (CIQUIBIC, UNC-CONICET), Departamento de Quımica Biologica, Universidad ´ Nacional de Cordoba, Argentina Keywords calcium; membrane potential; polyphosphoinositides; RAS; sialic acid Correspondence J L Daniotti, Centro de Investigaciones en ´ ´ ´ Quımica Biologica de Cordoba (CIQUIBIC, ´ UNC-CONICET), Departamento de Quımica ´ ´ Biologica, Facultad de Ciencias Quımicas, ´ Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad ´ Universitaria, X5000HUA, Cordoba, Argentina Fax: +54 351 4334074 Tel: +54 351 4334168 ⁄ 4171 E-mail: daniotti@dqb.fcq.unc.edu.ar (Received 28 November 2006, revised 16 February 2007, accepted 27 February 2007) doi:10.1111/j.1742-4658.2007.05758.x K-Ras is a small G-protein, localized mainly at the inner leaflet of the plasma membrane The membrane targeting signal of this protein consists of a polybasic C-terminal sequence of six contiguous lysines and a farnesylated cysteine Results from biophysical studies in model systems suggest that hydrophobic and electrostatic interactions are responsible for the membrane binding properties of K-Ras To test this hypothesis in a cellular system, we first evaluated in vitro the effect of electrolytes on K-Ras membrane binding properties Results demonstrated the electrical and reversible nature of K-Ras binding to anionic lipids in membranes We next investigated membrane binding and subcellular distribution of K-Ras after disruption of the electrical properties of the outer and inner leaflets of plasma membrane and ionic gradients through it Removal of sialic acid from the outer plasma membrane caused a redistribution of K-Ras to recycling endosomes Inhibition of polyphosphoinositide synthesis at the plasma membrane, by depletion of cellular ATP, resulted in a similar subcellular redistribution of K-Ras Treatment of cells with ionophores that modify transmembrane potential caused a redistribution of K-Ras to cytoplasm and endomembranes Ca2+ ionophores, compared to K+ ionophores, caused a much broader redistribution of K-Ras to endomembranes Taken together, these results reveal the dynamic nature of interactions between K-Ras and cellular membranes, and indicate that subcellular distribution of K-Ras is driven by electrostatic interaction of the polybasic region of the protein with negatively charged membranes Ras proteins are small GTPases localized mainly on the cytoplasmic leaflet of cellular membranes, where they operate as binary molecular switches between a GDP-bound inactive and GTP-bound active state, regulated by the concerted action of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins [1,2] There are three ubiquitous isoforms of Ras: K-Ras4B (referred to hereafter as K-Ras), H-Ras, and N-Ras These isoforms, encoded by different genes, are more than 90% homologous, and their functions are not redundant [3] Ras proteins share a conserved G-domain which contains a GTP-binding cassette and an effector sequence involved in interactions between Ras proteins and their prominent Abbreviations BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N ¢,N ¢-tetraacetic acid-acetoxymethyl ester; CFP, cyan fluorescent protein; Chel, chelators; CHO, chinese hamster ovary; Cyt, cytosol; ECS, extracellular solution; FP, fluorescent protein; GalNAc-T, UDP-GalNAc:LacCer ⁄ G3 ⁄ GD3 N-acetylgalactosaminyltransferase; Gal-T2, UDP-Gal:GA2 ⁄ G2 ⁄ GD2 ⁄ GT2 galactosyltransferase; GEF, guanine nucleotide exchange factor; GPI, glycosylphosphatidylinositol; GFP, green fluorescent protein; HA, hemagglutinin; hvr, hypervariable domain; Man II, mannosidase II; NANase, neuraminidase; PIM, protease inhibitor mixture; PIP2, phosphatidylinositol (4,5)-bisphosphate; PKC, protein kinase C; poly PI, phosphatidylinositol; PM, plasma membrane; PS, phosphatidylserine; Tf, transferrin; TGN, trans Golgi network; Try, trypsin; YFP, yellow fluorescent protein 2210 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS G A Gomez and J L Daniotti effectors, which include Raf, PI3-K, and RalGEF [4] Ras proteins also have, in their C-terminal sequence (19–20 amino acid residues), a nonconserved hypervariable domain (hvr) that operates as a membrane targeting signal [3,5] The membrane association of Ras proteins, which is necessary for proper function, depends on different post-translational modifications at the hvr [3,6–8] A CAAX motif (where C represents cysteine, A is aliphatic, and X is any other amino acid) at the C-terminal end of each Ras isoform is first modified in the cytosol by a farnesyl anchor to the cysteine residue The AAX sequence is then cleaved by an endopeptidase at the cytoplasmic leaflet of the endoplasmic reticulum (ER), and finally the newly formed free carboxyl group of the C-terminal farnesylcysteine is carboxylmethylated [3] An additional signal for membrane association is present in Ras isoforms H-Ras contains two (cysteines 181 and 184), while N-Ras contains one (cysteine 184), palmitoylation sites [7] K-Ras does not contain palmitoylation sites; instead, it contains a polybasic stretch of six contiguous lysines which is critical for targeting K-Ras to plasma membrane [8] Together, the CAAX motif and the second signal constitute the minimal plasma membrane targeting signal of these proteins [9,10] Recent studies have demonstrated that protein kinase C (PKC)-dependent phosphorylation on S181 at the hvr of K-Ras promotes translocation of this protein to mitochondria, where it induces cell death [11] Ras isoforms, by regulating different effectors as above, affect different signaling pathways Recent experimental evidence indicates that Ras signaling is restricted to particular plasma membrane microdomains (e.g., caveolae and cholesterol-dependent or -independent membrane domains) and to particular intracellular compartments (including Golgi complex, ER, mitochondria, and membranes from early and recycling endosomes) [11–18] Although recent studies have shown that subcellular distribution and ⁄ or membrane association dynamics of Ras isoforms are important for their proper function, underlying mechanisms of intracellular transport and distribution of these proteins is not completely understood Palmitoylation of H-Ras and N-Ras causes membrane trapping early in the classical secretory pathway, and subsequent transport to plasma membrane through association with exocytic vesicles [9,10] Unlike farnesylation, which is a stable lipid modification of proteins, depalmitoylation of H-Ras was shown to be a dynamic process [19–21] causing reduction of Ras membrane affinity Recent experiments showed that depalmitoylation of H- and N-Ras is responsible for Membrane targeting of K-Ras retrograde transport of these isoforms through desorption from the plasma membrane, followed by adsorption of the prenylated proteins to the endomembrane system Repalmitoylation in the secretory pathway causes kinetic trapping of these proteins in membrane carriers, and transport to the plasma membrane [22,23] An adsorption ⁄ desorption mechanism has also been proposed [24–27], and recently described for intracellular transport of K-Ras between subcellular compartments [28] In contrast to H- and N-Ras, K-Ras is not palmitoylated, but contains a polycationic domain required for anchoring to plasma membrane, which also operates as an electronegative surface potential probe [29,30] A reduction in the number of positively charged residues at the hvr of K-Ras was shown to be sufficient to redistribute this protein to endomembranes [27,29,31] On the other hand, complete replacement of lysine residues by arginine or d-lysine residues in the polybasic domain of K-Ras does not interfere with plasma membrane localization of this protein [30], suggesting that binding of K-Ras to plasma membrane does not depend on additional factors This idea is consistent with results of earlier biophysical and biochemical studies [8,25–27], and with recent observations in vivo [28,29,32], that prenylated polycationic peptides bind dynamically and reversibly with model and cellular membranes through electrostatic and hydrophobic interactions In the present study, we combined biochemical techniques and fluorescence confocal microscopy analysis to clarify the role of electrical properties of the plasma membrane in the subcellular distribution of K-Ras In particular, we investigated (a) the role of surface charge on inner and outer leaflet of plasma membrane and (b) effect of ionic gradients through plasma membrane on membrane binding and subcellular distribution of K-Ras in Chinese hamster ovary (CHO)-K1 cells At steady state, K-Ras is associated with plasma membrane, cytosol, and endosomal compartments, but not with ER or Golgi membranes Results from our in vitro experiments demonstrate the electrical and reversible nature of K-Ras binding to cellular membranes, consistent with a proposed model of K-Ras membrane association based on electrostatic interaction [33] Confocal microscopy analysis, in combination with live cell imaging, demonstrated that enzymatic removal of sialic acid from the outer leaflet caused a significant accumulation of K-Ras, but not H-Ras, in recycling endosome membranes Inhibition of synthesis of polyphosphoinositides (poly PIs) in live cells, by depletion of cellular ATP, resulted in significant accumulation of K-Ras in a perinuclear region, FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2211 Membrane targeting of K-Ras G A Gomez and J L Daniotti colocalizing with recycling endosome and Golgi complex markers Finally, the dependence of ionic strength on plasma membrane targeting of K-Ras was evaluated using a battery of ionophores Ionophores that modify transmembrane potential caused a rapid redistribution of K-Ras from plasma membrane to endomembranes Specifically, calcium ionophore induces a redistribution of K-Ras from plasma membrane to Golgi complex, recycling endosomes, cytoplasm and mitochondria, but not to ER while potassium ionophore redistributed K-Ras to recycling endosome Conversely, monensin, which alters pH gradients but not transmembrane potential, did not affect plasma membrane targeting of K-Ras Taken together, our results indicate that intracellular distribution of K-Ras in CHO-K1 cells is modulated by electrical properties of plasma membrane and endomembranes, which are relevant to K-Ras signaling Results Membrane association and subcellular distribution of full-length and C-terminal domain (14 amino acids) of K-Ras fused to spectral variants of green fluorescent protein Constructs expressing full-length and C-terminal (KKKKKKSKTKCVIM) domain of human K-Ras (K-Rasfull and K-RasC14) fused to green fluorescent protein (GFP) and to its spectral variants, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), were described and partially characterized in our previous study [16] In order to evaluate expression and subcellular distribution of these proteins, CHO-K1 cells were transiently transfected with corresponding DNA constructs, and expression was monitored by western blot analysis with an antibody directed to the fluorescent protein The antibody detected YFP and YFP-K-RasC14 as bands of 27 kDa and 27.5 kDa, respectively, and YFP-K-Rasfull as a band of  55 kDa according to the expected molecular mass (Fig 1A) Membrane association of the expressed fusion proteins was investigated by ultracentrifugation of extracts from mechanically lysed cells YFP-K-RasC14 and YFP-K-Rasfull were associated mainly with the particulate fraction (65% and 63%, respectively) (Fig 1B) To analyze the degree of post-translational modification, and to rule out possible association of these proteins with insoluble components such as cytoskeleton, nuclear remnants, or extracellular matrix, we performed Triton X-114 partitioning assay on particulate fractions of cells transiently expressing the fusion proteins [34,35] (Fig 1C) Fifty percent and 44% of 2212 YFP-K-RasC14 and YFP-K-Rasfull, respectively, were enriched in the detergent phase, indicating that a fraction of the expressed proteins are hydrophobic, and therefore post-translationally modified by lipidation To characterize expression of these proteins in CHO-K1 cells, subcellular distribution of YFP-KRasC14 and YFP-K-Rasfull was analyzed by confocal microscopy Detailed phenotypic analysis showed that 43% and 49% of CHO-K1 cells expressed K-RasC14 and K-Rasfull, respectively, mostly in plasma membrane (PM > Cyt); 10% and 14% of cells expressed them in both plasma membrane and a perinuclear compartment (Perinuclear) and 40% and 37% of cells expressed them mostly in cytosol (Cyt > PM) (Fig 1D) The phenotype Cyt > PM does not exclude the presence of K-Ras in plasma membrane, but the cytosolic concentration of K-Ras in this phenotype is higher than the others CFP-K-RasC14 and YFP-KRasfull were extensively colocalized in cells that expressed K-Ras mostly in plasma membrane (Fig 1E), as well as in the other phenotypes (data not shown) These findings indicate that the C-terminal domain of K-Ras operates as a membrane targeting motif when fused to a soluble protein, and that the polybasic region and post-translational modifications on this domain could be relevant for proper function of K-Ras At steady state, K-Ras is associated with plasma membrane, cytosol, and endosomal compartments In order to characterize subcellular distribution of K-Ras in CHO-K1 cells at steady state, we performed extensive colocalization analyses with markers of organelles (Fig and Fig S1) No colocalization was observed between YFP-K-RasC14 and major histocompatability complex class II invariant chain isoform lip33 fused to cyan fluorescent protein (lip33-CFP) and calnexin, two ER markers, suggesting that the diffuse pattern in the cytosol probably represents a soluble fraction of the expressed protein There was also no colocalization between K-RasC14 and mannosidase II (Man II), a medial Golgi marker or mitochondria (MitoTracker) In addition to plasma membrane, K-Ras was found distributed in peripheral structures, some of which were positive for mannose 6-phosphate receptor (Fig S1) This was probably due to a pool of K-Ras associated with late or recycling endosomes, because no colocalization was observed between this protein and N27GalNAc-T-CFP (N27GalNAc-T), a trans Golgi network (TGN) resident protein in CHO-K1 cells YFP-K-RasC14 was colocalized with endocytosed Alexa647-human transferrin (Tf), a marker FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS G A Gomez and J L Daniotti A Membrane targeting of K-Ras B C D E Fig Protein expression and subcellular localization of YFP-K-RasC14 and YFP-K-Rasfull in CHO-K1 cells (A) Homogenates from CHO-K1 cells expressing YFP, YFP-K-RasC14 or YFP-K-Rasfull were run in SDS ⁄ PAGE and immunoblotted with anti-GFP Sizes of the markers in kDa are indicated on the left (B) CHO-K1 cells expressing YFP-K-RasC14 or YFP-K-Rasfull were mechanically lysed, and the homogenates were centrifuged at 400 000 g The supernatant fraction (S) was removed, and the particulate fraction (P) was resuspended in lysis buffer Recombinant proteins both in S and P fractions were determined by western blot analysis as indicated in (A) The percentage of K-Ras membrane association is indicated in the figure (C) Triton X-114 partitioning assays P fractions from CHO-K1 cells expressing YFP-K-RasC14 or YFP-K-Rasfull were incubated with 1% (v ⁄ v) Triton X-114 for h Then, samples were incubated at 37 °C for to induce phase separation The aqueous phase (A) and detergent-enriched phase (D) were separated, and proteins were precipitated with chloroform ⁄ methanol previous to western blot analyses using anti-GFP The percentage of K-Ras recovered from the detergent phase is indicated (D) CHO-K1 cells expressing YFP-KRasC14 or YFP-K-Rasfull were fixed with paraformaldehyde and visualized by confocal microscopy Left, representative cell phenotypes showing YFP-KRasC14 subcellular distribution Right, frequency of phenotypes (%) showing YFP-K-RasC14 and YFP-K-Rasfull subcellular distribution Values are mean ± SEM for three or more experiments (300 cells analyzed for each condition) (E) CHO-K1 cells expressing both YFP-K-Rasfull (pseudocolored red) and CFP-K-RasC14 (pseudocolored green) Right panel is a merged image from YFP-K-Rasfull and CFP-K-RasC14 Scale bars ¼ 20 lm of recycling endosomes, in  10% of transfected CHO-K1 cells [36] (Fig 2; K-RasC14-perinuclear) Similar subcellular distributions were observed for the full-length version of K-Ras (data not shown) In summary, YFP-K-RasC14 and its full-length counterpart at steady state are associated mostly with plasma membrane and cytosol, and to a minor degree with membranes from recycling endosomes Membrane binding properties of K-Ras Results from model system experiments and theoretical analyses suggest that membrane association and plasma membrane targeting of K-Ras are a consequence of the electronegative sensing function of the C-terminal domain of this protein, and that membrane association depends on both electrostatic and hydro- phobic interactions between this domain and the plasma membrane [25,26,37] The models predict that electrostatic interactions and plasma membrane association are reduced when ionic strength of the medium increases or when negative surface charge density of membranes or net charge of the C-terminal domain decreases Mutagenesis experiments to reduce net charge of the polybasic region of K-Ras gave results consistent with the models [8,9,27,31,38] To better characterize the membrane binding properties of K-Ras to biological membranes we performed extensive biochemical experiments to evaluate effects of various electrolytes (including poly l-lysine, NaCl, and CaCl2) on membrane association of K-Ras We also investigated effects of these factors on membrane binding properties of CFP-H-RasC20 [16], which is dually palmitoylated and does not FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2213 Membrane targeting of K-Ras G A Gomez and J L Daniotti Fig At steady state, most of K-Ras is associated with plasma membrane, cytosol and to a minor extent to endosomal compartments CHOK1 cells transiently expressing YFP-K-RasC14 were fixed and immunostained with antibodies for Man II, a medial Golgi marker; or fixed and examined for the intrinsic fluorescence of CFP from lip33-CFP, an ER marker; N27GalNAc-T-CFP (N27GalNAc-T), a TGN marker or incubated with MitoTracker or Alexa647-Tf (Tf) and then fixed The expression of YFP-K-RasC14 was analyzed by the intrinsic fluorescence of YFP (pseudocolored green) All images corresponding to organelle markers are pseudocolored red Panels are merged images from YFP-K-RasC14 and the corresponding organelle marker Cells shown in this figure correspond to the PM > Cyt phenotype of subcellular distribution of YFP-RasC14, except for cells shown in the lower row, right panel (perinuclear phenotype) The insets in each image show details of the boxed area at higher magnification Scale bars ¼ lm contain a polybasic domain, and of GPI-YFP, a fluorescent protein containing a glycosylphosphatidylinositol (GPI) attachment signal When membrane fractions from cells expressing YFP-K-RasC14 or YFP-K-Rasfull were incubated in solutions with increasing concentration of poly l-lysine, significant dissociation of the expressed proteins was observed at higher concentrations (Fig 3Ai) In contrast, no significant change in the amount of CFP-H-RasC20 associated with particulate fraction was observed under the same conditions To test whether the effect of poly l-lysine on membrane binding of K-Ras depends on its electrical properties, we performed similar experiments in the presence of increasing concentrations of NaCl (Fig 3Aii) A significant membrane dissociation of both YFP-K-RasC14 and YFP-K-Rasfull ( 45%) was observed at 1.5 m NaCl, in accordance with the elec2214 trostatic model However, membrane dissociation of K-Ras at 1.5 m NaCl could be considered complete, because in these membrane extracts only 44% of K-Ras was accessible to protease digestion (Fig S2) Membrane dissociation of CFP-H-RasC20 was observed at low ionic strength, but was insignificant at high ionic strength Ca2+ is a central second messenger having a higher affinity for anionic than zwitterionic and neutral phospholipids [39] Ca2+ also promotes the formation of lateral domains of phosphatidylserine (PS) in bilayers of mixed phosphatidylcholine and PS because of the different affinities of these lipids [40–42] It was recently reported that the polybasic-prenyl motif of K-Ras acts as a Ca2+ ⁄ calmodulin-regulated molecular switch that controls plasma membrane concentration of K-Ras, and redistributes its activity to internal sites [43] In view of these previous findings, we studied the FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS G A Gomez and J L Daniotti Membrane targeting of K-Ras A i ii iii B C Fig Membrane binding properties of K-Ras (A) Membrane fractions of CHO-K1 cells expressing YFP-K-RasC14 or YFP-K-Rasfull or CFP-HRasC20 were obtained as described in Fig 1B and then incubated for h in solutions containing 0, 0.012 and 0.12 mgỈmL)1 poly L-lysine (i) or 0, · 10)6, 1.5 · 10)4, · 10)2, 15 · 10)2 and 1.5 M NaCl (ii) or 0.1 · 10)6, · 10)5, · 10)3, 0.05 and 0.5 M CaCl2 (iii) A soluble (S) and a particulate (P) fraction were obtained after centrifugation at 400 000 g Left, western blot analysis of protein expression in S and P fractions Right, densitometric analyses of results from western blot Data are mean ± SEM from three independent experiments Asterisks (*) and double asterisks (**) represent P < 0.1 and P < 0.05, resectively, versus control (without electrolyte) (B) Membrane and cytosolic fractions from nontransfected cells or cells expressing YFP, YFP-K-RasC14 or YFP-K-Rasfull were obtained as described in Fig Membrane fractions of transfected cells were incubated for h with cytosol from nontransfected cells and a soluble (S) and a particulate (P) fraction was obtained after ultracentrifugation and processed for western blot analysis with anti-GFP (membrane bound FP + cytosol) Conversely, membrane fractions from nontransfected cells were incubated with the cytosolic fraction of transfected cells for h and S and P fraction obtained by ultracentrifugation for western blot analysis with anti-GFP (cytosolic FP + membranes) (C) Membranes were obtained from nontransfected CHO-K1 cells, treated with 200 lgỈmL)1 proteinase K or BSA for 30 and further washed five times Proteinase PK- or BSAtreated membranes were then incubated for h with cytosol from YFP-K-RasC14 expressing CHO-K1 cells and centrifuged at 400 000 g The supernatant was removed (S) and the pellet (P) was resuspended in buffer and centrifuged twice Soluble fractions after washing were recovered (W1 and W2) YFP-K-RasC14 expression in W1, W2 and P fractions was analyzed by western blot Right lane shows 30% of the cytosolic YFP-K-RasC14 input Proteinase K activity was monitored by measuring the degradation of a-tubulin present in total CHO-K1 extracts (lower panel) effect of increasing CaCl2 concentration on membrane affinity of K-Ras The results (Fig 3Aiii) show that both YFP-K-RasC14 and YFP-K-Rasfull are dissociated from membrane at high Ca2+ concentration (0.5 m) However, the degree of this dissociation is not significantly different from that observed for NaCl, suggest- ing a nonspecific effect of Ca2+ on K-Ras membrane affinity CFP-H-RasC20 and GPI-YFP were not significantly dissociated under the same conditions Having demonstrated that K-Ras membrane association depends on electrostatic interaction, we analyzed in vitro the reversibility of such interaction Cytosolic FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2215 Membrane targeting of K-Ras G A Gomez and J L Daniotti and particulate fractions were prepared from cells expressing YFP, YFP-K-RasC14, and YFP-K-Rasfull, and from nontransfected cells Soluble fractions from transfected cells were incubated with membranes from nontransfected cells; conversely, membrane fractions from transfected cells were incubated with cytosol from nontransfected cells Samples were incubated for h at °C and then ultracentrifuged to separate soluble and particulate fractions Presence of fluorescent proteins in the fractions was evaluated by western blot analysis Results (Fig 3B) showed that cytosol from nontransfected cells caused 30% dissociation of membrane associated K-Ras Cytosolic YFP (a soluble protein) was recovered mostly in the soluble fraction, indicating that it was not associated with membranes from nontransfected cells In contrast,  50% of soluble K-RasC14 and K-Rasfull was associated with membranes from nontransfected cells The K-Ras fraction reassociated with membranes from nontransfected cells was completely dissociated when incubated in the presence of 1.5 m NaCl (Fig S2B) These results support the concept that K-Ras binds to cellular membranes through an electrostatic, reversible mechanism Results to this point indicated some involvement of lipid moieties and ⁄ or membrane-associated proteins in K-Ras binding to membranes Next, we analyzed the association of cytosolic K-RasC14 with membranes from nontransfected cells pretreated with BSA (control) or proteinase K (Fig 3C) The association of K-Ras was similar under both conditions, suggesting that membrane binding of K-Ras could be driven by electrostatic interaction of the polybasic region of the protein with negatively charged lipids Electrical properties of the outer leaflet of plasma membrane ) contribution to membrane targeting of K-Ras Biochemical studies as above demonstrate that membrane binding properties of K-Ras are due to electrostatic and reversible interactions To further characterize the mechanisms underlying plasma membrane targeting of this protein, we attempted to disrupt membrane surface potential of the outer leaflet of plasma membrane, and to analyze subcellular distribution of K-Ras following such disruption Sialic acid is a charged monosaccharide that contributes significantly to surface potential of the outer leaflet, and may also be involved in molecular rearrangement at the inner leaflet, and in cytosolic events [44,45] To evaluate the role of sialic acid in subcellular distribution of K-Ras, CHO-K1 cells were treated with neuraminidase (NANase) Neuraminidase activity was 2216 assayed by conversion of GD1a (disialoganglioside) to GM1 (monosialoganglioside) in a CHO-K1 clone stably expressing UDP-GalNAc:LacCer ⁄ G3 ⁄ GD3 N-acetylgalactosaminyltransferase (GalNAc-T) and UDP-Gal:GA2 ⁄ G2 ⁄ GD2 ⁄ GT2 galactosyltransferase (Gal-T2) glycosyltransferases [46] (Fig 4A) Live cell imaging analysis showed that neuraminidase treatment increased K-RasC14, but not H-RasC20, expression in a perinuclear compartment (Fig 4B), and that K-Ras colocalized with recycling endosome markers but not with cis ⁄ medial Golgi and TGN markers (Fig 4C) These changes were not due to modifications in shape of neuraminidase-treated cells (results not shown) Quantification of neuraminidase effect on subcellular distribution of K-Ras (Fig 4B) suggested that the increase in number of cells showing K-Ras at the perinuclear compartment is a consequence of a reduction in number of cells showing cytosolic K-Ras expression Taken together, these results suggest a dynamic interplay between the cytosolic, recycling endosome and plasma membrane fractions of K-Ras Independent of the mechanism ⁄ s involved in this subcellular distribution of K-Ras, our results reveal that outer leaflet membrane properties differentially regulate subcellular distribution of Ras isoforms Effect of ATP depletion on subcellular distribution of K-Ras To characterize the mechanisms underlying plasma membrane targeting of K-Ras, we reduced surface charge of the inner leaflet, by inhibiting poly PI synthesis through depletion of cellular ATP [29] (Fig S3), and analyzed resulting subcellular distribution of K-Ras ATP depletion also impairs aminophospholipid translocase activity, inhibiting the inward movement of PS from the outer to inner leaflet [47,48] This treatment was reported to inhibit PS internalization in live CHO cells [49] However, in ATP depleted cells there was not externalization of PS (Fig S3) Simultaneous impairment of glycolysis and mitochondrial respiration by 2-d-deoxyglucose and sodium azide caused a significant increase in cell phenotype showing accumulation of YFP-K-RasC14, but not H-RasC20, in a perinuclear region (Fig 5A) To identify the perinuclear organelle in which YFP-K-RasC14 localized in ATP-depleted CHO-K1 cells, we performed colocalization experiments with a TGN marker (N27GalNAc-T) and endocytosed human Alexa647-Tf, a recycling endosome marker [50] We observed colocalization of YFP-KRasC14 with endocytosed Tf, and with TGN marker, in ATP-depleted cells (Fig 5B) No colocalization was observed between K-Ras and N52Gal-T2-CFP FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS G A Gomez and J L Daniotti Membrane targeting of K-Ras A Fig Enzymatic release of sialic acid redistributes K-Ras to recycling endosomes (A) CHO-K1 cells or a parental clone stably expressing GalNAc-T and Gal-T2-HA were treated or not with 1.5 mL)1 NANase for h at 37 °C Then, cells were shifted to °C and incubated with cholera toxin for 30 Homogenates were analyzed by western blot using antibodies to reveal the A subunit of cholera toxin (CTx-A) and Gal-T2-HA (left) Densitometric analysis of western blots showed in the left panel normalized to control values (right) (B) Confocal microscopy of live cells expressing YFP-K-RasC14 and YFP-H-RasC20 treated or not with 1.5 mL)1 NANase Cells are representative of PM > Cyt (control) and perinuclear (NANase) phenotypes for K-Ras subcellular distribution (left) Frequency of phenotypes (%) showing YFP-K-RasC14 and YFP-H-RasC20 subcellular distribution (right) (C) CHO-K1 cells coexpressing YFP-K-RasC14 (K-RasC14, green) and N52Gal-T2-CFP (N52Gal-T2, red) or N27GalNAc-T-CFP (N27GalNAc-T, red) or cells expressing YFP-K-RasC14 (K-RasC14, green) and labeled with Alexa647-Tf (Tf; red) were treated (NANase) or not (control) with 1.5 mL)1 NANase for h, fixed and visualized by confocal microscopy Panels are merged images from YFP-K-RasC14 and the corresponding organelle marker The insets show details of the boxed area at higher magnification Scale bars ¼ 10 lm for (B) and lm for (C) B C (N52Gal-T2), a medial Golgi marker These results suggest that surface charges from poly PIs at the inner leaflet are necessary for proper membrane binding and subcellular distribution of K-Ras Calcium ionophore redistributes K-Ras to endomembrane In vitro experiments in this study and others have demonstrated that binding of lipid modified cationic peptides, YFP-K-RasC14 and YFP-K-Rasfull, depends on ionic strength of the medium To investigate the relationship between ionic composition of cytosol and plasma membrane targeting of K-Ras, we evaluated the effect of various ionophores in live cells We first analyzed the effect of ionophore A23187, which is selective for Ca2+ and to a minor degree for Mg2+ [51] A23187 forms a stable complex with Ca2+ which is membrane permeable (see subcellular distribution in Fig S4) Within the cell, Ca2+ ions are replaced by H+, and the protonated form of the ionophore is externalized [52,53] A23187 thus functions as a Ca2+ ⁄ H+ exchanger, and reduces both Ca2+ and H+ diffusion potentials Calcium affects membrane surface potential shielding negative charges of plasma membrane, stimulating PI hydrolysis and PS ‘flipping out’ in a Ca2+-scramblase dependent fashion (Fig S4) [25,40,42,54,55] Following treatment of YFP-K-RasC14-expressing CHO-K1 cells with A23187, live cell confocal microscopy showed a clear dissociation of this protein from plasma membrane (Fig 6A and Video S1) Perinuclear and scattered structures were also decorated with K-Ras Similar redistribution was observed for full-length K-Ras fused to YFP FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2217 Membrane targeting of K-Ras G A Gomez and J L Daniotti A Fig ATP depletion redistributes K-Ras to recycling endosomes and Golgi membranes (A) CHO-K1 cells expressing YFP-K-RasC14 or YFP-H-RasC20 were incubated for h in DMEM without glucose containing 50 mM 2-deoxiglucose and mM NaN3 (–ATP) or 50 mM D-(+)-glucose and vehicle (control) and visualized alive at 20 °C by confocal microscopy (left) Frequency of phenotypes (%) showing YFP-K-RasC14 and YFP-H-RasC20 subcellular distribution (right) Scale bars: 10 lm (B) CHO-K1 cells coexpressing YFP-K-RasC14 (K-RasC14; green) and N52 Gal-T2-CFP (N52Gal-T2; red) or N27GalNAcT-CFP (N27GalNAc-T; red) or cells expressing YFP-K-RasC14 (green) and labeled with Alexa647-Tf (Tf; red) were treated as described above, fixed and visualized by confocal microscopy Panels are merged images from YFP-K-RasC14 and the corresponding organelle marker The insets show details of the boxed area at higher magnification B (data not shown) In contrast, YFP-H-RasC20 and GPI-CFP showed no redistribution under the same conditions (Fig 6A and Video S1) A23187 function was evaluated using Lysotracker, a fluorescent acidotropic probe for labeling acidic organelles As expected, Lysotracker did not reveal any acidic intracellular compartments in A23187-treated cells (Fig S4) Ionophore A23187 is membrane permeable and could potentially alter intracellular calcium reservoirs We evaluated its effect on subcellular distribution of YFP-K-RasC14 in cells pretreated with EGTA (an impermeable calcium chelator) and with 1,2-bis(o-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; a permeable calcium chelator) Reduced calcium level caused an increase in cell phenotype showing clear plasma membrane expression of K-Ras, and a decrease in number of cells showing cytosolic distribution of K-Ras (Fig 6B) Restoring of Ca2+ and addition of A23187 to medium caused an increase of cells with cytosolic distribution of YFP-K-RasC14 (Fig 6B) Addition of calcium chelators together with Ca2+ and A23187 produced the same phenotypic distribution as observed in the 2218 absence of chelators, indicating that very low levels of extracellular calcium are sufficient to alter subcellular distribution of YFP-K-RasC14 Increase in cytosolic Ca2+ can cause PKC activation and consequent K-Ras phosphorylation [11] and ⁄ or Ca+2 ⁄ calmodulin binding to K-Ras [43] We evaluated membrane affinity of K-RasC14 under the conditions described in Fig 6B Membrane affinity of K-Ras was not changed by any of the experimental conditions (Fig 6C) These results suggest that redistribution of K-Ras from plasma membrane to endomembranes is not a consequence of further posttranslational modifications or association with cytosolic protein; rather, K-Ras responds to local changes in membrane properties which are lost during subcellular fractionation To further characterize the subcellular distribution of YFP-K-RasC14 under the different conditions shown in Fig 6B, we performed extensive colocalization experiments using organelle markers (Fig 6D) Changes in calcium level caused alterations in morphology of Golgi complex and ER This phenomenon was evident for both ectopically expressed markers and FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS G A Gomez and J L Daniotti A Membrane targeting of K-Ras B C +Ca2+ +A23187 -Ca2+ D Fig Ca2+ influx causes K-Ras to redistribute from plasma membrane to the endomembrane system (A) CHO-K1 cells expressing YFP-K-RasC14 or YFP-H-RasC20 were incubated in DMEM at 20 °C on the microscope stage and imaged (pretreatment) Then, cells were incubated with 30 lM A23187 and a time series was acquired Images obtained at after A23187 addition is shown (B) CHO-K1 cells expressing YFP-K-RasC14 were Ca2+ depleted and incubated for h in media without Ca2+ (–Ca2+) or containing mM Ca2+ (+Ca2+) or mM Ca2+ and 30 lM A23187 (+Ca2+ + A23187) or mM Ca2+, 30 lM A23187, 10 lM BAPTA-AM and 10 mM EGTA (+Ca2+ + A23187 + Chel) Non depleted cells correspond to cells maintained in normal media (DMEM) Graphic shows the frequency of Cyt > PM and PM > Cyt phenotypes for YFP-K-RasC14 expression (%) (C) Homogenates from cells expressing K-RasC14 were treated as described in (B), lysed and ultracentrifugated The supernatant (S) was recovered and the particulate fraction (P) resuspended in lysis buffer YFP-K-RasC14 expression was investigated by western blot The percentage of YFP-K-RasC14 associated to P fraction is indicated (D) CHO-K1 cells coexpressing CFP-K-RasC14 (K-RasC14) and lip33-YFP (lip33) or YFP-K-RasC14 and N52Gal-T2-CFP (N52Gal-T2) or N27GalNAc-TCFP (N27GalNAc-T) or cells expressing YFP-K-RasC14 and labeled with MitoTracker or endocyted Alexa647-Tf (Tf) were treated as described in (B), fixed and visualized by confocal microscopy Panels are merged images from K-RasC14 (pseudocolored green) and organelles markers (pseudocolored red) Insets show details of the boxed area at higher magnification Scale bars ¼ 20 lm for (A) and lm for (D) endogenous resident proteins (data not shown) K-Ras was colocalized to a minor extent with lip33-YFP, an ER marker, in Ca2+-depleted cells and Ca2+ + A23187 treated cells (Fig 6D) Similar results were obtained in Ca2+ and Ca2+ + A23187 + chelator treated cells (data not shown) YFP-K-RasC14 was partially colocalized with N52GalT2-CFP, a cis ⁄ medial Golgi marker, and with N27GalNAcT-CFP, a TGN marker, when cells were incubated in the presence of Ca+2 and A23187 (Fig 6D) Under the same conditions, YFP-K-RasC14 was colocalized with mitochondria (MitoTracker) and partially with endocytosed Tf Overall, these results show that alteration of intracellular calcium homeostasis in CHO-K1 cells induces a redistribution of YFP-K-RasC14 from plasma membrane to the endomembrane system, according probably to their physical and chemical properties Change in intracellular pH does not affect K-Ras subcellular distribution Because ionophore A23187 operates as a Ca2+ ⁄ H+ exchanger (see above), its observed effect on K-Ras distribution could conceivably result from modification of not only calcium homeostasis but also intracellular pH To test this possibility, we abolished pH gradients across the endomembrane system using the polyether ionophore monensin (a Na+⁄ H+ exchanger), and FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2219 Membrane targeting of K-Ras G A Gomez and J L Daniotti Disruption of K+ homeostasis alters subcellular distribution of K-Ras A Results of this and previous studies [11,12,29,43] indicate that bivalent cations affect plasma membrane association of K-Ras In order to investigate the role of the monovalent cation K+ on K-Ras subcellular distribution in CHO-K1 cells, we used the K+ ionophore valinomycin, which forms K+-selective pores through which K+ can flux across the cell membrane [57] K-RasC14 showed a rapid and significant accumulation (10% increment) in a perinuclear compartment defined as recycling endosome by colocalization with endocytosed Alexa647-human Tf (Fig 8A,B) This effect was enhanced (15% increment) when extracellular K+ was increased to 55 mm The results for valinomycin and for A23187 suggest that cytosolic ionic composition and transmembrane potential are relevant for plasma membrane targeting of K-Ras B Discussion Fig pH gradients does not affect subcellular distribution of K-Ras (A) CHO-K1 cells transiently expressing YFP-K-RasC14 (upper panels) and YFP-H-RasC20 (middle panels) were incubated with 10 lM monensin (Monensin) or vehicle (Control) for 30 at 37 °C and visualized alive by confocal microscopy Cells treated as described above and labeled with Lysotracker are shown at the bottom Images from control and monensin treated cells were acquired with identical acquisition settings (B) Cells were treated as described above, fixed and visualized by confocal microscopy Graphic shows the frequency of phenotypes (%) showing YFP-K-RasC14 subcellular distribution both in control and monensin treated cells Scale bars ¼ 20 lm observed the effect on subcellular distribution of YFPK-RasC14 Because the exchange of electrolytes is : 1, monensin alters pH gradient but not transmembrane potential [56] When YFP-K-RasC14-expressing CHOK1 were incubated in the presence of monensin, plasma membrane targeting of K-Ras was not altered (Fig 7), thus ruling out a possible role of H+ in intracellular transport and distribution of this protein As a control of monensin function, we observed loss of staining with Lysotracker in cells labeled with the dye (Fig 7) 2220 Membrane potential in biological membranes is determined by three main components: (a) transmembrane potential, (b) membrane dipole potential, and (c) membrane surface potential [58,59] Transmembrane potential is associated with gradients of electrical charge across the lipid bilayer and is well documented because of its role in normal function of excitable cells However, it is not relevant for plasma membrane binding of polybasic polypeptides (such as K-Ras) because these molecules not diffuse through biological membrane Moreover, this potential ranges in cells from 10 to 100 mV, with the inside compartment negative relative to the outside one For K-Ras, the subcellular localization suggests that transmembrane potential is not the main contribution for plasma membrane binding However, in hyperpolarized cells [60] the transmembrane potential could contribute to its endomembrane targeting (see above) The second component of membrane potential, membrane dipole potential, reflects molecular polarization or electrical dipoles associated with carbonyl groups and oxygen bound to phosphate groups [58,60] Structured water molecules at the membrane surface are also thought to contribute to this potential Dipole potential is not relevant to binding of polybasic peptides to membrane because (a) polybasic peptides not penetrate significantly into the leaflet of biological membranes [61,62]; (b) this potential is strongly dependent with distance [58]; (c) the overall sign of this potential is positive toward the inside of the membrane [63] However, it is possible that membrane dipole FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS G A Gomez and J L Daniotti Membrane targeting of K-Ras A B Fig K+ ionophore redistributes K-Ras from plasma membrane to recycling endosomes (A) CHO-K1 cells were incubated for 20 at 20 °C in DMEM and dimethylsulfoxide (5 mM KCl + DMSO) or DMEM and 10 lM valinomicyn (5 mM KCl + valinomicyn) or incubated in Locke’s, high K+, and dimethylsulfoxide (55 mM KCl + DMSO) or Locke’s, high K+, containing 10 lM valinomicyn (55 mM KCl + valynomicin) and visualized alive by confocal microscopy Images are representative from PM > Cyt phenotype (media plus dimethylsulfoxide) and perinuclear phenotype (media plus valinomicyn) of YFP-K-RasC14 subcellular distribution (left) Cells were treated as described above, fixed and visualized by confocal microscopy The graphic (right) shows the frequency of phenotypes (%) showing YFP-K-RasC14 subcellular distribution in cells incubated in mM KCl or mM KCl ⁄ 10 lM valinomicyn or 55 mM KCl ⁄ 10 lM valinomicyn (B) CHO-K1 cells coexpressing YFP-K-RasC14 (K-RasC14; green) and N27GalNAc-T-CFP (N27Gal-NAc-T; red) or cells expressing YFP-K-RasC14 and labeled with Alexa647-Tf (Tf; red) were incubated for 20 at 20 °C in Locke’s media and dimethylsulfoxide (55 mM KCl) or Locke’s, high K+, containing 10 lM valinomicyn (55 mM KCl + valynomicin) and visualized alive by confocal microscopy Panels are merged images from YFP-K-RasC14 and the corresponding organelle marker The insets show details of the boxed area at higher magnification Scale bars ¼ 10 lm potential regulates lateral distribution of K-Ras after it binds to membrane The third component of membrane potential, electrostatic membrane surface potential, is a consequence of incomplete quenching of the net excess of surface charge found in membrane surfaces [64] Strength of this potential depends on surface charge density, ionic strength, and the dielectric constant of the membrane surface [39,64] Transmembrane potential can promote an ion flux that indirectly affects surface potential [64,65] Surface potential has been shown to play a role in electrostatic interactions between lipid modified proteins containing a polybasic domain, and lipid bilayers [25,26,37,61] The degree of interaction between basic polypeptides and membrane depends on the content of anionic lipids in the bilayer, and salt concentration in the environment An increase in environmental ion content shields surface charge density and reduces electrostatic interaction between polybasic peptides and charged membranes [66,67] Surface charge density at the inner leaflet of the plasma membrane is due mainly to enrichment of PS in comparison to other intracellular membranes [54,68] The inner leaflet contains  30 mol% of PS and polyanionic lipids such as poly PIs (5–10%), which contribute to an electronegative surface potential [69–71] On the other hand, sialic acid and sulfate groups contribute to electronegative membrane surface potential at the outer leaflet Sialic acid content is important for electrophoretic properties of various cell types [45], and enzymatic release of sialic acid alters electrostatic binding of peripheral proteins to the cell surface [72] There FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2221 Membrane targeting of K-Ras G A Gomez and J L Daniotti are a few reports suggesting an effect of enzymatic removal of sialic acid on cytosolic events [73–75], but this topic remains largely unexplored Results of our biochemical experiments indicate that the association of K-Ras with biological membranes is driven by electrostatic and reversible interactions of its polybasic region with negatively charged lipids, in agreement with previous models [33,76] Translocation of K-Ras to intracellular compartments was recently reported to be controlled by its interaction with Ca2+ ⁄ calmodulin [43,77] These authors suggest that destabilization of K-Ras in plasma membrane by Ca2+ ⁄ calmodulin may result from disruption of electrostatic interaction between the polybasic region and negatively charged membrane phospholipids However, results from our biochemical experiments suggest that dissociation of K-Ras occurs in the presence of Ca2+ but in the absence of calmodulin with not significant differences from results from NaCl experiments, suggesting an unspecific effect of Ca2+ on membrane affinity of K-Ras A significant proportion of ectopically expressed YFP-K-RasC14 and YFP-K-Rasfull was found in the soluble fraction (35% and 37%, respectively) after ultracentrifugation This could be a consequence of (a) equilibrium between the soluble and particulate pools; (b) association with cytosolic escort proteins; and ⁄ or (c) post-translational modification that affect membrane binding of K-Ras Regarding possibility (c), recent studies showed that PKC-dependent phosphorylation of S181 within the hvr of oncogenic K-Ras leads to dissociation of K-Ras from plasma membrane [11] Our present results indicate that a significant proportion of soluble K-RasC14 and K-Rasfull was reversibly associated with membranes from nontransfected CHOK1 cells Thus, the soluble pool of K-Ras appears to undergo a dynamic exchange with the particulate pool under our experimental conditions Live cell imaging studies showed that enzymatic release of sialic acid increased K-Ras expression in membranes from recycling endosomes Incubation of cells in the presence of high calcium concentration (50 mm), a condition reported to reduce surface potential due to sialic acid residues [44], did not cause a significant redistribution of K-Ras to the pericentriolar recycling compartment (data not shown) Therefore, the role of sialic acid in plasma membrane targeting of K-Ras may involve a specific sialylated protein required for this process, rather than alteration of surface potential In summary, our studies indicate that composition of the outer leaflet affects membrane localization of K-Ras, and are likely to be of considerable relevance in K-Ras signaling in physiological and pathological cell conditions 2222 The role of cytoplasmic composition of anionic lipids in membrane binding of K-Ras was evaluated by studying the effect of ATP depletion, which inhibits inward movement of PS, with consequent loss of plasma membrane asymmetry, and depletes newly synthesized poly PIs The simultaneous impairment of glycolysis and mitochondrial respiration was accompanied by dissociation of K-Ras from the plasma membrane, and subsequent accumulation of K-Ras in recycling endosomes and Golgi complex membranes This redistribution of K-Ras was probably due to a reduction in phosphatidylinositol (4,5)-bisphosphate (PIP2) content at the plasma membrane and not an inhibition of PS ‘flipping in’, because we did not observe significant externalization of PS in ATPdepleted cells (Fig S3) The normal subcellular distribution of PIs is unclear [71], but it appears that PIP2 is located at the plasma membrane, while PI(3)P and PI(4)P are associated with membranes from endosomes and Golgi complex When synthesis of poly PIs is inhibited, PIP2 is first degraded to phosphatidylinositolphosphate by specific phosphatases, resulting in accumulation of these lipids in the cell This catabolism can shift to some extent the negative surface charge density gradient between plasma membrane and endosomal and Golgi membranes, causing K-Ras to localize in intracellular compartments Depletion of ATP led to cessation of kinase activity, and we speculate that phosphorylation on hvr (S181) of K-Ras is not operating under this experimental condition Because ATP is necessary for intracellular vesicular transport [78–80], K-Ras may translocate to endomembranes via a nonvesicular pathway following its dissociation from plasma membrane [15,25–28] This translocation could result from diffusion down an electronegative gradient, because negative charge density in normal cells is greater at the plasma membrane than in intracellular membranes (plasma membrane > recycling endosomes > Golgi complex > ER) [71,81] The dependence of ionic strength on plasma membrane targeting of K-Ras was evaluated using a battery of ionophores Changes in subcellular distribution of K-Ras were observed only for ionophores that modify transmembrane potential by changing cytosolic ionic strength, and these effects were more pronounced for bivalent than monovalent ions Ca2+ ionophore caused a rapid redistribution of K-Ras from plasma membrane to cytoplasm, Golgi complex, and mitochondria In contrast, K+ ionophore caused a more discrete redistribution, mainly to a pericentriolar compartment characterized as recycling endosomes This effect was probably due to calcium influx or changes FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS G A Gomez and J L Daniotti in transmembrane potential in response to K+ efflux in valinomycin-treated cells pH gradient through membranes was not relevant to K-Ras subcellular distribution, because monensin treatment had no effect on K-Ras localization Taken together, our findings indicate that the polybasic domain of K-Ras acts as a probe for electronegative surface membrane potential Subcellular distribution of K-Ras is dynamic, and depends on not only sequence and ⁄ or post-translational modifications of the membrane targeting domain, but also on electrical properties of cell membranes, which in turn depends on physiological and pathological status of the cell The dynamic interaction of K-Ras with membranes, and the fact that knockout of K-Ras, but not H-Ras or N-Ras is lethal in mice, suggest that the pleiotropic subcellular distribution of K-Ras is essential for its proper activity Membrane targeting of K-Ras Subcellular fractionation Cells were washed with cold NaCl ⁄ Pi and harvested by scraping in mm Tris ⁄ HCl (pH 7.0) (buffer T) Extracts were centrifuged at °C for at 13 000 g using a F-45-24-11 rotor in a 5415R centrifuge (Eppendorf, Hamburg, Germany) and resuspended in 400 lL of buffer T in the presence of lgỈmL)1 aprotinin, 0.5 lgỈmL)1 leupeptin and 0.7 lgỈmL)1 pepstatin (buffer T ⁄ protease inhibitor mixture; T-PIM) Pellets were dispersed by vortex and passed 60 times through a 25-gauge needle Nuclear fractions and unbroken cells were removed by centrifuging twice at °C for at 600 g using a F-45-24-11 rotor in a 5415R centrifuge (Eppendorf) Supernatants were then ultracentrifuged at °C for h at 400 000 g using a TLA 100.3 rotor (Beckman Coulter, Inc., Fullerton, CA) The supernatant (S fraction) was removed, and the pellet (P fraction) was resuspended in 400 lL of T-PIM for subsequent western blot analysis Triton X-114 partition assay Experimental procedures Plasmids Expression plasmids for yellow fluorescent protein (YFP)K-RasC14 and YFP-H-RasC20, N27GalNAc-T-CFP and N52 Gal-T2-CFP have been described previously [16,82] Plasmid encoding YFP-K-Rasfull was kindly supplied by M Philips (New York University School of Medicine, New York, NY) GPI-YFP fusion construct was kindly supplied by P Keller (Max-Plank Institute, Dresden, Germany) Plasmid encoding GFP-PH-PLCd1 was kindly supplied by M Lemmon (University of Pennsylvania School of Medicine, Philadelphia, PA) Cells lines, cell culture and DNA transfections The following CHO-K1 cell clones were used: wild-type CHO-K1 cells (ATCC, Manassas, VA) and clone 4, a stable double transfectant expressing GalNAc-T and GalT2 tagged at the C-terminal with the hemagglutinin (HA) epitope (YPYDVPDYA) Cells were maintained at 37 °C, 5% CO2, DMEM supplemented with 10% fetal bovine serum and antibiotics Cells were transfected with 0.6– 1.2 lg per 35 mm dish of expression plasmids using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations Twenty-four hours after transfection, cells were labeled with Tf, and Orange or Red MitoTracker (Molecular Probes, Eugene, OR) or treated under different conditions (neuraminidase treatment, calcium or ATP depletion or ionophores treatment), washed with cold phosphate buffered saline (140 mm NaCl, 8.4 mm Na2HPO4, 1.6 mm NaH2PO4, pH 7.5; NaCl ⁄ Pi) and harvested by scraping or fixed for microscopy P fractions were solubilized for h at °C in 1% Triton X-114 in NaCl ⁄ Pi-PIM Then, samples were incubated at 37 °C for and centrifuged at 13 000 g using a F-4524-11 rotor in a 5415R centrifuge (Eppendorf) The aqueous upper phase (A) and the detergent-enriched lower phase (D) were separated and extracted again with detergent and aqueous solutions, respectively The resulting samples were adjusted to equal volumes and detergent content and proteins were precipitated with chloroform ⁄ methanol (1 : v ⁄ v) for western blot analyses Poly l-lysine, NaCl and CaCl2 treatment of membranes P fractions were resuspended in buffer T and centrifuged again at 400 000 g using a TLA 100.3 rotor in an Optima TLX ultracentrifuge (Beckman Coulter, Fullerton, CA) Then, particulate fractions were resuspended and incubated on ice for h in buffer T supplemented with different concentrations of electrolytes (poly l-lysine, NaCl and CaCl2) After incubation samples were centrifuged at °C for h at 400 000 g using a TLA 100.3 rotor in an Optima TLX ultracentrifuge (Beckman Coulter) S and P fractions were normalized to the same amount of electrolyte, precipitated with trichloroacetic acid and subsequently analyzed by western blot Data above correspond to at least three independent experiments K-Ras membrane binding assays S and P fractions were obtained from K-Ras transfected and untransfected CHO-K1 cells S fractions were cleared and P fractions washed by ultracentrifugation at 400 000 g using a TLA 100.3 rotor in an Optima TLX ultracentrifuge FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2223 Membrane targeting of K-Ras G A Gomez and J L Daniotti (Beckman Coulter) Membrane binding of soluble K-Ras protein was assayed by incubation of S fractions obtained from transfected cells with P fractions obtained from untransfected cells during h at °C The mix was then centrifuged at 400 000 g using a TLA 100.3 rotor in an Optima TLX ultracentrifuge (Beckman Coulter), for h at °C K-Ras distribution in S and P fractions was analyzed by western blot Membrane dissociation of K-Ras was assayed in the same condition but using P fractions from K-Ras transfected CHO-K1 cells and S fractions from untransfected CHO-K1 cells Topology assays Membrane fractions from cells expressing YFP-K-RasC14 and membrane fractions obtained after incubation of particulate fractions from unstransfected cells and cytosol from K-Ras transfected cells were resuspended in 200 lL of buffer T containing 200 lgỈmL)1 BSA or 200 lgỈmL)1 trypsin (Try) and further incubated at 37 °C for h Reactions were stopped by addition of 10% (w ⁄ v, final concentration) trichroloacetic acid Proteins were then recovered by centrifugation at 13 000 g for 30 at °C using a F-45-2411 rotor in a 5415R centrifuge (Eppendorf), resuspended in sample buffer and analyzed by western blot Electrophoresis and western blot Proteins were resolved by electrophoresis through 12% (w ⁄ v) SDS ⁄ PAGE gels under denaturing conditions and then electroblotted onto nitrocellulose membranes for 80 at 300 mA Protein bands in nitrocellulose membranes were visualized by Ponceau staining For immunoblotting, nonspecific binding sites on the nitrocellulose membrane were blocked with 5% (w ⁄ v) nonfat dry milk in Tris-buffered saline (200 mm NaCl, 50 mm Tris ⁄ HCl, pH 7.5) Anti-GFP polyclonal IgGIj (Roche Diagnostics, Indianapolis, IN) was used at a dilution of : 1000 Bands were detected by protein A coupled to horseradish peroxidase combined with the chemioluminiscence detection kit (SuperSignalÒ West Pico Chemioluminiscent Substrate, Pierce, Rockford, IL) and Hyperfilm MP films (GE Healthcare, Fairfield, VT) The relative contribution of each band was measured using the computer software scion image (Scion Corporation, Frederick, MD, USA) on scanned films of low exposure images Statistical significances (P) between each condition and control were determined by t-student test.* for P < 0.1, ** for P < 0.05 Neuraminidase treatment Twenty-four hours after transfection, cells were incubated for h at 37 °C in DMEM containing 1.5 or mL)1 neuraminidase (NANase) type V from Clostridium perfrin- 2224 gens (Sigma Aldrich, St Louis, MO) or vehicle (control) Then, cells were directly visualized or washed with cold NaCl ⁄ Pi and fixed in 3% (v ⁄ v) paraformaldehyde (30 at °C) ATP depletion treatment ATP depletion in CHO-K1 cells was performed as described by [49] Briefly, 24 h after transfection cells were washed twice with DMEM without glucose (Gibco, Invitrogen, Carlsbad, CA) and incubated in the same media containing mm NaN3 and 50 mm 2-deoxi-d-glucose (ATP-depleted cells) or water (vehicle) and d-(+)-glucose (control cells) for h Then, cells were directly visualized or washed with NaCl ⁄ Pi and fixed in 3% (v ⁄ v) paraformaldehyde (30 at °C) Calcium depletion and A23187 treatment Twenty-four hours after transfection, cells were washed three times with extracellular solution [140 mm NaCl, mm KCl, mm MgCl2, 10 mm glucose, 0.1% BSA, 15 mm Hepes pH 7.4, extracellular solution (ECS)] without calcium and then incubated for h in the same media containing 10 lm BAPTA-AM (Molecular Probes) and 10 mm EGTA Then, cells were washed in the absence of chelators (Chel) and incubated for h with ECS without calcium (–Ca2+ treatment) or ECS containing mm Ca2+ (+Ca2+ treatment) or mm Ca2+ and 30 lm A23187 (Sigma Aldrich, +Ca2+ + A23187 treatment) or mm Ca2+, 30 lm A23187, 10 lm BAPTAAM and 10 mm EGTA (+Ca2+ + A23187 + Chel) Then, cells were washed in NaCl ⁄ Pi and fixed for visualization by fluorescence microscopy For live cells experiments, cells were incubated in DMEM for 20 at 20 °C on the microscope stage Time series were acquired during this period and then A23187 was added to a final concentration of 30 lm (in the presence or absence of chelators) and a time series was then acquired during 60 Ionophore treatment Stock solution of valinomycin (1.25 mm) was prepared in dimethylsulfoxide A stock solution of mm monensin was prepared in ethanol Cells transiently expressing the quimeric proteins were washed twice with DMEM and incubated for 15 with 10 lm valinomycin or 25 lm monensin or vehicle for control cells and then visualized alive or fixed for fluorescence microscopy For high K+ and valinomycin incubations, cells were washed with 1· buffer Lockes, high K+ (55 mm KCl, 85 mm NaCl, 2.4 mm NaHCO3, 1.8 mm CaCl2, mm Hepes pH 7.2) and then incubated for 20 in the same media containing 10 lm valinomycin FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS G A Gomez and J L Daniotti Live cell imaging Live cells experiments were performed at 20 °C on a Carl Zeiss LSM5 Pascal laser scanning confocal microscope (Carl Zeiss AG, Gottingen, Germany) or an Olympus ă FluoView FV1000 confocal microscope (Olympus Latin America, Miami, FL) equipped with a argon laser and a 63· Plan-Apochromat objective using a pinhole appropriate to obtain 0.8 lm optical slices Images for each experiment were taken during 30 Endocytosis of Alexa647-conjugated transferrin and MitoTracker staining To label the endocytic recycling compartment, CHO-K1 cells grown in coverslips were incubated in DMEM, 0.1% BSA for h in a CO2 incubator and then labeled in identical conditions with 14 lgỈmL)1 Alexa647-human transferrin (Molecular Probes) during 30 before and during the addition of NANase or 2-deoxi-d-glucose ⁄ NaN3 or ionophores for the indicated times After treatment, cells were washed three times with NaCl ⁄ Pi, and fixed for fluorescence microscopy For MitoTracker staining, cells were incubated during 10 with lm MitoTracker (Molecular Probes, Eugene, OR, USA) at 37 °C and then fixed or live cells were analyzed by confocal microscopy Confocal microscopy and image acquisition processing Confocal images were collected using a Carl Zeiss LSM5 Pascal laser scanning confocal microscope or an Olympus FluoView FV1000 confocal microscope Excitation wavelengths and filter set for CFP, GFP, YFP and Alexa647 were described previously [16] MitoTracker fluorescence was detected using the filters for rhodamine or Cy5 Images of fixed cells for quantitative purposes were acquired using a 63· Plan-Apochromat oil immersion objective using a pinhole appropriate to obtain an optical slice of 0.8 lm For colocalization experiments, images were taken using a 100· Plan-Apochromat NA 1.4 oil immersion objective Phenotypic analysis was performed using the metamorphÒ 4.5 (Molecular Devices, Sunnyvale, CA) software using the threshold function Statistical significance (P) (t-student test) was performed from at least three independent experiments (600 cells per experiment) Asterisks represent P < 0.1 versus control conditions Acknowledgements This work was supported in part by grants from Secretarı´ a de Ciencia y Tecnologı´ a, Universidad Nacional de ´ Cordoba (162 ⁄ 06); Consejo Nacional de Investigaci´ ones Cientı´ ficas y Tecnicas (CONICET), Argentina Membrane targeting of K-Ras ´ (PIP 5151); Fundacion Antorchas (14116-112) and ´ ´ Agencia Nacional de Promocion Cientı´ fica y Tecnologica (FONCYT), Argentina (01-13522) The authors thank the technical assistance of G Schachner, S Deza and C Mas, and Eduardo Guimaraes (Departamento ´ de Bioquı´ mica-Instituto de Ciencias Basicas da Saude, ˆ ´ Porto Alegre, Brazil) for his help in preliminary biochemical experiments GAG is the recipient of CONICET Fellowship JLD is a Career Investigator of CONICET (Argentina) We thank Dr S Anderson for editing GAG would like to thank M L Ferrari for encouragement References Zheng Y & Quilliam LA (2003) Activation of the Ras superfamily of small GTPases Workshop on exchange factors EMBO Rep 4, 463–468 Quilliam LA, Rebhun JF & Castro AF (2002) A growing family of guanine nucleotide exchange factors is responsible for activation 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Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS ... lipids Electrical properties of the outer leaflet of plasma membrane ) contribution to membrane targeting of K-Ras Biochemical studies as above demonstrate that membrane binding properties of K-Ras. .. electrical properties of the plasma membrane in the subcellular distribution of K-Ras In particular, we investigated (a) the role of surface charge on inner and outer leaflet of plasma membrane. .. underlying plasma membrane targeting of this protein, we attempted to disrupt membrane surface potential of the outer leaflet of plasma membrane, and to analyze subcellular distribution of K-Ras following