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

Electrical properties of plasma membrane modulate subcellular distribution of K-Ras Guillermo A. Gomez and Jose L. Daniotti

Centro de Investigaciones en Quı´mica Biolo´ gica de Co´ rdoba (CIQUIBIC, UNC-CONICET), Departamento de Quı´mica Biolo´ gica, Universidad Nacional de Co´ rdoba, Argentina

Keywords calcium; membrane potential; polyphosphoinositides; RAS; sialic acid

Correspondence J. L. Daniotti, Centro de Investigaciones en Quı´mica Biolo´ gica de Co´ rdoba (CIQUIBIC, UNC-CONICET), Departamento de Quı´mica Biolo´ gica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA, Co´ rdoba, 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 farnesyl- ated 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 mem- brane binding properties. Results demonstrated the electrical and reversible nature of K-Ras binding to anionic lipids in membranes. We next investi- gated membrane binding and subcellular distribution of K-Ras after dis- ruption 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 nucleo- tide 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 differ- ent 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 inter- actions 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.

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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 hyper- variable domain (hvr) that operates as a membrane targeting signal [3,5].

retrograde transport of these isoforms through desorp- tion from the plasma membrane, followed by adsorp- tion 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 intracellu- lar transport of K-Ras between subcellular compart- ments [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 to redistribute this protein to endomem- sufficient branes [27,29,31]. On the other hand, complete replace- ment 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 mem- brane does not depend on additional factors. This idea is consistent with results of earlier biophysical and bio- chemical studies [8,25–27], and with recent observa- tions in vivo [28,29,32], that prenylated polycationic peptides bind dynamically and reversibly with model and cellular membranes through electrostatic and hydrophobic interactions.

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 alipha- tic, 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 palmi- toylation 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].

plasma membrane

transport

to

cell

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 micro- domains (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 mem- brane association dynamics of Ras isoforms are important for their proper function, underlying mecha- intracellular transport and distribution of nisms of these proteins is not completely understood. Palmitoyl- ation of H-Ras and N-Ras causes membrane trapping early in the classical secretory pathway, and subse- through quent association with exocytic vesicles [9,10]. Unlike farn- esylation, 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

In the present study, we combined biochemical tech- niques and fluorescence confocal microscopy analysis to clarify the role of electrical properties of the plasma membrane in the subcellular distribution of K-Ras. In the role of surface particular, we investigated (a) charge on inner and outer leaflet of plasma membrane and (b) effect of ionic gradients through plasma mem- brane on membrane binding and subcellular distribu- tion 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 mem- branes, consistent with a proposed model of K-Ras membrane association based on electrostatic interac- tion [33]. Confocal microscopy analysis, in combina- imaging, demonstrated that tion with live 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 signifi- cant accumulation of K-Ras in a perinuclear region,

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To characterize expression of

YFP-K-RasC14 and YFP-K-Rasfull, respectively, were enriched in the detergent phase, indicating that a frac- tion of the expressed proteins are hydrophobic, and therefore post-translationally modified by lipidation. these proteins

them mostly

cytosol

in

colocalizing with recycling endosome and Golgi com- plex markers. Finally, the dependence of ionic strength on plasma membrane targeting of K-Ras was evalu- ated using a battery of ionophores. Ionophores that modify transmembrane potential caused a rapid redis- tribution of K-Ras from plasma membrane to endo- membranes. 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 iono- phore 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

in CHO-K1 cells, subcellular distribution of YFP-K- RasC14 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 mem- brane (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 (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-K- Rasfull were extensively colocalized in cells that expres- sed 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.

expressing

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 colocalization analyses with markers of extensive organelles (Fig. 2 and Fig. S1). No colocalization was observed between YFP-K-RasC14 and major histocom- patability 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

Constructs 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 fluor- escent 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 (cid:2) 55 kDa according to the expected molecular mass (Fig. 1A). Membrane association of the expressed fusion proteins investigated by ultracentrifugation of extracts was from mechanically lysed cells. YFP-K-RasC14 and YFP-K-Rasfull were associated mainly with the particu- late fraction (65% and 63%, respectively) (Fig. 1B). To analyze the degree of post-translational modifica- tion, and to rule out possible association of these pro- teins with insoluble components such as cytoskeleton, nuclear remnants, or extracellular matrix, we per- formed Triton X-114 partitioning assay on particulate fractions of cells transiently expressing the fusion proteins [34,35] (Fig. 1C). Fifty percent and 44% of

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B

C

A

D

E

Fig. 1. 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 indi- cated 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 pro- teins 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 1 h. Then, samples were incubated at 37 (cid:2)C for 3 min 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-K- RasC14 or YFP-K-Rasfull were fixed with paraformaldehyde and visualized by confocal microscopy. Left, representative cell phenotypes show- ing 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.

recycling endosomes,

in (cid:2) 10% of

[36]

transfected of (Fig. 2; K-RasC14-perinuclear). CHO-K1 cells Similar subcellular distributions were observed for the full-length version of K-Ras (data not shown). In sum- mary, 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.

phobic interactions between this domain and the plasma membrane [25,26,37]. The models predict that electrostatic interactions and plasma membrane associ- ation 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 to reduce net decreases. Mutagenesis experiments charge of the polybasic region of K-Ras gave results consistent with the models [8,9,27,31,38].

Membrane binding properties of K-Ras

suggest

To better characterize the membrane binding prop- erties of K-Ras to biological membranes we per- formed 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

Results from model system experiments and theoretical analyses that membrane association and plasma membrane targeting of K-Ras are a conse- quence of the electronegative sensing function of the C-terminal domain of this protein, and that membrane association depends on both electrostatic and hydro-

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Fig. 2. At steady state, most of K-Ras is associated with plasma membrane, cytosol and to a minor extent to endosomal compartments. CHO- K1 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 corres- ponding 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 magnifica- tion. Scale bars ¼ 5 lm.

dissociation

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). of CFP-H-RasC20 was Membrane observed at low ionic strength, but was insignificant at high ionic strength.

contain a polybasic domain, and of GPI-YFP, a fluorescent protein containing a glycosylphosphatidy- (GPI) attachment signal. When membrane linositol expressing YFP-K-RasC14 or fractions from cells 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 signi- ficant change in the amount of CFP-H-RasC20 associ- ated with particulate fraction was observed under the same conditions.

experiments

To test whether the effect of poly l-lysine on mem- brane binding of K-Ras depends on its electrical prop- erties, we performed similar in the presence of increasing concentrations of NaCl (Fig. 3- Aii). A significant membrane dissociation of both YFP-K-RasC14 and YFP-K-Rasfull ((cid:2) 45%) was observed at 1.5 m NaCl, in accordance with the elec-

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 bila- yers 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

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A

i

ii

iii

B

C

Fig. 3. Membrane binding properties of K-Ras. (A) Membrane fractions of CHO-K1 cells expressing YFP-K-RasC14 or YFP-K-Rasfull or CFP-H- RasC20 were obtained as described in Fig. 1B and then incubated for 1 h in solutions containing 0, 0.012 and 0.12 mgÆmL)1 poly L-lysine (i) or 0, 3 · 10)6, 1.5 · 10)4, 3 · 10)2, 15 · 10)2 and 1.5 M NaCl (ii) or 0.1 · 10)6, 5 · 10)5, 1 · 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. 1. Membrane fractions of transfected cells were incubated for 1 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 1 h and S and P fraction obtained by ultracentrifugation for western blot analysis with anti-GFP (cytosolic FP + membranes). (C) Membranes were obtained from non- transfected CHO-K1 cells, treated with 200 lgÆmL)1 proteinase K or BSA for 30 min and further washed five times. Proteinase PK- or BSA- treated membranes were then incubated for 1 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).

ing a nonspecific effect of Ca2+ on K-Ras membrane affinity. CFP-H-RasC20 and GPI-YFP were not signifi- cantly dissociated under the same conditions.

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 signifi- cantly different from that observed for NaCl, suggest-

Having demonstrated that K-Ras membrane associ- ation depends on electrostatic interaction, we analyzed in vitro the reversibility of such interaction. Cytosolic

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galactosyltransferase

compartment

(results not

Taken together,

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 1 h at 4 (cid:2)C and then ultracentrifuged to separate sol- uble 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 mem- brane associated K-Ras. Cytosolic YFP (a soluble pro- tein) was recovered mostly in the soluble fraction, indicating that it was not associated with membranes from nontransfected cells. In contrast, (cid:2) 50% of sol- uble K-RasC14 and K-Rasfull was associated with mem- branes from nontransfected cells. The K-Ras fraction reassociated with membranes from nontransfected cells was completely dissociated when incubated in the pres- ence of 1.5 m NaCl (Fig. S2B). These results support the concept that K-Ras binds to cellular membranes through an electrostatic, reversible mechanism.

assayed by conversion of GD1a (disialoganglioside) to GM1 (monosialoganglioside) in a CHO-K1 clone stably expressing UDP-GalNAc:LacCer ⁄ G3 ⁄ GD3 N-acetyl- galactosaminyltransferase (GalNAc-T) and UDP-Gal:- (Gal-T2) GA2 ⁄ G2 ⁄ GD2 ⁄ GT2 glycosyltransferases [46] (Fig. 4A). Live cell imaging analysis showed that neuraminidase treatment incre- ased K-RasC14, but not H-RasC20, expression in a peri- (Fig. 4B), and that K-Ras nuclear 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 shown). of neuraminidase-treated cells 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 peri- nuclear compartment is a consequence of a reduction in number of cells showing cytosolic K-Ras expression. these results suggest a dynamic interplay between the cytosolic, recycling endosome and plasma membrane fractions of K-Ras. Independ- ent of the mechanism ⁄ s involved in this subcellular dis- tribution 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

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 (con- trol) 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

and reversible

Biochemical studies as above demonstrate that mem- brane binding properties of K-Ras are due to elec- trostatic interactions. To further characterize the mechanisms underlying plasma mem- brane targeting of this protein, we attempted to disrupt membrane surface potential of the outer leaflet of plasma membrane, and to analyze subcellular distribu- tion of K-Ras following such disruption.

To characterize the mechanisms underlying plasma membrane targeting of K-Ras, we reduced surface charge of the inner leaflet, by inhibiting poly PI syn- thesis 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 treat- ment 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 signifi- cant 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 experi- ments with a TGN marker (N27GalNAc-T) and endo- cytosed human Alexa647-Tf, a recycling endosome marker [50]. We observed colocalization of YFP-K- RasC14 with endocytosed Tf, and with TGN marker, in ATP-depleted cells (Fig. 5B). No colocalization was observed between K-Ras and N52Gal-T2-CFP

Sialic acid is a charged monosaccharide that contri- butes significantly to surface potential of the outer leaflet, and may also be involved in molecular rear- rangement 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

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A

B

C

Fig. 4. Enzymatic release of sialic acid redis- tributes K-Ras to recycling endosomes. (A) CHO-K1 cells or a parental clone 4 stably expressing GalNAc-T and Gal-T2-HA were treated or not with 1.5 UÆmL)1 NANase for 2 h at 37 (cid:2)C. Then, cells were shifted to 4 (cid:2)C and incubated with cholera toxin for 30 min. 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) Con- focal microscopy of live cells expressing YFP-K-RasC14 and YFP-H-RasC20 treated or not with 1.5 UÆmL)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 (N27Gal- NAc-T, red) or cells expressing YFP-K-RasC14 (K-RasC14, green) and labeled with Alex- a647-Tf (Tf; red) were treated (NANase) or not (control) with 1.5 UÆmL)1 NANase for 2 h, fixed and visualized by confocal micros- copy. Panels are merged images from YFP-K-RasC14 and the corresponding organ- elle marker. The insets show details of the boxed area at higher magnification. Scale bars ¼ 10 lm for (B) and 5 lm for (C).

(N52Gal-T2), a medial Golgi marker. These results sug- gest that surface charges from poly PIs at the inner leaflet are necessary for proper membrane binding and subcellular distribution of K-Ras.

[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 ionophore redistributes K-Ras to endomembrane

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 struc- tures were also decorated with K-Ras. Similar redistri- bution was observed for full-length K-Ras fused to YFP

In vitro experiments in this study and others have dem- onstrated that binding of lipid modified cationic pep- tides, YFP-K-RasC14 and YFP-K-Rasfull, depends on ionic strength of the medium. To investigate the rela- tionship 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+

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A

B

Fig. 5. 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 1 h in DMEM without glucose containing 50 mM 2-deoxiglucose and 5 mM NaN3 (–ATP) or 50 mM D-(+)-glucose and vehicle (control) and visualized alive at 20 (cid:2)C by confocal microscopy (left). Frequency of pheno- types (%) showing YFP-K-RasC14 and YFP-H-RasC20 subcellular distribution (right). Scale bars: 10 lm. (B) CHO-K1 cells coex- pressing 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 (green) and labeled with Alexa647-Tf (Tf; red) were treated as des- cribed above, fixed and visualized by confo- cal 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.

absence of chelators, indicating that very low levels of extracellular calcium are sufficient to alter subcellular distribution of YFP-K-RasC14.

(data not shown). In contrast, YFP-H-RasC20 and GPI-CFP showed no redistribution under the same con- ditions (Fig. 6A and Video S1). A23187 function was evaluated using Lysotracker, a fluorescent acidotropic probe for labeling acidic organelles. As expected, Lyso- tracker did not reveal any acidic intracellular compart- ments in A23187-treated cells (Fig. S4).

ester

(BAPTA-AM; a permeable

Increase in cytosolic Ca2+ can cause PKC activa- tion 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 experimen- tal conditions (Fig. 6C). These results suggest that redistribution of K-Ras from plasma membrane to endomembranes is not a consequence of further post- translational modifications or association with cytoso- lic protein; rather, K-Ras responds to local changes in membrane properties which are lost during subcellular fractionation.

distribution

of K-Ras

cytosolic

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-ami- nophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid-acetoxy- methyl calcium chelator). Reduced calcium level caused an increase in cell phenotype showing clear plasma membrane expres- sion of K-Ras, and a decrease in number of cells show- ing (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 che- lators together with Ca2+ and A23187 produced the same phenotypic distribution as observed in the

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). Chan- ges in calcium level caused alterations in morphology of Golgi complex and ER. This phenomenon was evi- for both ectopically expressed markers and dent

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B

A

C

D

+ 2 a C

-

+ 2 a C +

7 8 1 3 2 A +

(S) was recovered and the particulate fraction (P)

lysed and ultracentrifugated. The supernatant

Fig. 6. 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 (cid:2)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 5 min after A23187 addition is shown. (B) CHO-K1 cells expressing YFP-K-RasC14 were Ca2+ depleted and incubated for 1 h in media without Ca2+ (–Ca2+) or containing 5 mM Ca2+ (+Ca2+) or 5 mM Ca2+ and 30 lM A23187 (+Ca2+ + A23187) or 5 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), 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-T- CFP (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 mark- ers (pseudocolored red). Insets show details of the boxed area at higher magnification. Scale bars ¼ 20 lm for (A) and 5 lm for (D).

redistribution of YFP-K-RasC14

from plasma a membrane to the endomembrane system, according probably to their physical and chemical properties.

Change in intracellular pH does not affect K-Ras subcellular distribution

endogenous resident proteins (data not shown). K-Ras was colocalized to a minor extent with lip33-YFP, in Ca2+-depleted cells and Ca2+ + an ER marker, A23187 treated cells (Fig. 6D). Similar results were obtained in Ca2+ and Ca2+ + A23187 + chelator treated cells (data not shown). YFP-K-RasC14 was par- tially 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 condi- tions, YFP-K-RasC14 was colocalized with mitochon- dria (MitoTracker) and partially with endocytosed Tf. Overall, these results show that alteration of intracellu- lar calcium homeostasis in CHO-K1 cells induces

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

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A

Disruption of K+ homeostasis alters subcellular distribution of K-Ras

Results of this and previous studies [11,12,29,43] indi- cate 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+ iono- phore valinomycin, which forms K+-selective pores through which K+ can flux across the cell membrane [57]. K-RasC14 showed a rapid and significant accumu- lation (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 extracellu- lar K+ was increased to 55 mm. The results for valino- mycin and for A23187 suggest that cytosolic ionic composition and transmembrane potential are relevant for plasma membrane targeting of K-Ras.

B

Discussion

that

localization suggests is not

Membrane potential in biological membranes is deter- mined by three main components: (a) transmembrane potential, (b) membrane dipole potential, and (c) mem- brane surface potential [58,59]. Transmembrane poten- tial 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. How- ever, it is not relevant for plasma membrane binding of polybasic polypeptides (such as K-Ras) because these molecules do not diffuse through biological membrane. Moreover, this potential ranges in cells from 10 to 100 mV, with the inside compartment neg- ative relative to the outside one. For K-Ras, the sub- cellular transmembrane the main contribution for plasma potential membrane binding. However, in hyperpolarized cells [60] the transmembrane potential could contribute to its endomembrane targeting (see above).

Fig. 7. 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 min at 37 (cid:2)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.

this potential

[61,62];

(b)

is

The second component of membrane potential, membrane dipole potential, reflects molecular polariza- tion 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 pep- tides to membrane because (a) polybasic peptides do not penetrate significantly into the leaflet of biological membranes strongly dependent with distance [58]; (c) the overall sign of this potential is positive toward the inside of the membrane it is possible that membrane dipole [63]. However,

observed the effect on subcellular distribution of YFP- K-RasC14. Because the exchange of electrolytes is 1 : 1, monensin alters pH gradient but not transmembrane potential [56]. When YFP-K-RasC14-expressing CHO- K1 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 pro- tein. As a control of monensin function, we observed loss of staining with Lysotracker in cells labeled with the dye (Fig. 7).

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A

B

Fig. 8. K+ ionophore redistributes K-Ras from plasma membrane to recycling endosomes. (A) CHO-K1 cells were incubated for 20 min at 20 (cid:2)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 perinu- clear phenotype (media plus valinomicyn) of YFP-K-RasC14 subcellular distribution (left). Cells were treated as described above, fixed and visu- alized by confocal microscopy. The graphic (right) shows the frequency of phenotypes (%) showing YFP-K-RasC14 subcellular distribution in cells incubated in 5 mM KCl or 5 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 incu- bated for 20 min at 20 (cid:2)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 organ- elle 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.

concentration in the environment. An increase in envi- ronmental ion content shields surface charge density and reduces electrostatic interaction between polybasic peptides and charged membranes [66,67].

[25,26,37,61]. The degree of

The third component of membrane potential, elec- trostatic 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 interaction bilayers between basic polypeptides and membrane depends on the content of anionic lipids in the bilayer, and salt

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 (cid:2) 30 mol% of PS and poly- anionic lipids such as poly PIs (5–10%), which contri- bute 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 elec- trophoretic properties of various cell types [45], and enzymatic release of sialic acid alters electrostatic bind- ing of peripheral proteins to the cell surface [72]. There

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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 agree- ment with previous models [33,76]. Translocation of K-Ras to intracellular compartments was recently repor- ted to be controlled by its interaction with Ca2+ ⁄ calmo- dulin [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 mem- brane binding of K-Ras. Regarding possibility (c), recent studies showed that PKC-dependent phosphory- lation 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 propor- tion of soluble K-RasC14 and K-Rasfull was reversibly associated with membranes from nontransfected CHO- K1 cells. Thus, the soluble pool of K-Ras appears to undergo a dynamic exchange with the particulate pool under our experimental conditions.

Live cell

The role of cytoplasmic composition of anionic li- pids 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 accom- panied 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 ATP- depleted cells (Fig. S3). The normal subcellular distri- bution 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 endo- somes and Golgi complex. When synthesis of poly PIs is inhibited, PIP2 is first degraded to phosphatidy- linositolphosphate by specific phosphatases, resulting in accumulation of these lipids in the cell. This cata- bolism 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 spe- culate 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 endo- membranes 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 den- sity in normal cells is greater at the plasma mem- (plasma than in intracellular membranes brane membrane > recycling endosomes > Golgi com- plex > ER) [71,81].

The dependence of ionic strength on plasma mem- brane targeting of K-Ras was evaluated using a bat- tery of ionophores. Changes in subcellular distribution of K-Ras were observed only for ionophores that mod- ify 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 mito- chondria. In contrast, K+ ionophore caused a more discrete redistribution, mainly to a pericentriolar com- partment characterized as recycling endosomes. This effect was probably due to calcium influx or changes

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 poten- tial due to sialic acid residues [44], did not cause a sig- nificant 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 sur- face potential. In summary, our studies indicate that composition of the outer leaflet affects membrane localization of K-Ras, and are likely to be of consider- able relevance in K-Ras signaling in physiological and pathological cell conditions.

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Subcellular fractionation

in transmembrane potential in response to K+ efflux in valinomycin-treated cells. pH gradient through membranes was not relevant to K-Ras subcellular dis- tribution, because monensin treatment had no effect on K-Ras localization.

leupeptin aprotinin, 0.5 lgÆmL)1 5 lgÆmL)1

Cells were washed with cold NaCl ⁄ Pi and harvested by scra- ping in 5 mm Tris ⁄ HCl (pH 7.0) (buffer T). Extracts were centrifuged at 4 (cid:2)C for 5 min at 13 000 g using a F-45-24-11 rotor in a 5415R centrifuge (Eppendorf, Hamburg, Ger- many) and resuspended in 400 lL of buffer T in the presence and of 0.7 lgÆmL)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 4 (cid:2)C for 5 min at 600 g using a F-45-24-11 rotor in a 5415R centrifuge (Eppen- dorf). Supernatants were then ultracentrifuged at 4 (cid:2)C for 1 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.

Taken together, our findings indicate that the poly- basic domain of K-Ras acts as a probe for electro- negative 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 electri- cal properties of cell membranes, which in turn depends on physiological and pathological status of the cell. The dynamic interaction of K-Ras with mem- branes, and the fact that knockout of K-Ras, but not H-Ras or N-Ras is lethal in mice, suggest that the plei- otropic subcellular distribution of K-Ras is essential for its proper activity.

Triton X-114 partition assay

Experimental procedures

Plasmids

P fractions were solubilized for 1 h at 4 (cid:2)C in 1% Triton X-114 in NaCl ⁄ Pi-PIM. Then, samples were incubated at 37 (cid:2)C for 3 min and centrifuged at 13 000 g using a F-45- 24-11 rotor in a 5415R centrifuge (Eppendorf). The aque- ous upper phase (A) and the detergent-enriched lower phase (D) were separated and extracted again with deter- gent and aqueous solutions, respectively. The resulting sam- ples were adjusted to equal volumes and detergent content and proteins were precipitated with chloroform ⁄ methanol (1 : 4 v ⁄ v) for western blot analyses.

Expression plasmids for yellow fluorescent protein (YFP)- K-RasC14 and YFP-H-RasC20, N27GalNAc-T-CFP and N52Gal-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 (Max-Plank Institute, Dresden, supplied by P. Keller Germany). Plasmid encoding GFP-PH-PLCd1 was kindly supplied by M. Lemmon (University of Pennsylvania School of Medicine, Philadelphia, PA).

Poly l-lysine, NaCl and CaCl2 treatment of membranes

Cells lines, cell culture and DNA transfections

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 1 h in buffer T supplemented with different con- centrations of electrolytes (poly l-lysine, NaCl and CaCl2). After incubation samples were centrifuged at 4 (cid:2)C for 1 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 inde- pendent experiments.

K-Ras membrane binding assays

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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 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 Gal- T2 tagged at the C-terminal with the hemagglutinin (HA) epitope (YPYDVPDYA). Cells were maintained at 37 (cid:2)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 Lipo- fectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. Twenty-four hours after transfection, cells were labeled with Tf, and Orange or (Molecular Probes, Eugene, OR) or Red MitoTracker treated under different conditions (neuraminidase treat- ment, calcium or ATP depletion or ionophores treat- ment), 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.

G. A. Gomez and J. L. Daniotti

Membrane targeting of K-Ras

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 min at 4 (cid:2)C).

ATP depletion treatment

(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 un- transfected cells during 1 h at 4 (cid:2)C. The mix was then cen- trifuged at 400 000 g using a TLA 100.3 rotor in an Optima TLX ultracentrifuge (Beckman Coulter), for 1 h at 4 (cid:2)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 un- transfected CHO-K1 cells.

Topology assays

ATP depletion in CHO-K1 cells was performed as des- cribed by [49]. Briefly, 24 h after transfection cells were washed twice with DMEM without glucose (Gibco, Invitro- gen, Carlsbad, CA) and incubated in the same media containing 5 mm NaN3 and 50 mm 2-deoxi-d-glucose (ATP-depleted cells) or water (vehicle) and d-(+)-glucose (control cells) for 1 h. Then, cells were directly visualized or washed with NaCl ⁄ Pi and fixed in 3% (v ⁄ v) paraformalde- hyde (30 min at 4 (cid:2)C).

Calcium depletion and A23187 treatment

Membrane fractions from cells expressing YFP-K-RasC14 and membrane fractions obtained after incubation of par- ticulate fractions from unstransfected cells and cytosol from K-Ras transfected cells were resuspended in 200 lL of buf- fer T containing 200 lgÆmL)1 BSA or 200 lgÆmL)1 trypsin (Try) and further incubated at 37 (cid:2)C for 1 h. Reactions were stopped by addition of 10% (w ⁄ v, final concentration) trichroloacetic acid. Proteins were then recovered by cen- trifugation at 13 000 g for 30 min at 4 (cid:2)C using a F-45-24- 11 rotor in a 5415R centrifuge (Eppendorf), resuspended in sample buffer and analyzed by western blot.

Electrophoresis and western blot

Twenty-four hours after transfection, cells were washed three times with extracellular solution [140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 10 mm glucose, 0.1% BSA, 15 mm Hepes pH 7.4, extracellular solution (ECS)] without calcium and then incubated for 1 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 incu- bated for 1 h with ECS without calcium (–Ca2+ treatment) or ECS containing 5 mm Ca2+ (+Ca2+ treatment) or 5 mm Ca2+ and 30 lm A23187 (Sigma Aldrich, +Ca2+ + A23187 treatment) or 5 mm Ca2+, 30 lm A23187, 10 lm BAPTA- AM 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 min at 20 (cid:2)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 pres- ence or absence of chelators) and a time series was then acquired during 60 min.

Ionophore treatment

Proteins were resolved by electrophoresis through 12% (w ⁄ v) SDS ⁄ PAGE gels under denaturing conditions and for then electroblotted onto nitrocellulose membranes 80 min at 300 mA. Protein bands in nitrocellulose mem- branes were visualized by Ponceau staining. For immuno- blotting, nonspecific binding sites on the nitrocellulose membrane were blocked with 5% (w ⁄ v) nonfat dry milk in (200 mm NaCl, 50 mm Tris ⁄ HCl, Tris-buffered saline pH 7.5). Anti-GFP polyclonal IgGIj (Roche Diagnostics, Indianapolis, IN) was used at a dilution of 1 : 1000. Bands were detected by protein A coupled to horseradish per- oxidase combined with the chemioluminiscence detection kit (SuperSignal(cid:3) West Pico Chemioluminiscent Substrate, Pierce, Rockford, IL) and Hyperfilm MP films (GE Health- care, 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

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Twenty-four hours after transfection, cells were incubated for 2 h at 37 (cid:2)C in DMEM containing 1.5 or 3 UÆmL)1 neuraminidase (NANase) type V from Clostridium perfrin- Stock solution of valinomycin (1.25 mm) was prepared in dimethylsulfoxide. A stock solution of 5 mm monensin was prepared in ethanol. Cells transiently expressing the qui- meric proteins were washed twice with DMEM and incu- bated for 15 min 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, 5 mm Hepes pH 7.2) and then incubated for 20 min in the same media containing 10 lm valinomycin.

G. A. Gomez and J. L. Daniotti

Membrane targeting of K-Ras

Live cell imaging

Live cells experiments were performed at 20 (cid:2)C on a Carl Zeiss LSM5 Pascal laser scanning confocal microscope (Carl Zeiss AG, Go¨ ttingen, Germany) or an Olympus 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 min.

(PIP 5151); Fundacio´ n Antorchas (14116-112) and Agencia Nacional de Promocio´ n Cientı´ fica y Tecnolo´ g- ica (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 Cieˆ ncias Ba´ sicas da Sau´ de, Porto Alegre, Brazil) for his help in preliminary bio- chemical experiments. GAG is the recipient of CONI- CET 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.

Endocytosis of Alexa647-conjugated transferrin and MitoTracker staining

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The following supplementary material online: Fig. S1. Subcellular distribution of K-Ras. Fig. S2. Topological distribution of K-Ras in mem- branes from CHO-K1 cells. Fig. S3. Effect of ATP depletion on PIP2 content and PS externalization in CHO-K1 cells. Fig. S4. Subcellular distribution of A23187 and its effect on H+ homeostasis, PS externalization and PIP2 content in CHO-K1 cells. Video S1. Ca2+ influx causes K-Ras, but not GPI anchored protein, to redistribute from plasma mem- brane to the endomembrane system.

This material is available as part of the online article

from http://www.blackwell-synergy.com

78 Smalley KS, Koenig JA, Feniuk W & Humphrey PP (2001) Ligand internalization and recycling by human recombinant somatostatin type 4 h sst (4) receptors expressed in CHO-K1 cells. Br J Pharmacol 132, 1102–1110. 79 Troyanovsky RB, Sokolov EP & Troyanovsky SM

(2006) Endocytosis of cadherin from intracellular junc- tions is the driving force for cadherin adhesive dimer disassembly. Mol Biol Cell 17, 3484–3493.

Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corres- ponding author for the article.

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