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

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:
(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. Ca
2+
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.
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
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].
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].
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-
nisms of intracellular transport and distribution 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-
quent transport to plasma membrane through
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
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
sufficient to redistribute this protein to endomem-
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.
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
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 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-
tion 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 signifi-
cant accumulation of K-Ras in a perinuclear region,
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2211
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
Constructs expressing full-length and C-terminal
(KKKKKKSKTKCVIM) domain of human K-Ras
(K-Ras
full
and K-Ras
C14
) 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-Ras
C14
as bands of 27 kDa and 27.5 kDa,
respectively, and YFP-K-Ras
full
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-Ras
C14
and
YFP-K-Ras
full
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

YFP-K-Ras
C14
and YFP-K-Ras
full
, 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.
To characterize expression of these proteins in
CHO-K1 cells, subcellular distribution of YFP-K-
Ras
C14
and YFP-K-Ras
full
was analyzed by confocal
microscopy. Detailed phenotypic analysis showed that
43% and 49% of CHO-K1 cells expressed K-Ras
C14
and K-Ras
full
, 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 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-Ras
C14

and YFP-K-
Ras
full
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.
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. 2 and Fig. S1). No colocalization was
observed between YFP-K-Ras
C14
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-Ras
C14
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
N27
GalNAc-T-CFP (
N27
GalNAc-T),
a trans Golgi network (TGN) resident protein in
CHO-K1 cells. YFP-K-Ras
C14
was colocalized with
endocytosed Alexa
647
-human transferrin (Tf), a marker
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2212 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
of recycling endosomes, in  10% of transfected
CHO-K1 cells [36] (Fig. 2; K-Ras
C14
-perinuclear).
Similar subcellular distributions were observed for the
full-length version of K-Ras (data not shown). In sum-
mary, YFP-K-Ras
C14
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 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-
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
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 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 CaCl
2
) on membrane association of
K-Ras. We also investigated effects of these factors
on membrane binding properties of CFP-H-Ras
C20
[16], which is dually palmitoylated and does not
A

D
E
BC
Fig. 1. Protein expression and subcellular localization of YFP-K-Ras
C14
and YFP-K-Ras
full
in CHO-K1 cells. (A) Homogenates from CHO-K1 cells
expressing YFP, YFP-K-Ras
C14
or YFP-K-Ras
full
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-Ras
C14
or YFP-K-Ras
full
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-Ras
C14
or YFP-K-Ras
full
were
incubated with 1% (v ⁄ v) Triton X-114 for 1 h. Then, samples were incubated at 37 °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-
Ras
C14

or YFP-K-Ras
full
were fixed with paraformaldehyde and visualized by confocal microscopy. Left, representative cell phenotypes show-
ing YFP-KRas
C14
subcellular distribution. Right, frequency of phenotypes (%) showing YFP-K-Ras
C14
and YFP-K-Ras
full
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-Ras
full
(pseudocolored red) and CFP-K-Ras
C14
(pseudocolored green). Right panel is a merged image from YFP-K-Ras
full
and CFP-K-Ras
C14
. Scale
bars ¼ 20 lm.
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2213
contain a polybasic domain, and of GPI-YFP, a
fluorescent protein containing a glycosylphosphatidy-
linositol (GPI) attachment signal. When membrane
fractions from cells expressing YFP-K-Ras
C14
or
YFP-K-Ras
full

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-Ras
C20
associ-
ated with particulate fraction was observed under the
same conditions.
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 experiments in the
presence of increasing concentrations of NaCl (Fig. 3-
Aii). A significant membrane dissociation of both
YFP-K-Ras
C14
and YFP-K-Ras
full
( 45%) was
observed at 1.5 m NaCl, in accordance with the elec-
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-Ras
C20
was
observed at low ionic strength, but was insignificant at
high ionic strength.
Ca

2+
is a central second messenger having a higher
affinity for anionic than zwitterionic and neutral
phospholipids [39]. Ca
2+
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 Ca
2+
⁄ 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
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-Ras
C14
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;
N27
GalNAc-T-CFP (
N27
GalNAc-T), a TGN marker or incubated with
MitoTracker or Alexa
647
-Tf (Tf) and then fixed. The expression of YFP-K-Ras
C14
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-Ras
C14
and the corres-
ponding organelle marker. Cells shown in this figure correspond to the PM > Cyt phenotype of subcellular distribution of YFP-Ras
C14
, 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.
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2214 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
effect of increasing CaCl
2
concentration on membrane
affinity of K-Ras. The results (Fig. 3Aiii) show that
both YFP-K-Ras
C14
and YFP-K-Ras
full
are dissociated
from membrane at high Ca
2+
concentration (0.5 m).
However, the degree of this dissociation is not signifi-
cantly different from that observed for NaCl, suggest-
ing a nonspecific effect of Ca
2+
on K-Ras membrane
affinity. CFP-H-Ras
C20
and GPI-YFP were not signifi-

cantly dissociated under the same conditions.
Having demonstrated that K-Ras membrane associ-
ation depends on electrostatic interaction, we analyzed
in vitro the reversibility of such interaction. Cytosolic
A
i
ii
iii
BC
Fig. 3. Membrane binding properties of K-Ras. (A) Membrane fractions of CHO-K1 cells expressing YFP-K-Ras
C14
or YFP-K-Ras
full
or CFP-H-
Ras
C20
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 CaCl
2
(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-Ras
C14
or YFP-K-Ras
full
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-Ras
C14
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-Ras
C14
expression in W1, W2 and P fractions was analyzed by western blot. Right lane shows 30% of the
cytosolic YFP-K-Ras
C14

input. Proteinase K activity was monitored by measuring the degradation of a-tubulin present in total CHO-K1
extracts (lower panel).
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2215
and particulate fractions were prepared from cells
expressing YFP, YFP-K-Ras
C14
, and YFP-K-Ras
full
,
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 °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,  50% of sol-
uble K-Ras
C14
and K-Ras
full
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.
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-Ras
C14
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
Biochemical studies as above demonstrate that mem-
brane binding properties of K-Ras are due to elec-
trostatic and reversible 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.
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
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:-
GA2 ⁄ G2 ⁄ GD2 ⁄ GT2 galactosyltransferase (Gal-T2)
glycosyltransferases [46] (Fig. 4A). Live cell imaging
analysis showed that neuraminidase treatment incre-
ased K-Ras
C14
, but not H-Ras
C20
, expression in a peri-
nuclear 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 peri-
nuclear 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. 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
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-Ras
C14
, but not H-Ras
C20
, in a perinuclear
region (Fig. 5A). To identify the perinuclear organelle
in which YFP-K-Ras
C14
localized in ATP-depleted
CHO-K1 cells, we performed colocalization experi-

ments with a TGN marker (
N27
GalNAc-T) and endo-
cytosed human Alexa
647
-Tf, a recycling endosome
marker [50]. We observed colocalization of YFP-K-
Ras
C14
with endocytosed Tf, and with TGN marker,
in ATP-depleted cells (Fig. 5B). No colocalization
was observed between K-Ras and
N52
Gal-T2-CFP
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2216 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
(
N52
Gal-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.
Calcium ionophore redistributes K-Ras
to endomembrane
In vitro experiments in this study and others have dem-
onstrated that binding of lipid modified cationic pep-
tides, YFP-K-Ras
C14
and YFP-K-Ras
full

, 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 Ca
2+
and to a minor degree for Mg
2+
[51]. A23187 forms a stable complex with Ca
2+
which
is membrane permeable (see subcellular distribution in
Fig. S4). Within the cell, Ca
2+
ions are replaced by
H
+
, and the protonated form of the ionophore is
externalized [52,53]. A23187 thus functions as a
Ca
2+
⁄ H
+
exchanger, and reduces both Ca
2+
and H
+
diffusion potentials.

Calcium affects membrane surface potential shielding
negative charges of plasma membrane, stimulating PI
hydrolysis and PS ‘flipping out’ in a Ca
2+
-scramblase
dependent fashion (Fig. S4) [25,40,42,54,55]. Following
treatment of YFP-K-Ras
C14
-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
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 °C. Then, cells were shifted to
4 °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-Ras
C14
and YFP-H-Ras
C20
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-Ras
C14
and
YFP-H-Ras
C20
subcellular distribution (right).
(C) CHO-K1 cells coexpressing YFP-K-Ras
C14
(K-Ras
C14
, green) and
N52
Gal-T2-CFP
(
N52
Gal-T2, red) or

N27
GalNAc-T-CFP (
N27
Gal-
NAc-T, red) or cells expressing YFP-K-Ras
C14
(K-Ras
C14
, green) and labeled with Alex-
a
647
-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-Ras
C14
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).
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2217
(data not shown). In contrast, YFP-H-Ras
C20
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).
Ionophore A23187 is membrane permeable and
could potentially alter intracellular calcium reservoirs.
We evaluated its effect on subcellular distribution of
YFP-K-Ras
C14
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 ester (BAPTA-AM; a permeable 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 cytosolic distribution of K-Ras (Fig. 6B).
Restoring of Ca
2+
and addition of A23187 to medium
caused an increase of cells with cytosolic distribution
of YFP-K-Ras
C14
(Fig. 6B). Addition of calcium che-
lators together with Ca
2+
and A23187 produced the
same phenotypic distribution as observed in the
absence of chelators, indicating that very low levels of
extracellular calcium are sufficient to alter subcellular

distribution of YFP-K-Ras
C14
.
Increase in cytosolic Ca
2+
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-Ras
C14
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.
To further characterize the subcellular distribution
of YFP-K-Ras
C14
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-

dent for both ectopically expressed markers and
A
B
Fig. 5. ATP depletion redistributes K-Ras to
recycling endosomes and Golgi membranes.
(A) CHO-K1 cells expressing YFP-K-Ras
C14
or YFP-H-Ras
C20
were incubated for 1 h in
DMEM without glucose containing 50 m
M
2-deoxiglucose and 5 mM NaN
3
(–ATP) or
50 m
MD-(+)-glucose and vehicle (control)
and visualized alive at 20 °C by confocal
microscopy (left). Frequency of pheno-
types (%) showing YFP-K-Ras
C14
and
YFP-H-Ras
C20
subcellular distribution (right).
Scale bars: 10 lm. (B) CHO-K1 cells coex-
pressing YFP-K-Ras
C14
(K-Ras
C14

; green) and
N52
Gal-T2-CFP (
N52
Gal-T2; red) or
N27
GalNAc-
T-CFP (
N27
GalNAc-T; red) or cells expressing
YFP-K-Ras
C14
(green) and labeled with
Alexa
647
-Tf (Tf; red) were treated as des-
cribed above, fixed and visualized by confo-
cal microscopy. Panels are merged images
from YFP-K-Ras
C14
and the corresponding
organelle marker. The insets show details of
the boxed area at higher magnification.
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2218 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
endogenous resident proteins (data not shown). K-Ras
was colocalized to a minor extent with lip33-YFP,
an ER marker, in Ca
2+
-depleted cells and Ca

2+
+
A23187 treated cells (Fig. 6D). Similar results were
obtained in Ca
2+
and Ca
2+
+ A23187 + chelator
treated cells (data not shown). YFP-K-Ras
C14
was par-
tially colocalized with
N52
GalT2-CFP, a cis ⁄ medial
Golgi marker, and with
N27
GalNAcT-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-Ras
C14
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
a redistribution of YFP-K-Ras
C14
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 Ca
2+
⁄ 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
AB
C
D
-Ca
2+
+Ca
2+
+A23187
Fig. 6. Ca
2+
influx causes K-Ras to redistribute from plasma membrane to the endomembrane system. (A) CHO-K1 cells expressing
YFP-K-Ras

C14
or YFP-H-Ras
C20
were incubated in DMEM at 20 °C on the microscope stage and imaged (pretreatment). Then, cells were
incubated with 30 l
M A23187 and a time series was acquired. Images obtained at 5 min after A23187 addition is shown. (B) CHO-K1
cells expressing YFP-K-Ras
C14
were Ca
2+
depleted and incubated for 1 h in media without Ca
2+
(–Ca
2+
) or containing 5 mM Ca
2+
(+Ca
2+
)
or 5 m
M Ca
2+
and 30 lM A23187 (+Ca
2+
+ A23187) or 5 mM Ca
2+
,30lM A23187, 10 lM BAPTA-AM and 10 mM EGTA (+Ca
2+
+ 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-Ras
C14
expression (%). (C) Homogenates from cells expressing K-Ras
C14
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-Ras
C14
expression was investigated by western blot. The percentage of YFP-K-Ras
C14
associated to P fraction is indicated. (D)
CHO-K1 cells coexpressing CFP-K-Ras
C14
(K-Ras
C14
) and lip33-YFP (lip33) or YFP-K-Ras
C14
and
N52
Gal-T2-CFP (
N52
Gal-T2) or
N27
GalNAc-T-
CFP (
N27
GalNAc-T) or cells expressing YFP-K-Ras
C14
and labeled with MitoTracker or endocyted Alexa
647

-Tf (Tf) were treated as described
in (B), fixed and visualized by confocal microscopy. Panels are merged images from K-Ras
C14
(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).
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2219
observed the effect on subcellular distribution of YFP-
K-Ras
C14
. Because the exchange of electrolytes is 1 : 1,
monensin alters pH gradient but not transmembrane
potential [56]. When YFP-K-Ras
C14
-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).
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-Ras
C14
showed a rapid and significant accumu-
lation (10% increment) in a perinuclear compartment
defined as recycling endosome by colocalization with
endocytosed Alexa
647
-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.
Discussion
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 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 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 [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
A

B
Fig. 7. pH gradients does not affect subcellular distribution of
K-Ras. (A) CHO-K1 cells transiently expressing YFP-K-Ras
C14
(upper
panels) and YFP-H-Ras
C20
(middle panels) were incubated with
10 l
M monensin (Monensin) or vehicle (Control) for 30 min 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-Ras
C14
subcellular distribution both in control and monensin
treated cells. Scale bars ¼ 20 lm.
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2220 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
potential regulates lateral distribution of K-Ras after it
binds to membrane.
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
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 envi-
ronmental 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 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
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 °C in DMEM and dimethylsulfoxide (5 m

M 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 l M 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-Ras
C14
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-Ras
C14
subcellular distribution in
cells incubated in 5 m
M KCl or 5 mM KCl ⁄ 10 lM valinomicyn or 55 mM KCl ⁄ 10 lM valinomicyn. (B) CHO-K1 cells coexpressing YFP-K-Ras
C14
(K-Ras
C14
; green) and
N27
GalNAc-T-CFP (
N27
Gal-NAc-T; red) or cells expressing YFP-K-Ras
C14
and labeled with Alexa
647
-Tf (Tf; red) were incu-
bated for 20 min at 20 °C in Locke’s media and dimethylsulfoxide (55 m
M 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-Ras
C14
and the corresponding organ-
elle marker. The insets show details of the boxed area at higher magnification. Scale bars ¼ 10 lm.
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2221
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 Ca
2+
⁄ calmo-
dulin [43,77]. These authors suggest that destabilization
of K-Ras in plasma membrane by Ca
2+
⁄ 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 Ca
2+
but in the absence

of calmodulin with not significant differences from
results from NaCl experiments, suggesting an unspecific
effect of Ca
2+
on membrane affinity of K-Ras.
A significant proportion of ectopically expressed
YFP-K-Ras
C14
and YFP-K-Ras
full
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-Ras
C14
and K-Ras
full
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 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.
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-
brane than in intracellular membranes (plasma
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. Ca
2+
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
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2222 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
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.
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.
Experimental procedures
Plasmids
Expression plasmids for yellow fluorescent protein (YFP)-
K-Ras
C14
and YFP-H-Ras
C20
,
N27
GalNAc-T-CFP and
N52
Gal-T2-CFP have been described previously [16,82].
Plasmid encoding YFP-K-Ras
full
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 Gal-

T2 tagged at the C-terminal with the hemagglutinin (HA)
epitope (YPYDVPDYA). Cells were maintained at 37 °C,
5% CO
2,
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
Red MitoTracker (Molecular Probes, Eugene, OR) or
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 Na
2
HPO
4
, 1.6 mm NaH
2
PO
4
,
pH 7.5; NaCl ⁄ P
i
) and harvested by scraping or fixed for
microscopy.
Subcellular fractionation
Cells were washed with cold NaCl ⁄ P
i

and harvested by scra-
ping in 5 mm Tris ⁄ HCl (pH 7.0) (buffer T). Extracts were
centrifuged at 4 °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
of 5 lgÆmL
)1
aprotinin, 0.5 lgÆmL
)1
leupeptin and
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 °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 °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.
Triton X-114 partition assay
P fractions were solubilized for 1 h at 4 °C in 1% Triton
X-114 in NaCl ⁄ P
i
-PIM. Then, samples were incubated at
37 °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.
Poly l-lysine, NaCl and CaCl
2
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 1 h in buffer T supplemented with different con-
centrations of electrolytes (poly l-lysine, NaCl and CaCl
2
).
After incubation samples were centrifuged at 4 °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
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
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras

FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2223
(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 °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 °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
Membrane fractions from cells expressing YFP-K-Ras
C14
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 °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 °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

Proteins were resolved by electrophoresis through 12%
(w ⁄ v) SDS ⁄ PAGE gels under denaturing conditions and
then electroblotted onto nitrocellulose membranes for
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
Tris-buffered saline (200 m m NaCl, 50 mm Tris ⁄ HCl,
pH 7.5). Anti-GFP polyclonal IgG
Ij
(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 (SuperSignalÒ 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
Twenty-four hours after transfection, cells were incubated
for 2 h at 37 °C in DMEM containing 1.5 or 3 UÆmL
)1
neuraminidase (NANase) type V from Clostridium perfrin-
gens (Sigma Aldrich, St Louis, MO) or vehicle (control).
Then, cells were directly visualized or washed with cold
NaCl ⁄ P

i
and fixed in 3% (v ⁄ v) paraformaldehyde (30 min
at 4 °C).
ATP depletion treatment
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 NaN
3
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 ⁄ P
i
and fixed in 3% (v ⁄ v) paraformalde-
hyde (30 min at 4 °C).
Calcium depletion and A23187 treatment
Twenty-four hours after transfection, cells were washed three
times with extracellular solution [140 mm NaCl, 5 mm KCl,
1mm MgCl
2
,10mm 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 (–Ca
2+
treatment)

or ECS containing 5 mm Ca
2+
(+Ca
2+
treatment) or 5 mm
Ca
2+
and 30 lm A23187 (Sigma Aldrich, +Ca
2+
+ A23187
treatment) or 5 mm Ca
2+
,30lm A23187, 10 lm BAPTA-
AM and 10 mm EGTA (+Ca
2+
+ A23187 + Chel). Then,
cells were washed in NaCl ⁄ P
i
and fixed for visualization by
fluorescence microscopy.
For live cells experiments, cells were incubated in
DMEM for 20 min 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 pres-
ence or absence of chelators) and a time series was then
acquired during 60 min.
Ionophore treatment
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 NaHCO
3
, 1.8 mm CaCl
2
,5mm Hepes pH 7.2) and
then incubated for 20 min in the same media containing
10 lm valinomycin.
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2224 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
Live cell imaging
Live cells experiments were performed at 20 °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.
Endocytosis of Alexa

647
-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 1 h in a CO
2
incubator and then labeled in identi-
cal conditions with 14 lgÆmL
)1
Alexa
647
-human transferrin
(Molecular Probes) during 30 min before and during the
addition of NANase or 2-deoxi-d-glucose ⁄ NaN
3
or ionoph-
ores for the indicated times. After treatment, cells were
washed three times with NaCl ⁄ P
i
, and fixed for fluorescence
microscopy. For MitoTracker staining, cells were incubated
during 10 min with 4 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 wave-

lengths and filter set for CFP, GFP, YFP and Alexa
647
were described previously [16]. MitoTracker fluorescence
was detected using the filters for rhodamine or Cy5. Ima-
ges 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 meta-
morph
Ò
4.5 (Molecular Devices, Sunnyvale, CA) software
using the threshold function. Statistical significance (P)
(t-student test) was performed from at least three independ-
ent experiments (600 cells per experiment). Asterisks repre-
sent P < 0.1 versus control conditions.
Acknowledgements
This work was supported in part by grants from Secre-
tarı
´
a de Ciencia y Tecnologı
´
a, Universidad Nacional de
Co
´
rdoba (162 ⁄ 06); Consejo Nacional de Investigaci-
ones Cientı
´

ficas y Te
´
cnicas (CONICET), Argentina
(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.
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Supplementary material
The following supplementary material is available
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. Ca
2+
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
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.
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2228 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS