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 gr
Trang 1subcellular 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: 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
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.
Trang 2variable 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
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 subcelluintracellu-lar 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 memmem-brane 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,
Trang 3colocalizing 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-Rasfull and K-RasC14) fused to green fluorescent
protein (GFP) and to its spectral variants, cyan
fluor-escent protein (CFP) and yellow fluorfluor-escent 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-Rasfullas a band of 55 kDa
according to the expected molecular mass (Fig 1A)
Membrane association of the expressed fusion proteins
was investigated by ultracentrifugation of extracts
from mechanically lysed cells YFP-K-RasC14 and
YFP-K-Rasfullwere 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-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
To characterize expression of these proteins 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 them mostly in cytosol (Cyt > PM) (Fig 1D) The phenotype Cyt > PM does not exclude the presence of K-Ras in plasma membrane, but the cytosolic concentration of K-Ras in this phenotype is higher than the others CFP-K-RasC14 and YFP-K-Rasfullwere 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-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
Trang 4of recycling endosomes, in 10% of transfected
CHO-K1 cells [36] (Fig 2; K-RasC14-perinuclear)
Similar subcellular distributions were observed for the
full-length version of K-Ras (data not shown) In
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
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 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
D
E
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-RasC14or YFP-K-Rasfullwere 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-RasC14(pseudocolored green) Right panel is a merged image from YFP-K-Rasfulland CFP-K-RasC14 Scale bars ¼ 20 lm.
Trang 5contain 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-RasC14 or
YFP-K-Rasfull were incubated in solutions with
increasing concentration of poly l-lysine, significant
dissociation of the expressed proteins was observed at
higher concentrations (Fig 3Ai) In contrast, no
signi-ficant change in the amount of CFP-H-RasC20
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-RasC14 and YFP-K-Rasfull ( 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-RasC20 was observed at low ionic strength, but was insignificant at high ionic strength
Ca2+ is a central second messenger having a higher affinity for anionic than zwitterionic and neutral phospholipids [39] Ca2+also promotes the formation
of lateral domains of phosphatidylserine (PS) in 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
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-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.
Trang 6effect of increasing CaCl2 concentration on membrane
affinity of K-Ras The results (Fig 3Aiii) show that
both YFP-K-RasC14and YFP-K-Rasfullare 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-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
Having demonstrated that K-Ras membrane associ-ation depends on electrostatic interaction, we analyzed
in vitro the reversibility of such interaction Cytosolic
ii
iii
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)1poly L -lysine (i)
or 0, 3 · 10)6, 1.5 · 10)4, 3 · 10)2, 15 · 10)2and 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-RasC14or YFP-K-Rasfullwere 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)1proteinase 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-RasC14expressing 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-RasC14 input Proteinase K activity was monitored by measuring the degradation of a-tubulin present in total CHO-K1 extracts (lower panel).
Trang 7and 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 4C 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-RasC14and K-Rasfullwas 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-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
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 disrupdistribu-tion
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-RasC14, but not H-RasC20, 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-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
Trang 8(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
Calcium ionophore redistributes K-Ras
to endomembrane
In vitroexperiments 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+
[51] A23187 forms a stable complex with Ca2+which
is membrane permeable (see subcellular distribution in Fig S4) Within the cell, Ca2+ ions are replaced by
H+, and the protonated form of the ionophore is externalized [52,53] A23187 thus functions as a
Ca2+⁄ H+exchanger, and reduces both Ca2+and H+ diffusion potentials
Calcium affects membrane surface potential shielding negative charges of plasma membrane, stimulating PI hydrolysis and PS ‘flipping out’ in a Ca2+-scramblase dependent fashion (Fig S4) [25,40,42,54,55] Following treatment of YFP-K-RasC14-expressing CHO-K1 cells with A23187, live cell confocal microscopy showed a clear dissociation of this protein from plasma membrane (Fig 6A and Video S1) Perinuclear and scattered struc-tures were also decorated with K-Ras Similar redistri-bution was observed for full-length K-Ras fused to YFP
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)1NANase 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)1NANase Cells are
representative of PM > Cyt (control) and
perinuclear (NANase) phenotypes for K-Ras
subcellular distribution (left) Frequency of
phenotypes (%) showing YFP-K-RasC14and
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
(N52Gal-T2, red) orN27GalNAc-T-CFP (N27
Gal-NAc-T, red) or cells expressing YFP-K-Ras C14
(K-Ras C14 , green) and labeled with
Alex-a647-Tf (Tf; red) were treated (NANase) or
not (control) with 1.5 UÆmL)1NANase 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).
Trang 9(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)
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 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 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
absence of chelators, indicating that very low levels of extracellular calcium are sufficient to alter subcellular distribution of YFP-K-RasC14
Increase in cytosolic Ca2+ can cause PKC activa-tion and consequent K-Ras phosphorylaactiva-tion [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
To further characterize the subcellular distribution
of YFP-K-RasC14under 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-RasC14
or YFP-H-Ras C20 were incubated for 1 h in DMEM without glucose containing 50 m M
2-deoxiglucose and 5 m M NaN 3 (–ATP) or
50 m M D -(+)-glucose and vehicle (control) and visualized alive at 20 C by confocal microscopy (left) Frequency of pheno-types (%) showing YFP-K-RasC14and YFP-H-Ras C20 subcellular distribution (right) Scale bars: 10 lm (B) CHO-K1 cells coex-pressing YFP-K-RasC14(K-RasC14; green) and
N52 Gal-T2-CFP ( N52 Gal-T2; red) or N27 GalNAc-T-CFP ( N27 GalNAc-T; red) or cells expressing YFP-K-RasC14(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.
Trang 10endogenous resident proteins (data not shown) K-Ras
was colocalized to a minor extent with lip33-YFP,
an ER marker, in Ca2+-depleted cells and Ca2+ +
A23187 treated cells (Fig 6D) Similar results were
obtained in Ca2+ and Ca2+ + A23187 + chelator
treated cells (data not shown) YFP-K-RasC14was
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
a redistribution of YFP-K-RasC14 from plasma membrane to the endomembrane system, according probably to their physical and chemical properties
Change in intracellular pH does not affect K-Ras subcellular distribution
Because ionophore A23187 operates as a Ca2+⁄ H+
exchanger (see above), its observed effect on K-Ras distribution could conceivably result from modification
of not only calcium homeostasis but also intracellular
pH To test this possibility, we abolished pH gradients across the endomembrane system using the polyether ionophore monensin (a Na+⁄ H+ exchanger), and
C
D
Fig 6 Ca2+ 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-RasC14were Ca2+depleted and incubated for 1 h in media without Ca2+(–Ca2+) or containing 5 m M Ca2+(+Ca2+)
or 5 m M Ca 2+ and 30 l M A23187 (+Ca 2+ + A23187) or 5 m M Ca 2+ , 30 l M A23187, 10 l M BAPTA-AM and 10 m M 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-RasC14 expression (%) (C) Homogenates from cells expressing K-RasC14 were treated as described in (B), lysed and ultracentrifugated The supernatant (S) was recovered and the particulate fraction (P) resuspended in lysis buffer YFP-K-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 (N27GalNAc-T) or cells expressing YFP-K-RasC14and 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-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).