Tài liệu Báo cáo khoa học: Specific membrane binding of the carboxypeptidase Y inhibitor IC, a phosphatidylethanolamine-binding protein family member doc

Specific membrane binding of the carboxypeptidase Y
inhibitor I
C
, a phosphatidylethanolamine-binding protein
family member
Joji Mima*, Hiroaki Fukada, Mitsuru Nagayama and Mitsuyoshi Ueda
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan
Endogenous protein inhibitors of lysosomal ⁄ vacuolar
proteases are found in the cytoplasm of various euk-
aryotic organisms, from microorganisms to mammals.
Lysosomal ⁄ vacuolar proteases are responsible for the
majority of intracellular protein degradation and turn-
over, but no definitive information on the physio-
logical roles of cytoplasmic inhibitors has been
reported. I
C
, carboxypeptidase Y (CPY) inhibitor, was
isolated as an endogenous cytoplasmic inhibitor of
vacuolar CPY in the yeast Saccharomyces cerevisiae
[1–3]. Recent biochemical and mutational studies of I
C
[4–8] and the crystal structure of the complex of I
C
with CPY (I
C
–CPY) [8,9] have provided information
on the nature of the inhibition. The N-terminal acetyl
group of I
C
is essential for inhibitory function, and
the inhibitor forms an equimolecular complex with

the cognate protease through dual binding sites, an
N-terminal inhibitory reactive site and a secondary
Keywords
I
C
; membrane binding; PEBP;
phosphatidylserine; phosphoinositide
Correspondence
J. Mima, Division of Applied Life Sciences,
Graduate School of Agriculture,
Kyoto University, Kitashirakawa, Sakyo-ku,
Kyoto 606-8502, Japan
Fax: +81 75 753 6112
Tel: +81 75 753 6125
E-mail:
*Present address
Department of Biochemistry, Dartmouth
Medical School, Hanover, NH, USA
(Received 7 July 2006, revised 4 October
2006, accepted 9 October 2006)
doi:10.1111/j.1742-4658.2006.05530.x
I
C
, an endogenous cytoplasmic inhibitor of vacuolar carboxypeptidase Y in
the yeast Saccharomyces cerevisiae, is classified as a member of the phos-
phatidylethanolamine-binding protein family. The binding of I
C
to phos-
pholipid membranes was first analyzed using a liposome-binding assay and
by surface plasmon resonance measurements, which revealed that the affin-

ity of this inhibitor was not for phosphatidylethanolamine but for anionic
phospholipids, such as phosphatidylserine, phosphatidylinositol 3-phos-
phate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol
3,4,5-trisphosphate, with K
D
values below 100 nm. The liposome-binding
assay and surface plasmon resonance analyses of I
C
, when complexed with
carboxypeptidase Y, and the mutant forms of I
C
further suggest that the
N-terminal segment (Met1–His18) in its carboxypeptidase Y-binding sites
is involved in the specific and efficient binding to anionic phospholipid
membranes. The binding of I
C
to cellular membranes was subsequently
analyzed by fluorescence microscopy of yeast cells producing the green
fluorescent protein-tagged I
C
, suggesting that I
C
is specifically targeted to
vacuolar membranes rather than cytoplasmic membranes, during the sta-
tionary growth phase. The present findings provide novel insights into the
membrane-targeting and biological functions of I
C
and phosphatidyletha-
nolamine-binding proteins.
Abbreviations

CPY, carboxypeptidase Y; FM4-64, N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide; GFP, green
fluorescent protein; I
C
, carboxypeptidase Y inhibitor; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEBP, phosphatidyl-
ethanolamine-binding protein; PG, phosphatidylglycerol; PS, phosphatidylserine; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol
3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(3,4)P
2
, phosphatidylinositol
3,4-bisphosphate; PtdIns(3,5)P
2
, phosphatidylinositol 3,5-bisphosphate; PtdIns(4,5)P
2
, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P
3
,
phosphatidylinositol 3,4,5-trisphosphate; SPR, surface plasmon resonance.
5374 FEBS Journal 273 (2006) 5374–5383 ª 2006 The Authors Journal compilation ª 2006 FEBS
CPY-binding site [6–8]. In addition to its function as a
protease inhibitor, it has also been shown that I
C
is
identical to Tfs1p [4], a multicopy suppressor of the
cdc25-1 mutant [10], and that it inhibits and interacts
with the y east Ras GTPase-activating protein, Ira2p [11].
The amino acid sequence of I
C
shows similarity to
sequences of, not other known protease inhibitors, but
rather members of the phosphatidylethanolamine-bind-
ing protein (PEBP) family, which is highly conserved

among many organisms, such as mammals, plants,
worms, and bacteria [4,12]. A variety of molecular
functions of PEBPs in mammals have been reported to
date, and include the association with phospholipids
and membranes [13–16], the inhibition of Raf1 kinase
[17,18], thrombin [19], and G-protein-coupled receptor
kinase 2 [20], and the N-terminal fragment serving as
the hippocampal cholinergic neurostimulating peptide
[21,22]. In plants, two homologs of PEBP from Arabid-
opsis thaliana, FT and TFL1, were identified as floral
regulators that may interact with FD, a bZIP tran-
scription factor [23–26]. The crystal structures of
PEBPs from several organisms, including the structure
of I
C
–CPY, have also been determined [8,27–32]. These
structures demonstrate that PEBPs contain two repre-
sentative structural features, a central b-sheet fold and
a conserved anion-binding site that may recognize
phosphate groups of phospholipids and ⁄ or phosphor-
ylated residues in potential binding partners [8,27–32],
whereas the molecular mechanisms for the putative
functions of PEBPs, except for CPY inhibition by I
C
[8], remain obscure.
In the present study, we report on a detailed study
of the membrane-binding mode of I
C
, a PEBP family
member. A liposome-binding assay and surface plas-

mon resonance (SPR) analysis indicate that I
C
specific-
ally binds to membranes containing anionic
phospholipids, rather than phosphatidylethanolamine
(PE). A cellular localization analysis of I
C
by fluores-
cence microscopy, using the green fluorescent protein
(GFP), subsequently revealed the localization of this
inhibitor at vacuolar membranes.
Results
Membrane-binding properties of I
C
In an attempt to detect and characterize the membrane
binding of I
C
, a member of the PEBP family, we first
performed a liposome-binding assay of this inhibitor
for the phosphatidylcholine (PC)-based liposomes
(Fig. 1). As shown in Fig. 1A,C, SDS ⁄ PAGE analysis
of the precipitates, which were mixtures of I
C
and lipo-
somes, indicated that considerably larger amounts of
this inhibitor were sedimented with phosphatidylserine
(PS) ⁄ PC and phosphatidylinositol (PtdIns)⁄ PC than
with PC and PE ⁄ PC. This experiment provided an esti-
mate of the affinity of binding of I
C

to phospholipid
membranes, and demonstrated that I
C
has an affinity
for anionic phospholipids such as PS and PtdIns,
rather than for zwitterionic phospholipids, such as PE
and PC. In addition to free I
C
,I
C
–CPY was subjected
to the binding assay with PS ⁄ PC and PtdIns ⁄ PC lipo-
somes. As shown in Fig. 1B,C, neither I
C
nor CPY in
I
C
–CPY was sedimented with these liposomes, indica-
ting that the affinity of I
C
for anionic phospholipids
disappeared upon complex formation with CPY.
A
B
C
Fig. 1. Liposome-binding assay for I
C
and I
C
–CPY. I

C
(A) or I
C
–CPY
(B), the final concentration of which was 2 l
M, was added to PC-
based liposomes (0.5 mgÆmL
)1
of PE ⁄ PC, PC, PS ⁄ PC, and
PtdIns ⁄ PC) in 20 m
M Hepes (pH 7.2) containing 0.15 M NaCl, and
the suspension was incubated at 30 °C for 1 h. After centrifugation
of the samples, proteins bound to liposomes were analyzed by
SDS ⁄ PAGE of the resulting pellets. (C) The amounts of I
C
in the
pellets. The amounts of I
C
were quantitated with the UN-SCAN-IT gel
program (Silk Scientific Corporation, Orem, UT) using the band of
purified I
C
(2 lg) as a standard control. Error bars indicate SD from
two or more determinations.
J. Mima et al. Membrane binding of I
C
FEBS Journal 273 (2006) 5374–5383 ª 2006 The Authors Journal compilation ª 2006 FEBS 5375
Therefore, these results suggest that the binding inter-
face for CPY in the I
C

molecule is involved in its spe-
cific binding to anionic phospholipid membranes.
To further quantitatively evaluate the affinity and spe-
cificity of I
C
for phospholipid membranes, we next per-
formed SPR measurements, using this inhibitor as an
analyte and a number of the PC-based liposomes as a
ligand immobilized on the sensor surface of the L1 chip
[33]. Representative sensorgrams for the binding of I
C
to
the phospholipid liposomes showed that the inhibitor
has an affinity not only for PS ⁄ PC and PtdIns ⁄ PC,
which had been determined by the liposome-binding
assay, but also for other anionic phospholipid
liposomes, including phosphatidylglycerol (PG) ⁄ PC,
phosphatidylinositol 3-phosphate [PtdIns(3)P] ⁄ PC, phos-
phatidylinositol 4-phosphate [PtdIns(4)P] ⁄ PC, phos -
phatidylinositol 3,4-bisphosphate [PtdIns(3,4)P
2
] ⁄ PC,
phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P
2
] ⁄ PC,
phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P
2
] ⁄
PC, and phosphatidylinositol 3,4,5-trisphosphate
[PtdIns(3,4,5)P

3
] ⁄ PC (Fig. 2). In contrast to the findings
with these liposomes, no binding was detected of I
C
to
the zwitterionic phospholipid liposomes PE ⁄ PC and PC,
or to one of the anionic phospholipid liposomes, phos-
phatidylinositol 5-phosphate [PtdIns(5)P] ⁄ PC (data not
shown). In accordance with the liposome-binding assay
with I
C
–CPY (Fig. 1B,C), SPR responses of the complex
could not be detected toward all the phospholipid lipo-
somes (Fig. 2). These SPR analyses, as well as the lipo-
some-binding assay, demonstrated that I
C
, when
complexed with CPY, loses its intrinsic affinity for ani-
onic phospholipid membranes, and that the CPY-bind-
ing sites of I
C
[8] may be responsible for its phospholipid
recognition.
Using SPR sensorgrams for various concentrations
(0.1–10 lm)ofI
C
, the membrane association rate con-
stants (k
a
), dissociation rate constants (k

d
), and equi-
librium dissociation constants (K
D
) for the interaction
between the protein and PC-based liposomes, except
for PE, PC, and PtdIns(5)P (Table 1), were deter-
mined. A comparison of the membrane-binding
parameters indicates that I
C
exhibits a broad specificity
for a wide variety of anionic phospholipid membranes
with K
D
values below 600 nm, but has a slightly higher
affinity for PS, PtdIns(3)P, PtdIns(3,4)P
2
, and
PtdIns(3,4,5)P
3
(K
D
values of 75–97 nm) than for
PtdIns, PG, and the other phosphoinositides (K
D
val-
ues of 200–550 nm) (Table 1). The lower affinity of I
C
for PtdIns and PG results mainly from the smaller
k

a
values, whereas the lower affinity for phosphoino-
sitides other than PtdIns(3)P, PtdIns(3,4)P
2
and
PtdIns(3,4,5)P
3
results from the higher k
d
values.
Recent SPR studies of membrane–protein interactions
have shown that k
a
and k
d
are influenced by nonspe-
cific electrostatic interactions and proximal specific
interactions, respectively [34,35]. Those findings there-
fore suggest that nonspecific electrostatic interactions
between the negatively charged head groups of the
phospholipids, which include the carboxyl group of PS
and the phosphoryl groups of phosphoinositides, and
Fig. 2. SPR sensorgrams for membrane binding of I
C
and I
C
–CPY.
I
C
(bold solid lines) or I

C
–CPY (solid lines), the concentration of
which was 4 l
M, was injected for 90 s over the surface of the L1
sensor chip coated with the phospholipid liposomes of PS ⁄ PC
(black), PtdIns(4,5)P
2
⁄ PC (green), PtdIns(3,4,5)P
3
⁄ PC (brown),
PG ⁄ PC (lime), PtdIns(3,5)P
2
⁄ PC (cyan), PtdIns(3)P ⁄ PC (yellow),
PtdIns(4)P ⁄ PC (blue), PtdIns(3,4)P
2
⁄ PC (pink), or PtdIns ⁄ PC (red).
All sensorgrams were obtained by SPR measurements in 20 m
M
Hepes (pH 7.2) containing 0.15 M NaCl at 30 °C, with a flow rate of
60 lLÆmin
)1
.
Table 1. Membrane-binding parameters for I
C
determined by SPR
analysis. Parameters represent mean ± SD from three or more
determinations. All SPR measurements were performed in 20 m
M
Hepes (pH 7.2) containing 0.15 M NaCl at 30 °C, with a flow rate of
60 llÆmin

)1
. PC-based liposomes (0.5 mgÆmL
)1
) were immobilized
on the L1 sensor chip. ND, not detectable.
Liposomes
k
a
(10
2
M
)1
Æs
)1
)
k
d
(10
)5
s
)1
)
K
D
(10
)9
M)
PE ⁄ PC ND ND ND
PC ND ND ND
PS ⁄ PC 80 ± 11 59 ± 3.9 75 ± 13

PtdIns ⁄ PC 34 ± 9.8 70 ± 18 230 ± 94
PG ⁄ PC 43 ± 2.4 85 ± 14 200 ± 24
PtdIns(3)P ⁄ PC 64 ± 23 60 ± 19 97 ± 26
PtdIns(4)P ⁄ PC 35 ± 12 180 ± 13 550 ± 190
PtdIns(5)P ⁄ PC ND ND ND
PtdIns(3,4)P
2
⁄ PC 68 ± 13 60 ± 14 88 ± 7.6
PtdIns(3,5)P
2
⁄ PC 68 ± 30 170 ± 42 280 ± 130
PtdIns(4,5)P
2
⁄ PC 45 ± 8.4 89 ± 8.4 210 ± 57
PtdIns(3,4,5)P
3
⁄ PC 67 ± 3.9 50 ± 3.8 75 ± 5.3
Membrane binding of I
C
J. Mima et al.
5376 FEBS Journal 273 (2006) 5374–5383 ª 2006 The Authors Journal compilation ª 2006 FEBS
the positively charged residues of I
C
initially attract
the inhibitor to the membrane surface, and that the
membrane–protein interactions are then further stabil-
ized by short-range specific interactions, resulting in
the higher affinity for PS, PtdIns(3)P, PtdIns(3,4)P
2
,

and PtdIns(3,4,5)P
3
.
Involvement of the CPY-binding sites of I
C
in its
membrane binding
As I
C
–CPY has no affinity for phospholipid membranes,
to obtain additional information on the involvement of
the CPY-binding sites of I
C
in its phospholipid recogni-
tion, we determined the membrane-binding parameters
for the mutant forms of I
C
, d1–7I
C
and d1–18I
C
, with
the N-terminal seven (Ac-MNQAIDF) and 18 (Ac-MN
QAIDFAQASIDSYKKH) residues, respectively, dele-
ted (Table 2). d1–7I
C
and d1–18I
C
lack the N-terminal
inhibitory reactive site (Ac-Met1–Phe7) [8] alone, and

both the N-terminal site and, in part, the secondary
CPY-binding site (Ala10–Gln70 and Phe133–Glu137)
[8], respectively. Prior to the SPR analyses, amino acid
sequencing, MS and CD spectroscopic analyses con-
firmed that the N-terminal residues were deleted in the
purified mutants of I
C
and that the mutant proteins were
correctly folded, forming the b-type gross structures
similar to the native protein (data not shown). SPR ana-
lyses of d1–7I
C
and d1–18I
C
showed that these mutants
of I
C
, as well as the native protein, were associated
with the anionic phospholipid liposomes of PS ⁄ PC,
PtdIns ⁄ PC and PG ⁄ PC, and also the liposomes contain-
ing phosphoinositides rather than zwitterionic liposomes
of PE ⁄ PC and PC (Table 2). However, the elimination
of the N-terminal residues significantly affects the bind-
ing parameters of I
C
with respect to these anionic
phospholipid liposomes. No binding of d1–7I
C
to
PtdIns(3)P ⁄ PC was detected, and the K

D
value of
d1–7I
C
binding to PtdIns(3,4)P
2
⁄ PC was increased
13-fold. For the other liposomes, the K
D
values of the
mutant were also increased more than four-fold over
those of the native protein (Table 2). In contrast to
those of d1–7I
C
, the K
D
value of d1–18I
C
for
PtdIns(3,4)P
2
⁄ PC was increased 2.4-fold, whereas the
K
D
values for PS ⁄ PC, PtdIns ⁄ PC, PG ⁄ PC and
PtdIns(3)P ⁄ PC were increased 4.0–4.7-fold, and that for
PtdIns(3,4,5)P
3
⁄ PC was increased 8.1-fold (Table 2).
These results demonstrate that the N-terminal segment

of I
C
(Ac-Met1–His18) is essential for its binding effi-
ciency and specificity for phospholipid membranes and
suggest that the phospholipid recognition site of I
C
is
composed of residues in and adjacent to this N-terminal
segment.
Association of I
C
with cellular membranes
To gain insights into the association of I
C
with cellular
membranes, we subsequently examined the intracellular
localization of the inhibitor by fluorescence microscopy
of living yeast cells producing I
C
–GFP (Fig. 3). The
yeast cells were also labeled with N-(3-triethylammoni-
umpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridi-
nium dibromide (FM4-64), a fluorescent dye used for
Table 2. Membrane-binding parameters for the mutant forms of I
C
with the N-terminal residues deleted, determined by SPR analysis.
Parameters represent mean ± SD from three or more determinations. All SPR measurements were performed in 20 m
M Hepes (pH 7.2)
containing 0.15
M NaCl at 30 °C, with a flow rate of 60 llÆmin

)1
. PC-based liposomes (0.5 mgÆmL
)1
) were immobilized on the L1 sensor
chip. Increase in K
D
, K
D
for d1–7I
C
or d1–18I
C
⁄ K
D
for I
C
. ND, not detectable.
Proteins Liposomes
k
a
(10
2
M
)1
Æs
)1
)
k
d
(10

)5
Æs
)1
)
K
D
(10
)9
M)
Increase in K
D
(fold)
d1–7I
C
PE ⁄ PC ND ND ND –
PC ND ND ND –
PS ⁄ PC 16 ± 3.7 56 ± 2.5 350 ± 93 4.7
PtdIns ⁄ PC 10 ± 1.6 220 ± 36 2200 ± 630 9.6
PG ⁄ PC 5.9 ± 0.44 91 ± 3.0 1600 ± 170 8.0
PtdIns(3)P ⁄ PC ND ND ND –
PtdIns(3,4)P
2
⁄ PC 15 ± 9.1 150 ± 66 1100 ± 230 13
PtdIns(3,4,5)P
3
⁄ PC 16 ± 0.71 65 ± 7.6 400 ± 30 5.3
d1–18I
C
PE ⁄ PC ND ND ND –
PC ND ND ND –

PS ⁄ PC 24 ± 1.1 82 ± 23 350 ± 110 4.7
PtdIns ⁄ PC 17 ± 5.3 150 ± 48 910 ± 290 4.0
PG ⁄ PC 18 ± 3.3 160 ± 4.0 890 ± 160 4.5
PtdIns(3)P ⁄ PC 46 ± 4.4 200 ± 14 430 ± 68 4.4
PtdIns(3,4)P
2
⁄ PC 49 ± 24 80 ± 10 200 ± 110 2.4
PtdIns(3,4,5)P
3
⁄ PC 25 ± 1.7 150 ± 20 610 ± 110 8.1
J. Mima et al. Membrane binding of I
C
FEBS Journal 273 (2006) 5374–5383 ª 2006 The Authors Journal compilation ª 2006 FEBS 5377
staining vacuolar membranes that was taken up by
endocytosis. A western blotting analysis using an anti-
body to GFP showed that the full-length protein of I
C
–
GFP was correctly produced in the yeast cells at com-
parable levels during both the logarithmic (12 h and
24 h) and stationary (48 h and 72 h) growth phases
(data not shown). The observed fluorescence of I
C
–
GFP was in the extravacuolar cytoplasmic fraction in
the logarithmic growth phase (the left panels of
Fig. 3A). However, in the stationary growth phase, the
fluorescence of I
C
–GFP was observed at the FM4-64-

stained vacuolar membranes and also the vacuolar
lumens in the majority of yeast cells (70% of the cells
grown at 72 h; right panels of Fig. 3A,B). Therefore,
the fluorescence microscopic analyses clearly demon-
strate that I
C
–GFP present in the cytoplasm during the
logarithmic growth phase was selectively relocalized at
the vacuolar membranes and lumens during the station-
ary phase.
Discussion
PEBP from bovine brain, a mammalian homolog of I
C
,
was originally isolated as a 23 kDa cytoplasmic protein
A
B
Fig. 3. Fluorescence microscopic analyses
of yeast cells producing I
C
–GFP. (A) Repre-
sentative fluorescence images. S. cerevisiae
BY4741icD cells producing I
C
–GFP were
labeled with the vacuolar membrane fluores-
cent dye FM4-64, and harvested at the log-
arithmic (12–24 h) and stationary (48–72 h)
growth phases. The localization of I
C

–GFP
and FM4-64 was visualized and compared
by fluorescence microscopy. (B) Quantitation
of intracellular localization of I
C
–GFP. Cells
(n > 100 ⁄ group) at the logarithmic and sta-
tionary phases were scored for the localiza-
tion of I
C
–GFP at the vacuolar membrane
and lumen or in the cytoplasm. Error bars
indicate SE.
Membrane binding of I
C
J. Mima et al.
5378 FEBS Journal 273 (2006) 5374–5383 ª 2006 The Authors Journal compilation ª 2006 FEBS
associated with PE [13,14]. The crystal structure of this
protein, complexed with phosphorylethanolamine, the
polar head group of PE, was also determined, and
the data suggest that a conserved anion-binding site at
the protein surface may correspond to the recognition
site of PE [28]. However, it was recently reported that
the bovine PEBP had an affinity, not for PE-containing
membranes, but rather for anionic membranes contain-
ing PG [16], and little information is available regard-
ing the phospholipid and membrane binding of the
other members of the PEBP family. Consequently, the
binding characteristics of PEBP proteins, including
those of the binding of I

C
to phospholipid membranes,
are currently unclear and remain to be clarified.
The present in vitro membrane-binding analyses of I
C
permitted the phospholipid specificity and phospholipid
recognition mode to be determined during its membrane
targeting. This inhibitor cannot bind to zwitterionic
phospholipids of PE and PC but shows an affinity for a
wide variety of anionic phospholipids, especially PS,
PtdIns(3)P, PtdIns(3,4)P
2
, and PtdIns(3,4,5)P
3
(Table 1). The two further findings that (a) I
C
–CPY
completely loses its ability to bind to membranes
(Figs 1B and 2) and (b) the removal of the N-terminal
residues (Ac-Met1–Phe7 or Ac-Met1–His18) affects
both the binding affinity and specificity (Table 2) clearly
suggest that the CPY-binding sites [8] and the phosphol-
ipid recognition site of I
C
overlap, and that the N-ter-
minal segment at the CPY-binding sites participates in
regulation of the specific binding of I
C
to the anionic
phospholipid membranes (Fig. 4A). The participation

of the N-terminal region of bovine PEBP in its mem-
brane binding was also suggested by the binding experi-
ments with a synthetic peptide corresponding to the
N-terminal 12 residues and model membranes [16]. On
the other hand, the binding specificity for anionic
phospholipids suggests that I
C
contains a positively
charged residue at the phospholipid recognition site
and is targeted to membranes through electrostatic
interactions between a positively charged residue and an
anionic head group of lipid molecules in membranes,
similar to the well-known membrane targeting domains
PH, FYVE, PX, ENTH, C1, and C2 [35–37]. Consider-
ing the present mutational studies on the membrane
binding of I
C
and the disposition of the I
C
residues that
are positively charged and make up the CPY-binding
sites (Fig. 4A,B), the basic residues in the vicinity of the
N-terminal segment, such as Lys16, Lys17, His18,
Lys101, and Arg162, could be candidates for a residue
that directly interacts with negatively charged groups of
anionic phospholipids in membranes. The K
D
values of
d1–18I
C

further suggest that the three basic residues in
AB
Secondary
CPY-binding site
Secondary
CPY-binding site
Phe7
Phe7
N-Terminal inhibitory
reactive site
N-Terminal inhibitory
reactive site
Met1
Met1
Fig. 4. Phospholipid recognition through the CPY-binding sites of I
C
. The crystal structure of I
C
in the complex with CPY is represented as a
surface model. (A) The binding interface between I
C
and CPY. The I
C
residues at the buried surface in the complex with CPY constitute the
N-terminal inhibitory reactive site (Ac-Met1–Phe7) and the secondary CPY-binding site (Ala10–Gln70 and Phe133–Glu137) [8], and are colored
green. These two binding sites, Met1 and Phe7 in the N-terminal inhibitory reactive site, and His18 in the secondary CPY-binding site are
labeled. (B) The basic (Arg, His, and Lys), acidic (Asp and Glu) and polar (Asn, Gln, Ser, Thr, and Tyr) residues of I
C
are colored blue, red, and
orange, respectively. The N-terminal inhibitory reactive site, the secondary CPY-binding site, Met1, Phe7, and His18 of I

C
are labeled. The
N-terminal segment (Met1–His18) of I
C
and basic residues in or adjacent to the segment may participate in recognition of anionic phospho-
lipids, such as PS and phosphoinositides.
J. Mima et al. Membrane binding of I
C
FEBS Journal 273 (2006) 5374–5383 ª 2006 The Authors Journal compilation ª 2006 FEBS 5379
the N-terminal segment of I
C
, Lys16, Lys17, and His18,
are essential for the targeting toward PtdIns(3,4,5)P
3
and that the other two basic residues, Lys101 and
Arg162, might be responsible for the targeting toward
PtsIns(3,4)P
2
rather than the three residues in the N-ter-
minal segment (Table 2). Although the K
D
value of
d1–7I
C
for PtdIns(3,4)P
2
was significantly increased, as
no basic residue is located in the N-terminal seven resi-
dues, the low affinity of d1–7I
C

could be caused by the
conformational change in the vicinity of the five basic
residues in this mutant protein.
The present in vitro membrane-binding studies using
a liposome-binding assay and SPR measurements
revealed a high affinity of I
C
for membranes contain-
ing anionic phospholipids such as PS, PtdIns(3)P,
PtdIns(3,4)P
2
, and PtdIns(3,4,5)P
3
, whereas this inhib-
itor is generally known to reside in the soluble cyto-
plasmic fraction [2,3,38]. Our fluorescence microscopic
analyses using I
C
–GFP revealed the cellular localiza-
tion of I
C
–GFP at the vacuolar membranes and
lumens during the stationary growth phase, suggesting
that I
C
is specifically associated with the vacuolar
membranes rather than the other cellular membranes.
The lipid composition of subcellular membranes in the
yeast S. cerevisiae has been reported [39,40], but little
precise information about the content of phosphoinosi-

tides in vacuolar membranes and the variation of
phospholipid compositions at different growth phases
is available. Thus, it remains to be resolved if the tar-
geting of I
C
to vacuolar membranes depends upon the
ability of this inhibitor to bind to the anionic phos-
pholipids, including PS and phosphoinositides. The
relocation of I
C
leads us to propose a working model
in which I
C
in the cytoplasm is specifically targeted to
anionic phospholipid molecules in the vacuolar mem-
branes during the stationary phase, and is subsequently
sorted into the lumens to regulate the vacuolar CPY
activities through complex formation with the cognate
protease. The interaction of I
C
with the yeast Ras
GTPase-activating protein, Ira2p, reported recently
[11], could regulate the cytoplasmic localization of I
C
in the logarithmic-phase cells. Previous work on the
inhibitory properties of I
C
in vitro, in which I
C
was

shown to inactivate and interact with CPY under aci-
dic conditions below pH 5 [9], support a scenario
involving CPY inhibition by I
C
in the acidic vacuoles
of yeast cells.
In conclusion, the present study reveals that I
C
binds
to anionic phospholipid membranes, the involvement
of the CPY-binding sites of I
C
in its phospholipid
recognition, and the intracellular localization of this
inhibitor at vacuolar membranes. Although the biolo-
gical significance of these membrane-binding properties
of I
C
is still obscure, these findings provide novel
insights into the membrane targeting of I
C
and PEBPs
and will be useful in terms of understanding the
diverse cellular functions of the PEBP family members.
Experimental procedures
Protein production and purification
CPY was purified from bakers’ yeast (Oriental Yeast,
Osaka, Japan) as described in a previous report [41]. I
C
was

produced using the S. cerevisiae expression system with the
vacuolar proteases-deficient strain BJ2168 (ATCC, Manas-
sas, VA) and the expression vector pYTF1 [5], and was
purified by a previously described method [5]. I
C
–CPY was
prepared by mixing equimolar amounts of purified I
C
and
CPY. The expression vectors for d1–7I
C
and d1–18I
C
were
constructed using a QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA) and pETF1 as a template
vector [6]. d1–7I
C
and d1–18I
C
were produced using the
Escherichia coli expression system with the constructed vec-
tors and BL21(DE3) strain (Novagen, Madison, WI), and
were purified by a previously described method [6].
Liposome-binding assay
The assay for the binding of I
C
and I
C
–CPY to phospho-

lipid liposomes was performed using a previously described
method [42], with minor modifications. PC (Sigma, St
Louis, MO) and mixtures of PC with a weight equivalent
to PE (Sigma), PS (Sigma), and PtdIns (Sigma) were
dissolved in chloroform and dried by evaporation with
nitrogen gas. The dried lipids were suspended in 20 mm
Hepes (pH 7.2), containing 0.15 m NaCl, to final concentra-
tions of 0.5 mgÆmL
)1
, and were then vortexed for 5 min
and sonicated for 10 min, to prepare the PC-based lipo-
somes [42,43]. I
C
and I
C
–CPY (2 lm final concentrations)
were added to the liposome-containing solutions, and the
solutions were then incubated at 30 °C for 1 h. After sedi-
mentation of the liposomes and the associated proteins
by centrifugation at 100 000 g for 30 min at 30 °C with
Sorvall RC28S and F-28 ⁄ 36 rotor (Thermo Electron
Corporation, Asheville, NC) the resulting pellets were sus-
pended in the sample buffer, 50 mm Tris ⁄ HCl (pH 6.8),
containing 2% SDS, 5% 2-mercaptoethanol, and 25%
glycerol. The suspensions were immediately boiled at
100 °C for 10 min and subjected to SDS ⁄ PAGE analysis.
SPR analysis
SPR measurements for binding of I
C
,I

C
–CPY, d1–7I
C
and d1–18I
C
to the PC-based liposomes were performed
at 30 °C in the running buffer (20 mm Hepes, pH 7.2,
containing 0.15 m NaCl) using the Biacore X system
Membrane binding of I
C
J. Mima et al.
5380 FEBS Journal 273 (2006) 5374–5383 ª 2006 The Authors Journal compilation ª 2006 FEBS
(Biacore AB, Uppsala, Sweden) and Sensor chip L1 (Bia-
core AB) [33], basically according to the reported proce-
dure [34]. The liposomes used were prepared as described
earlier, from PC and mixtures of PC with an equivalent
weight of PE, PS, PtdIns, PG (Sigma), PtdIns(3)P (Cay-
man, Ann Arbor, MI), PtdIns(4)P (Cayman), PtdIns(5)P
(Cayman), PtdIns(3,4)P
2
(Cayman), PtdIns(3,5)P
2
(Sigma),
PtdIns(4,5)P
2
(Cayman), and PtdIns(3,4,5)P
3
(Cayman).
The sensor surface of the L1 chip was coated with the
liposomes (0.5 mgÆmL

)1
) at a flow rate of 5 lLÆmin
)1
for
20 min, and this was followed by the injection of 50 mm
NaOH, to wash the surface, 0.1 mgÆmL
)1
BSA for block-
ing the exposed lipophilic groups, and 50 mm NaOH for
rewashing. The control sensor surface was coated with
0.1 mgÆmL
)1
BSA and then washed with 50 mm NaOH.
In the kinetic SPR measurements, at least five concentra-
tions (0.1–10 lm)ofI
C
,I
C
–CPY, d1–7I
C
and d1–18I
C
were injected onto the liposome-coated sensor surface at a
flow rate of 60 lLÆmin
)1
for 90 s. The bound proteins
were subsequently dissociated from the surface by passing
running buffer at 60 lLÆmin
)1
for 240 s, and were then

completely removed with 50 mm NaOH before the next
protein injection. For the acquisition of a new dataset, the
sensor surface was regenerated by injecting 40 mm
Chaps (Nacalai tesque, Kyoto, Japan) at a flow rate of
5 lLÆmin
)1
for 5 min and recoating with fresh liposomes.
All sensorgrams obtained were corrected by subtracting
the responses of the control surface. Kinetic parameters,
the association rate constant k
a
, and the dissociation rate
constant k
d
, were determined by the global fitting of the
sensorgrams to a 1 : 1 Langmuir binding model using
biaevaluation 3.0 software (Biacore AB), as described
previously [34]. The dissociation constant, K
D
, was then
calculated from the equation, K
D
¼ k
d
⁄ k
a
.
Fluorescence microscopy
For producing the C-terminal GFP-tagged I
C

(I
C
–GFP),
the DNA fragment encoding GFP was inserted downstream
of the I
C
-encoding gene in pYTF1 [5], and the S. cerevisiae
strain BY4741icD (MATa tfs1D::kanMX4 his3D leu2D
met15D ura3D) (Euroscarf, Frankfurt, Germany) was trans-
formed with the generated vector. The transformed cells
were grown to the early stationary phase in SD medium
(0.67% yeast nitrogen base without amino acids, 2.0% glu-
cose) supplemented with histidine, leucine, and methionine
(500 lgÆmL
)1
). The cultured cells were then suspended at
an A
600
of 1.0–1.2 in nutrient-rich YPGal medium (2%
galactose, 2% bactopeptone, 1% yeast extract) containing
1 lgÆmL
)1
FM4-64 (Molecular Probes, Eugene, OR), a
fluorescent dye used for staining vacuolar membranes [44].
During the cultivation at 30 °C for 72 h, the labeled cells
producing I
C
–GFP were collected at the logarithmic and
stationary growth phases and resuspended in the same med-
ium at an A

600
of 100–200. Fluorescence images of the
resuspended cells were obtained using an IX71 inverted
microscope (Olympus, Tokyo, Japan) equipped with
U-MNIBA2 and U-MWIG2 mirror units (Olympus), a
digital charge-coupled device camera C4742-95–12ER
(Hamamatsu Photonics, Hamamatsu, Japan), and aqua-
cosmos 2.0 software (Hamamatsu Photonics). Figures were
prepared using the corel photo-paint 9 software program
(Corel, Ottawa, Canada).
Acknowledgements
This work was supported, in part, by the Japan Foun-
dation for Applied Enzymology.
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