The N-terminal acetyl group of IC is essential for inhibitory function, and the inhibitor forms an equimolecular complex with the cognate protease through dual binding sites, an N-termin
Trang 1Specific membrane binding of the carboxypeptidase Y
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 IC, 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 IC [4–8] and the crystal structure of the complex of IC with CPY (IC–CPY) [8,9] have provided information
on the nature of the inhibition The N-terminal acetyl group of IC 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: mima@kais.kyoto-u.ac.jp
*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
IC, 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 IC 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-bisphos3-phos-phate, and phosphatidylinositol 3,4,5-trisphosphate, with KD values below 100 nm The liposome-binding assay and surface plasmon resonance analyses of IC, when complexed with carboxypeptidase Y, and the mutant forms of IC 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 IC to cellular membranes was subsequently analyzed by fluorescence microscopy of yeast cells producing the green fluorescent protein-tagged IC, suggesting that IC 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 IC and phosphatidyletha-nolamine-binding proteins
Abbreviations
CPY, carboxypeptidase Y; FM4-64, N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide; GFP, green fluorescent protein; IC, 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)P2, 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.
Trang 2CPY-binding site [6–8] In addition to its function as a
protease inhibitor, it has also been shown that IC is
identical to Tfs1p [4], a multicopy suppressor of the
cdc25-1mutant [10], and that it inhibits and interacts
with the yeast Ras GTPase-activating protein, Ira2p [11]
The amino acid sequence of IC 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 IC–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 IC
[8], remain obscure
In the present study, we report on a detailed study
of the membrane-binding mode of IC, a PEBP family
member A liposome-binding assay and surface
plas-mon resonance (SPR) analysis indicate that IC
specific-ally binds to membranes containing anionic
phospholipids, rather than phosphatidylethanolamine
(PE) A cellular localization analysis of IC 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 IC
In an attempt to detect and characterize the membrane
binding of IC, 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 ICand
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 IC to phospholipid membranes, and demonstrated that IC 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 IC, IC–CPY was subjected
to the binding assay with PS⁄ PC and PtdIns ⁄ PC lipo-somes As shown in Fig 1B,C, neither ICnor CPY in
IC–CPY was sedimented with these liposomes, indica-ting that the affinity of IC 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 ICwere 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.
Trang 3Therefore, these results suggest that the binding
inter-face for CPY in the ICmolecule is involved in its
spe-cific binding to anionic phospholipid membranes
To further quantitatively evaluate the affinity and
spe-cificity of ICfor 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 ICto
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)P2]⁄ PC,
phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]⁄ PC,
phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]⁄
PC, and phosphatidylinositol 3,4,5-trisphosphate
[PtdIns(3,4,5)P3]⁄ PC (Fig 2) In contrast to the findings
with these liposomes, no binding was detected of ICto
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 IC–CPY (Fig 1B,C), SPR responses of the complex
could not be detected toward all the phospholipid
somes (Fig 2) These SPR analyses, as well as the lipo-some-binding assay, demonstrated that IC, when complexed with CPY, loses its intrinsic affinity for ani-onic phospholipid membranes, and that the CPY-bind-ing sites of IC[8] may be responsible for its phospholipid recognition
Using SPR sensorgrams for various concentrations (0.1–10 lm) of IC, the membrane association rate con-stants (ka), dissociation rate constants (kd), and equi-librium dissociation constants (KD) 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 ICexhibits a broad specificity for a wide variety of anionic phospholipid membranes with KDvalues below 600 nm, but has a slightly higher affinity for PS, PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (KD values of 75–97 nm) than for PtdIns, PG, and the other phosphoinositides (KD val-ues of 200–550 nm) (Table 1) The lower affinity of IC for PtdIns and PG results mainly from the smaller
ka values, whereas the lower affinity for phosphoino-sitides other than PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 results from the higher kd values Recent SPR studies of membrane–protein interactions have shown that ka and kd are influenced by nonspe-cific electrostatic interactions and proximal spenonspe-cific 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 ICand IC–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)P2⁄ PC (green), PtdIns(3,4,5)P3⁄ 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 )1Æs)1)
k d (10)5s)1)
K D (10)9M )
PtdIns(4)P ⁄ PC 35 ± 12 180 ± 13 550 ± 190
PtdIns(3,4)P2⁄ PC 68 ± 13 60 ± 14 88 ± 7.6 PtdIns(3,5)P2⁄ 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
Trang 4the positively charged residues of IC 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)P2,
and PtdIns(3,4,5)P3
Involvement of the CPY-binding sites of ICin its
membrane binding
As IC–CPY has no affinity for phospholipid membranes,
to obtain additional information on the involvement of
the CPY-binding sites of ICin its phospholipid
recogni-tion, we determined the membrane-binding parameters
for the mutant forms of IC, d1–7IC and d1–18IC, with
the N-terminal seven (Ac-MNQAIDF) and 18 (Ac-MN
QAIDFAQASIDSYKKH) residues, respectively,
dele-ted (Table 2) d1–7ICand d1–18IClack 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 ICand 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 d1–7ICand d1–18ICshowed that these mutants
of IC, 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 IC with respect to these anionic phospholipid liposomes No binding of d1–7IC to PtdIns(3)P⁄ PC was detected, and the KD value of d1–7IC binding to PtdIns(3,4)P2⁄ PC was increased 13-fold For the other liposomes, the KDvalues of the mutant were also increased more than four-fold over those of the native protein (Table 2) In contrast to those of d1–7IC, the KD value of d1–18IC for PtdIns(3,4)P2⁄ PC was increased 2.4-fold, whereas the
KD 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)P3⁄ PC was increased 8.1-fold (Table 2) These results demonstrate that the N-terminal segment
of IC (Ac-Met1–His18) is essential for its binding effi-ciency and specificity for phospholipid membranes and suggest that the phospholipid recognition site of IC is composed of residues in and adjacent to this N-terminal segment
Association of ICwith cellular membranes
To gain insights into the association of ICwith cellular membranes, we subsequently examined the intracellular localization of the inhibitor by fluorescence microscopy
of living yeast cells producing IC–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 KD, KDfor d1–7I C or d1–18I C ⁄ K D for I C ND, not detectable.
ka (102M )1Æs)1)
kd (10)5Æs)1)
KD (10)9M )
Increase in KD (fold)
Trang 5staining 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 IC–
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 IC–
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 IC–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 IC–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 IC, was originally isolated as a 23 kDa cytoplasmic protein
A
B
Fig 3 Fluorescence microscopic analyses
of yeast cells producing IC–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.
Trang 6associated 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 bindregard-ing of the
other members of the PEBP family Consequently, the
binding characteristics of PEBP proteins, including
those of the binding of ICto phospholipid membranes,
are currently unclear and remain to be clarified
The present in vitro membrane-binding analyses of IC
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)P2, and PtdIns(3,4,5)P3
(Table 1) The two further findings that (a) IC–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 ICoverlap, and that the N-ter-minal segment at the CPY-binding sites participates in regulation of the specific binding of IC 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 IC 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 ICand the disposition of the ICresidues 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 KDvalues of d1–18ICfurther suggest that the three basic residues in
Secondary CPY-binding site
Secondary CPY-binding site
N-Terminal inhibitory reactive site
N-Terminal inhibitory reactive site
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 ICand basic residues in or adjacent to the segment may participate in recognition of anionic phospho-lipids, such as PS and phosphoinositides.
Trang 7the N-terminal segment of IC, Lys16, Lys17, and His18,
are essential for the targeting toward PtdIns(3,4,5)P3
and that the other two basic residues, Lys101 and
Arg162, might be responsible for the targeting toward
PtsIns(3,4)P2rather than the three residues in the
N-ter-minal segment (Table 2) Although the KD value of
d1–7ICfor PtdIns(3,4)P2was significantly increased, as
no basic residue is located in the N-terminal seven
resi-dues, the low affinity of d1–7ICcould 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 IC for membranes
contain-ing anionic phospholipids such as PS, PtdIns(3)P,
PtdIns(3,4)P2, and PtdIns(3,4,5)P3, whereas this
inhib-itor is generally known to reside in the soluble
cyto-plasmic fraction [2,3,38] Our fluorescence microscopic
analyses using IC–GFP revealed the cellular
localiza-tion of IC–GFP at the vacuolar membranes and
lumens during the stationary growth phase, suggesting
that IC 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 ICto vacuolar membranes depends upon the
ability of this inhibitor to bind to the anionic
phos-pholipids, including PS and phosphoinositides The
relocation of IC leads us to propose a working model
in which IC 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 IC with the yeast Ras
GTPase-activating protein, Ira2p, reported recently
[11], could regulate the cytoplasmic localization of IC
in the logarithmic-phase cells Previous work on the
inhibitory properties of IC in vitro, in which IC was
shown to inactivate and interact with CPY under
aci-dic conditions below pH 5 [9], support a scenario
involving CPY inhibition by IC in the acidic vacuoles
of yeast cells
In conclusion, the present study reveals that ICbinds
to anionic phospholipid membranes, the involvement
of the CPY-binding sites of IC 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 IC is still obscure, these findings provide novel insights into the membrane targeting of ICand 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] ICwas 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] IC–CPY was prepared by mixing equimolar amounts of purified IC and CPY The expression vectors for d1–7ICand d1–18ICwere constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and pETF1 as a template vector [6] d1–7IC and d1–18IC were produced using the Escherichia coliexpression 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 IC and IC–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] IC and IC–CPY (2 lm final concentrations) were added to the liposome-containing solutions, and the solutions were then incubated at 30C for 1 h After sedi-mentation of the liposomes and the associated proteins
by centrifugation at 100 000 g for 30 min at 30C 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
100C for 10 min and subjected to SDS ⁄ PAGE analysis
SPR analysis SPR measurements for binding of IC, IC–CPY, d1–7IC and d1–18IC to the PC-based liposomes were performed
at 30C in the running buffer (20 mm Hepes, pH 7.2, containing 0.15 m NaCl) using the Biacore X system
Trang 8(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)P2 (Cayman), PtdIns(3,5)P2 (Sigma),
PtdIns(4,5)P2 (Cayman), and PtdIns(3,4,5)P3 (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) of IC, IC–CPY, d1–7IC and d1–18IC
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 ka, and the dissociation rate
constant kd, 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, KD, was then
calculated from the equation, KD¼ kd⁄ ka
Fluorescence microscopy
For producing the C-terminal GFP-tagged IC (IC–GFP),
the DNA fragment encoding GFP was inserted downstream
of the IC-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 transtrans-formed 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 A600 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 30C for 72 h, the labeled cells
producing IC–GFP were collected at the logarithmic and
stationary growth phases and resuspended in the same
med-ium at an A600 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-cosmos2.0 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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