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: 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 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]. 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Specific membrane binding of the carboxypeptidase Y inhibitor I C , a phosphatidylethanolamine -binding protein family member Joji Mima*, Hiroaki Fukada,. an endogenous cytoplasmic inhibitor of vacuolar carboxypeptidase Y in the yeast Saccharomyces cerevisiae, is classified as a member of the phos- phatidylethanolamine-binding