Báo cáo khoa học: Secretion of the mammalian Sec14p-like phosphoinositide-binding p45 protein pptx

Secretion of the mammalian Sec14p-like phosphoinositide-binding p45 protein Maria Merkulova 1 , Huong Huynh 2 , Vitaly Radchenko 1 , Kan Saito 2 , Valery Lipkin 1 , Tatiana Shuvaeva 1 and Tomas Mustelin 2 1 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian, Academy of Sciences, Moscow, Russia 2 Program of Inflammation, Infectious and Inflammatory Disease Center and Program of Signal Transduction, Cancer Center, The Burnham Institute, La Jolla, CA, USA Protein–lipid interactions are important for protein targeting, signal transduction, lipid transport, and the maintenance of cellular compartments and mem- branes. The yeast PtdIns transfer protein Sec14p is the prototype for a large family of protein modules referred to as the SEC14 domain (Smart entry: smart00516) or CRAL ⁄ TRIO domain (Pfam entry: pfam00650) for cellular retinaldehyde binding protein⁄ Trio protein homology. There are presently about 500 proteins with this domain in the conserved domains database (http://www.ncbi.nlm.nih.gov/) and this number is still growing. It is now apparent that this is an evolutionary ancient and widespread domain found in plants, yeast, invertebrates, and mammals [1]. About two thirds of all proteins that contain a Sec14p homology domain, consist only of this domain, while the rest are multidomain proteins with additional protein–protein interaction or catalytic domains. The prototypical Sec14p from Saccharomyces cerevisiae is two-lobed globular protein with a large hydrophobic pocket [2], which binds the entire PtdIns molecule. While the same overall topology is found in all Sec14p-like proteins, they differ from each other mostly in the region predicted to bind the head group of PtdIns. Other known ligands for these proteins include phosphatidylcholine [3], trans-retinaldehyde [4], and PtdIns(3,4,5)P 3 [5]. It seems that Sec14p-like pro- teins have been adapted during evolution to fulfill a number of different functions that depend on protein– lipid interactions. Keywords CRAL ⁄ TRIO domain; GOLD domain; nonclassical protein secretion; phosphoinositides; Sec14p Correspondence T. Mustelin, Program of Signal Transduction, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA Fax: +1 858 713–6274 Tel: +1 858 713–6270 E-mail: tmustelin@burnham.org (Received 11 March 2005, revised 23 August 2005, accepted 2 September 2005) doi:10.1111/j.1742-4658.2005.04955.x Protein–lipid interactions are important for protein targeting, signal trans- duction, lipid transport, and the maintenance of cellular compartments and membranes. Specific lipid-binding protein domains, such as PH, FYVE, PX, PHD, C2 and SEC14 homology domains, mediate interactions between proteins and specific phospholipids. We recently cloned a 45-kDa protein from rat olfactory epithelium, which is homologous to the yeast Sec14p phosphatidylinositol (PtdIns) transfer protein and we report here that this protein binds to PtdIns(3,4,5)P 3 and far weaker to less phosphoryl- ated derivatives of PtdIns. Expression of the p45 protein in COS-1 cells resulted in accumulation of the protein in secretory vesicles and in the extracellular space. The secreted material contained PtdIns(3,4,5)P 3 . Our findings are the first report of a Sec14p-like protein involved in transport out of a cell and, to the best of our knowledge, inositol-containing phos- pholipids have not previously been detected in the extracellular space. Our findings suggest that p45 and phosphoinositides may participate in the formation of the protective mucus on nasal epithelium. Abbreviations Btk, Bruton’s tyrosine kinase; EEA1, early endosomal antigen-1; FYVE, Fab1, YotB,Vac1, and EEA1 homology; GFP, green fluorescent protein; GOLD, Golgi dynamics; HA, hemagglutinin; PH, pleckstrin homology; PI3K, phosphatidylinositol 3¢-kinase; PITP, PtdIns transfer proteins; PtdIns, phosphatidylinositol; PTP, protein tyrosine phosphatase; PX, phox homology; s, secretory; SPF, supernatant protein factor; TAP, tocopherol-associated protein. FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS 5595 The mammalian Sec14p-like protein p45 was origin- ally thought to be a GTP-binding protein present in rat olfactory epithelium [6,7]. The determination of its sequence in 1999 [8] showed that it is homologous to yeast Sec14p and related lipid-binding proteins. A clo- sely related bovine protein was reported by Stocker and coworkers in the same year [9]. A human ortholog of this protein was later cloned and termed toco- pherol-associated protein (TAP) [10]. This protein is identical to supernatant protein factor (SPF), a protein with squalene transfer activity [11]. The human gen- ome contains three genes for TAP within 100 kb of each other on chromosome 22q12.1 [12], designated hTAP1, hTAP2, and hTAP3. The two latter are 86% and 80% identical to hTAP1 ⁄ SPF [12]. The rat p45 protein is the ortholog of hTAP2. The crystal structure of hTAP1 ⁄ SPF [13] revealed a two-domain topology very similar to yeast Sec14p. In hTAP1 ⁄ SPF, the 275-residue Sec14p homology domain has a large horse shoe-shaped ligand-binding pocket between the central b-sheet and the surrounding a-heli- ces [13]. The C-terminal 115 residues form a separate domain consisting of eight b strands organized as jelly roll barrel [13]. This globular domain was referred to as a Golgi dynamics (GOLD) domain [12], a putative protein–protein interaction domain predicted to be involved in Golgi function or trafficking [14]. GOLD domains are frequently found in combination with domains known to interact with lipids, i.e. SEC14, pleckstrin homology (PH), and Fab1p ⁄ YotB ⁄ Vac1p ⁄ EEA1 (FYVE) domains [14]. Such two-domain pro- teins may function as adapters that assemble protein complexes on membranes or aid in packaging of cargo proteins into membrane vesicles. The ligand specificity of hTAP1 ⁄ SPF is rather con- tradictory. First, it was reported to bind tocopherol [9,10] and squalene [11], then the three-dimensional crystal structure in complex with a-tocopheryl quinone was reported [15]. Recent studies with a variety of hydrophobic ligands showed that recombinant hTAP1 ⁄ SPF can bind a-, b-, c-, and d-tocopherols, a -toco- pheryl quinone, squalene, phosphatidylcholine, and PtdIns, with the lowest dissociation constants for the latter [16]. This absence of a clear ligand preference leaves the question of the true physiological ligand unresolved. The ligand(s) for TAP2 (p45) and TAP3 are unknown. To begin to address the biological function of p45 ⁄ TAP2 protein we have performed lipid binding experiments and we studied the subcellular localization of p45. We report that this protein binds to PtdIns(3,4,5)P 3 and far weaker to less phosphorylated derivatives of PtdIns in vitro. When expressed in COS-1 cells, p45 accumulated in secretory vesicles and the extracellular space along with PtdIns(3,4,5)P 3 . This could represent a novel mechanism of export of PtdIns-derived molecules. The significance of this is discussed. Results P45 binds PtdIns(3,4,5)P 3 in vitro Several proteins containing the lipid-binding SEC14 domain have now been shown to bind phospholipids [3,5,12]. For example, the SEC14 domain of PTP- MEG2 was shown to bind PtdIns(3,4,5)P 3 in vitro and colocalized with it in intact cells [5]. We therefore deci- ded to study the binding of the SEC14-containing p45 to phospholipids using a filter-binding assay [17]. Both natural purified and recombinant p45 proteins were used to eliminate possible artifacts due to contamin- ation with bacterial lipids, endogenous lipid occu- pancy, issues with misfolding, or other potential problems with either preparation. Both preparations were of high purity (Fig. 1A). Nitrocellulose filters with 15 immobilized phospholipids (PIP-Strips TM , Ech- elon) were overlaid with recombinant or natural p45 in solution at a final concentration of 0.5 lgÆmL )1 (10 nm), washed extensively, and detected by immuno- blotting. As shown in Fig. 1B, natural p45 bound best to PtdIns(3,4,5)P 3 and, significantly weaker, to PtdIns(3)P, PtdIns(4)P, PtdIns(3,5)P 2 , PtdIns(4,5)P 2 , PtdIns(3,4)P 2 and PA. Other phospholipids did not bind at all to natural p45 protein. Recombinant p45 protein gave very similar results: it also bound best to PtdIns(3,4,5)P 3 and far weaker to the same other phospholipids as natural p45 did (Fig. 1C). We con- clude from these experiments that p45, like the SEC14 domain PTP-MEG2 [5], binds to phosphoinositides with highest affinity for PtdIns(3,4,5)P 3 . Subcellular localization of p45 protein Previous studies [7] using immunohistochemistry and electron microscopy indicated that endogenous p45 in the olfactory epithelium is found in secretory granules of sustentacular cells and in the adjacent layer of the surrounding mucus. p45 was also detected in the wash- out medium of nasal mucosa [6]. However, it was not clear from these studies if p45 was actively secreted or only released from disrupted nasal cells [6]. To study the subcellular localization of p45, we cloned its cDNA into the pEF3HA eukaryotic expres- sion vector, which adds an HA epitope tag to the C-terminus of the insert. COS-1 cells were transfected Secretion of p45 Sec14p-like protein M. Merkulova et al. 5596 FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS with either empty pEF3HA vector or the pEF3- HA_p45 construct, fixed 48 h later, permeabilized, stained with a TRITC-conjugated 12CA5 anti-HA mAb, and viewed under a confocal microscope. While vector-transfected cells did not show any staining (Fig. 2A, upper panel), the HA-tagged p45 was readily detected in the cells transfected with pEF3HA_p45 as a staining of intracellular vesicles, as granular plasma membrane-associated structures, and as irregular aggregates in the extracellular space (Fig. 2A, lower panel). Immunoblotting of the culture medium as well as lysates of the transfected cells with the 16B12 anti- HA tag antibody showed that a protein of the correct M r (molecular mass calculated to be 48 192 Da) was present in both locations (Fig. 2B). Thus, transfected COS-1 cells produce HA-tagged p45, package it into vesicles, and secrete much of it into the medium. To further demonstrate that the intracellular p45 was indeed concentrated in secretory vesicles, we coex- pressed p45 in COS-1 cells with a fusion protein con- sisting of GFP plus two tandem FYVE domains from early endosomal antigen-1 (EEA1). This GFP-EEA1- (FYVE) 2 constructs binds to PtdIns(3)P [18,19], a phospholipid most abundant on endosomes and secre- tory vesicles, but also found on Golgi and post-Golgi membranes and in small quantities at the plasma membrane. In these transfected cells, the intense red immunofluorescence staining of intracellular p45 was surrounded by rings of green fluorescence (Fig. 2C). There were also smaller vesicles marked by GFP- EEA1-(FYVE) 2 , without p45 inside, as well as a strong green fluorescence in the nucleus. While the former probably represent lysosomes and ⁄ or endosomes, the nuclear staining is presumably nonspecific as GFP alone also tends to accumulate in the nucleus [5]. We conclude from these experiments that p45, which is synthesized in these cells, translocates into the Golgi system (perhaps directly from the rough endoplasmic reticulum through cis-Golgi) and is subsequently pack- aged in the trans-Golgi network into secretory vesicles. Thus, it appears that many of the vesicles with high p45 content are cytosolic secretory vesicles destined for exocytosis (‘secretory vesicle’ in Fig. 2C). Indeed, many cells contained vesicles in the process of being emptied to the extracellular environment (‘exocytosis’ in Fig. 2C). Extracellular aggregates of p45 were detec- ted in every transfection experiment (‘extracellular’ in Fig. 2C). In contrast, numerous other proteins with a HA tags remain completely intracellular [5,20,21]. The SEC14 domain, but not GOLD domain, is sufficient for secretion of p45 Secreted proteins are usually synthesized as precursors with a cleavable N-terminal signal peptide, composed of a positively charged region, a hydrophobic core, and a proteolytic cleavage site [22]. No such cleavable signal sequence was found in p45 using the hidden Markov model (HMM)-based signalip software, which is available on the internet [23]. However, many AB C Fig. 1. Phospholipid binding by p45 protein. (A) SDS ⁄ PAGE gel stained with Coomassie blue of purified recombinant (lane 1) and natural (lane 2) p45 proteins. (B, C) Protein lipid overlay assay using PIP Strips TM from Echelon and the indicated concentrations of natural (B) and recombinant (C) p45 proteins. PE, phosphatidylethanolamine, PC, phosphatidylcholine, PS, phosphatidylserine, LPA, lysophosphatidic acid, LPC, lysophosphatidylcholine, S1P, sphingosine-1-phosphate, PA, phosphatidic acid. M. Merkulova et al. Secretion of p45 Sec14p-like protein FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS 5597 other well documented secretory proteins lack signal sequence and cannot currently be identified computa- tionally as secreted proteins. These proteins have been proposed to contain nonlinear or unrecognized signal regions, which can be N-terminal, internal, or C-terminal [24]. In fibroblast growth factor-16, a A C B Secretion of p45 Sec14p-like protein M. Merkulova et al. 5598 FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS hydrophobic motif was shown to be important for translocation of the protein into the endoplasmic reti- culum membrane and mutation of this region abro- gated secretion [25]. In order to determine if p45 contains such hydrophobic motifs, we analyzed its hydrophilicity ⁄ hydrophobicity using the method of Kyte and Doolittle [26]. The resulting plot (Fig. 3A) showed that p45 contains several strongly hydrophobic motifs, which could be signaling for secretion. How- ever, these motifs were located within the SEC14 and GOLD domains of p45 (Fig. 3A), which probably fold as independent units. Thus, simply mutating the hydrophobic segments would likely impair the proper folding of these domains. Therefore, we instead chose to make deletion mutants of p45. In the first deletion mutant, we cloned amino acid residues 76–246 of p45 comprising the SEC14 domain into the pEF3HA vec- tor. The second mutant consisted of the GOLD domain, amino acid residues 265–381. When these con- structs were expressed in COS-1 cells and visualized by anti-HA staining, it was clear that the SEC14 construct behaved like full-length p45 (Fig. 3B) and was found both in cytoplasmic vesicles and in the extracellular space (Fig. 3C), while the GOLD domain construct was only seen inside cells (Fig. 3D). By immunoblot- ting, the 19-kDa SEC14 protein was readily detectable in the culture supernatant (Fig, 3E, lane 4), while the 13-kDa GOLD protein was present only in trace amounts (lane 3) despite being well expressed by the cells (lane 1). Thus, all the information necessary for secretion of p45 appears to be contained within the SEC14 domain. Co-localization of p45 with PtdIns(3,4,5)P 3 in intact cells Based on the finding that p45 bound to PtdIns(3,4,5)P 3 in vitro we decided to examine if p45 was associated with this phospholipid in intact cells. COS-1 cells were transfected with the HA- tagged p45 plasmid and then immunostained with an anti-PtdIns(3,4,5)P 3 mAb, Alexa FluorÒ 488 goat anti-(mouse Ig) Ig, and last with the TRITC-conju- gated anti-HA mAb. As shown in Fig. 4, the resulting green fluorescence was found to colocalize with p45 inside secretory vesicles and to a lesser extent at the plasma membrane and in the extracellular space. The weak staining of extracellular material is not surprising given that detergents are included in the staining pro- tocol. Thus, the staining for PtdIns(3,4,5)P 3 is partly overlapping with p45, particularly in the secretory vesi- cles, which usually do not stain specifically with this mAb [5]. Since p45 appears to reside mostly inside a post- Golgi vesicular compartment, we decided to test if a cytosolic marker for PtdIns(3,4,5)P 3 , a fusion between GFP and the pleckstrin homology (PH) domain of the Btk kinase (GFP-Btk-PH) [27], would colocalize with p45. When COS-1 cells cotransfected with GFP-Btk- PH and HA-p45 were stained for p45 and viewed under a confocal microscope, it was clear that the green and red fluorescence did not colocalize at all. While p45 was confined to discrete spots, GFP-Btk-PH excluded these spots and was instead diffusely cyto- plasmic and accumulated at parts of the plasma mem- brane (Fig. 4B). Thus, unlike PTP-MEG2 [5] which also binds PtdIns(3,4,5)P 3 via its SEC14 domain [5], p45 is inaccessible to a cytoplasmic marker for this lipid. This is in agreement with the notion that p45 with bound PtdIns(3,4,5)P 3 resides in the secretory vesicle compartment. Finally, we stained cells for the secretory vesicle marker carboxypeptidase E, which largely colocalized with p45 (Fig. 5). Discussion Taken together, our findings indicate that the mam- malian Sec14p-like protein p45 (TAP2) is a secreted protein that binds PtdIns(3,4,5)P 3 and perhaps other highly phosphorylated inositol phospholipids. Al- though this transport function is not too different from that of S. cerevisiae Sec14p, which transports PtdIns between cellular membranes, our findings are the first to report that phosphoinositides can be trans- ported out by TAP2 into the extracellular environment. Because p45 was purified from olfactory epithelium Fig. 2. Endogenous p45 is located in granular cytoplasmic structures and extracellular space in COS-1 cells. (A) Confocal microscopy COS-1 cells transfected with empty pEF3HA vector (upper panels) or pEF3HA_p45 (lower panels) and then immunostained for HA-tagged p45 with TRITC-conjugated anti-HA mAb (red). Right hand panels are Nomarski differential interference contrast images of the same field. The shown cells are representative of the majority of stained cells. (B) Immunoblot with anti-HA mAb of conditioned medium (lanes 1 and 2) and cell ly- sates (lanes 3 and 4) from COS-1 cells transfected with pEF3HA_p45 (lanes 1 and 3) or empty vector pEF3HA (lanes 2 and 4) for 48 h. p45 is indicated by an arrow. (C) Confocal microscopy of a COS-1 cells transfected with cotransfected with GFP-EEA1-(FYVE) 2 (green) and pEF3- HA_p45 and stained as in (A). Note that overexpressed p45 protein is localized inside secretory vesicles and in extracellular space, while GFP-EEA1-(FYVE) 2 is enriched around secretory vesicles and other organelles. There is no direct colocalization of GFP-EEA1-(FYVE) 2 with p45. M. Merkulova et al. Secretion of p45 Sec14p-like protein FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS 5599 A B E C D Secretion of p45 Sec14p-like protein M. Merkulova et al. 5600 FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS and can be detected in secretory vesicles of sustentacu- lar cells in the nasal mucosa, as well as in the mucus produced by these cells, it seems that the secretion of p45 we observe represents the physiological behavior of this protein. The mRNA for p45 was also detected in the surface layer of epithelial tissues [8]. p45 and the B A Fig. 4. p45 colocalizes with PtdIns(3,4,5)P 3 , but not with GFP-Btk-PH, inside secretory vesicles. (A) Confocal microscopy of COS-1 cells transfected with pEF3HA_p45 and double stained for PtdIns(3,4,5)P 3 with the specific mAb plus Alexa FluorÒ 488 goat anti-(mouse Ig) Ig (green), and then for p45 with the TRITC-conjugated anti-HA mAb (red). (B) Confocal microscopy of COS-1 cells cotransfected with GFP-Btk- PH construct (green) plus pEF3HA_p45 and stained with the TRITC-conjugated anti-HA mAb (red). Differential interference contrast images of the same cells are shown in the far right panels. The shown cells are representative of the majority of stained cells. Note that PtdIns(3,4,5)P 3 colocalizes with p45 whereas the GFP-Btk-PH cannot penetrate the secretory vesicles and therefore cannot colocalize with p45. Fig. 3. Secretion of the SEC14 domain of p45. (A) The hydrophilicity plot of p45 protein and location of the SEC14 and GOLD domains. Note that the strongly hydrophobic motifs in p45 lie within these two domains. (B–D) Confocal microscopy of COS-1 cells transfected with pEF3- HA_p45, pEF3HA_SEC14, and pEF3HA_GOLD constructs, as indicated, and stained with the TRITC-conjugated anti-HA mAb (red). Two rep- resentative fields with accompanying Nomarski differential interference contrast images are shown. (E) Anti-HA immunoblot of cell lysates (lanes 1 and 2) or conditioned medium (lanes 3 and 4) of COS-1 cells transfected with pEF3HA_GOLD (lanes 1 and 3) or pEF3HA_SEC14 (lanes 2 and 4). Note that only the SEC14 protein is present at high levels in the medium. M. Merkulova et al. Secretion of p45 Sec14p-like protein FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS 5601 bound PtdIns(3,4,5)P 3 may play important roles in the protective nasal mucosa, perhaps as components of the extracellular matrix. p45 may also release PtdIns(3,4,5)P 3 to serve a function of its own in the mucosa. Interestingly, plants also use a similar exocyto- sis of lipid transfer proteins as part of the formation of a protective surface wax [28,29]. We have found that secretion of p45 does not occur via the conventional targeting mechanism of N-ter- minal signal sequence recognition and cleavage. Rather, p45 secretion is determined by the SEC14 domain of p45, which is well secreted as an isolated protein (Fig. 3). The SEC14 domain is an ancient lipid-binding domain found in both plants and animals [1]. While the prototypical yeast Sec14p binds and transports PtdIns [2], this function has been taken over in higher eukary- otes by structurally unrelated PtdIns transfer proteins (PITPs) [30]. Dictyostelium discoideum represents a transition stage in evolution with orthologs of both S. cerevisiae Sec14p and mammalian PITPs involved in PtdIns transport in the same cell [31]. In higher eukary- otes, SEC14 domain-containing proteins have appar- ently evolved to carry out other functions, such as transport of tocopherol [10] or retinaldehyde [4] and allosteric regulation of enzymes like the PTP-MEG2 tyrosine phosphatase [5,32] or Dbl family regulators of small Ras-related G-proteins [1]. The metabolism of inositol phospholipids has been intensely investigated during the last two decades [33]. The D3 hydroxyl group of the inositol ring is phos- phorylated by a family of phosphoinositide 3-kinases (PI3Ks), which can be subdivided into three classes [34]. While class I PI3Ks mainly phosphorylate PtdIns(4,5)P 3 to produce PtdIns(3,4,5)P 3 in response to activation of many cell-surface receptors [35], class III PI3Ks phosphorylate only PtdIns to produce PtdIns(3)P [36]. Mammalian class III PI3K are ortho- logs of the yeast vacuolar sorting protein Vps34p [37]. They are located primarily in the Golgi and other internal membranes, where they produce PtdIns(3)P and are thought to play role in protein sorting and vesicle transport. Their function is mediated through a number of effector proteins, containing FYVE domains, which specifically bind PtdIns(3)P [18,19], such as EEA1 [38,39], Hrs [40], PIKfyve (a vesicle- bound PtdIns-5-kinase) [41]. In addition, phosphoryla- tion of the D4 and D5 positions of the inositol ring also play important roles in a variety of site-specific recruitment and activation events in membrane traf- ficking [42,43]. PtdIns(3,4,5)P 3 was also detected recently on the cytosolic face of the enclosing mem- brane of secretory vesicles [5]. However, it remains unclear how PtdIns(3,4,5)P 3 gains access to the lumen of secretory vesicles. Is it synthesized in this location or is it transported there by p45? It also remains unknown if PtdIns(3,4,5) P 3 bound to p45 was synthes- ized using the class III PI3K (plus D4 and D5 kinases) pathway on intracellular membranes or if it was syn- thesized by class I PI3K and then transported to the Golgi and ⁄ or secretory pathway from plasma mem- brane. In the latter case, p45 could be involved in the transport process. These questions, as well as the role of p45-mediated export of phosphoinositides, will require further studies. Experimental procedures Antibodies and reagents The anti-influenza hemagglutinin (HA) tag epitope mAb 12CA5 conjugated to tetramethyl rhodamine isothiocyanate (TRITC) was from Roche Molecular Biochemicals (Indi- anapolis, IN, USA). The 16B12 anti-HA from Covance (Richmond, CA, USA), was used for immunoblotting. Alexa FluorÒ 594 goat anti-(mouse IgG H + L) Ig and Alexa FluorÒ 488 goat anti-(mouse IgG H + L) Ig were Fig. 5. p45 colocalizes with carboxypeptidase E, a secretory vesicle marker. Confocal microscopy of COS-1 cells transfected with pEF3- HA_p45 and double stained for carboxypeptidase E with a specific mAb plus quantum dot-605-conjugated anti-(mouse Ig) Ig (green), and then for p45 with rat anti-HA mAb plus quantum dot-655-conju- gated anti-(rat Ig) Ig (red). DNA was stained with DAPI and the fourth panel is a merge of the three colors. Note that carboxypepti- dase E and p45 colocalize extensively. Secretion of p45 Sec14p-like protein M. Merkulova et al. 5602 FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS from Molecular Probes (Eugene, OR, USA). Rabbit poly- clonal antiserum against native rat p45 protein was raised as described previously [7]. All phospholipids, the anti- PtdIns(3,4,5)P 2 mAb, and commercial PIP Strips TM with 15 different phospholipids were from Echelon (Salt Lake City, UT, USA). Each PIP Strip TM had 100 pmol of PtdIns, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P 2 , PtdIns(3,5)P 2 , PtdIns(4,5)P 2 , PtdIns(3,4,5)P 3 , phosphatidyl- ethanolamine, phosphatidylcholine, phosphatidylserine, lysophosphatidic acid, lysophosphatidylcholine, sphingo- sine-1-phosphate, and phosphatidic acid spotted onto a nitrocellulose membrane. Preparation of water-soluble extract and purification of natural p45 protein Preparation of water-soluble extract of rat olfactory epithe- lium was done as before [7]. Briefly, rat olfactory epithe- lium was homogenized in 10 mm sodium phosphate buffer, containing 150 mm NaCl and 0.5 mm phenylmethanesulfo- nyl fluoride, pH 7.4. The homogenates were centrifuged at 20 000 g for 1 h, and supernatants (total protein concentra- tion approximately 1 mgÆmL )1 ) were subjected consequently to DEAE-Sepharose and Sephacryl S-200 chromatography as described [7]. Purity of the protein was about 90% as judged by SDS ⁄ PAGE followed by Coomassie blue stain- ing. Immunoblots with rabbit polyclonal antiserum showed a single band of the expected size ( 45 kDa). This protein preparation will be referred here to as ‘natural p45 protein’. Prokaryotic expression and purification of recombinant p45 protein For protein overexpression p45 cDNA was subcloned into the prokaryotic expression vector pET-11a (Novagen, Darmstadt, Germany) and then transformed into Escheri- chia coli strain Rosetta TM (Novagen) which was designed to enhance the expression of eukaryotic proteins that con- tain codons rarely used in E. coli [44]. Expression procedure was done according to conventional technique [45]. The recombinant protein was purified by a two-step chromato- graphic procedure using DEAE-Sepharose and Sephacryl S-200 as described earlier [7]. Purity of the protein was about 90% as judged by SDS ⁄ PAGE followed by Coomas- sie blue staining. Immunoblot with rabbit polyclonal anti- serum showed a single band of expected size ( 45 kDa). Protein lipid overlay Assay The lipid binding specificity of the p45 protein was assayed as described [5,17]. Nitrocellulose filters spotted with 100 pmol of phospholipids (PIP Strips TM ) were blocked in 3% (w ⁄ v) fatty acid-free BSA in 10 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, and 0.1% (v ⁄ v) Tween-20 for 1 h and incu- bated with 0.5 lg ÆmL )1 (10 nm) of the p45 protein, natural or recombinant, overnight at 4.C. The membrane was washed three times for 10 min with 3% fatty acid-free BSA in 10 m m Tris ⁄ HCl, pH 8.0, 150 mm NaCl, and 0.1% Tween-20, and then incubated for 1 h with a 1 : 750-diluted anti-p45 rabbit polyclonal antiserum at 37.C. The mem- brane was washed as before, and incubated for 1 h with 1 : 2,000-diluted anti-rabbit ⁄ HRP conjugate. Finally, the membranes were washed and p45 protein bound to the membrane by virtue of its interaction with phospholipids was detected by enhanced chemiluminescence using a kit from Amersham (Arlington Heights, IL, USA). Immunoblots Immunoblotting was performed as before [5]. All immuno- blots were developed by the enhanced chemiluminescence technique according to the manufacturer’s instructions. Transient transfection, immunofluorescence, and confocal microscopy For transient expression, p45 cDNA was subcloned into the mammalian expression vector pEF3HA [20], in-frame with a carboxy terminal HA tag. COS-1 cells seeded on 100-mm Petri dishes (for immunoblotting) or glass cover slips (for microscopy) were transfected with a pEF3HA_p45 construct in the presence of the Lipofectamine TM transfec- tion reagent (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. 48 h after transfection, cells were washed in phosphate-buffered saline and fixed in freshly made 4% (v ⁄ v) formaldehyde in phos- phate-buffered saline. Fixed cells were permeabilized with 0.1% (w ⁄ v) saponin in phosphate-buffered saline, then blocked in 2.5% (v ⁄ v) normal goat serum in 0.1% (w ⁄ v) saponin in phosphate-buffered saline for 30 min at room temperature, and then incubated with primary and secon- dary Ab diluted in the same buffer for 1 h each at room temperature. For double immunostaining, the permeabilized cells were first incubated with anti-PtdIns(3,4,5)P 3 mAb, then with an Alexa FluorÒ 488 goat anti-(mouse IgG H + L) Ig and then with the TRITC- conjugated anti-HA mAb. After three washes with phosphate-buffered saline, cover slips with cells were mounted onto glass slides and viewed under a confocal laser scanning microscope (MRC- 1024; Bio-Rad, Hercules, CA, USA) with a 60· oil immers- ion objective. A differential interference contrast image was also taken of most cells. Acknowledgements We are grateful to Lewis C. Cantley for GFP con- structs. This work was supported by grants from the U.S. Civilian Research & Development Foundation M. Merkulova et al. Secretion of p45 Sec14p-like protein FEBS Journal 272 (2005) 5595–5605 ª 2005 FEBS 5603 (RB1-2338-MO-02, to TS and TM), the Russian Foun- dation of Basic Research (02-04-48364, to MM), the Scientific School (312-2003-4) and the Molecular and Cellular Biology (no.200101, to VL), grants AG00252 (to HH), AI55741 (to TM), and CA96949 (to TM) from the National Institutes of Health. 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Fig. 3. Secretion of the SEC14 domain of p45. (A) The hydrophilicity plot of p45 protein. physiological behavior of this protein. The mRNA for p45 was also detected in the surface layer of epithelial tissues [8]. p45 and the B A Fig. 4. p45 colocalizes