Secretion of the mammalian Sec14p-likephosphoinositide-binding p45 proteinMaria Merkulova1, Huong Huynh2, Vitaly Radchenko1, Kan Saito2, Valery Lipkin1,Tatiana Shuvaeva1 and Tomas Mustelin2

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)P3 [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)P3 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)P3. 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 quinonewas 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)P3 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)P3. 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)P3 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)P3 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-StripsTM, 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)P3 and, significantly weaker, to

PtdIns(3)P, PtdIns(4)P, PtdIns(3,5)P2, PtdIns(4,5)P2,

PtdIns(3,4)P2 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)P3 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)P3.

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

Mr (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

A B 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 StripsTM 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)P3 in

intact cells

Based on the finding that p45 bound to

PtdIns(3,4,5)P3 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)P3 mAb, Alexa Fluor� 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)P3 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)P3, 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)P3 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)P3 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)P3 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)P3, 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)P3 with the specific mAb plus Alexa Fluor� 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)P3 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)P3 may play important roles in

the protective nasal mucosa, perhaps as components

of the extracellular matrix. p45 may also release

PtdIns(3,4,5)P3 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)P3 to produce PtdIns(3,4,5)P3 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)P3 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)P3 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)P3 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 Fluor� 594 goat anti-(mouse IgG H + L) Ig and

Alexa Fluor� 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)P2 mAb, and commercial PIP StripsTM with 15

different phospholipids were from Echelon (Salt Lake City,

UT, USA). Each PIP StripTM had 100 pmol of PtdIns,

PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2,

PtdIns(3,5)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3, 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 RosettaTM (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 StripsTM) 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 lgÆmL)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 mm 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 LipofectamineTM 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)P3 mAb,

then with an Alexa Fluor� 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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