QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 17, Issue 6, 2498-2512, June 2006

This Article Abstract Full Text (PDF) Supplemental Material All Versions of this Article: E06-01-0089v1 17/6/2498    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Phillips, S. E. Articles by Bankaitis, V. A. Search for Related Content PubMed PubMed Citation Articles by Phillips, S. E. Articles by Bankaitis, V. A.

Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein -Isoform to the Mammalian Trans-Golgi Network

Scott E. Phillips^*,, Kristina E. Ile^*,, Malika Boukhelifa^*, Richard P.H. Huijbregts, and Vytas A. Bankaitis^*

*Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7090; and Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0021

Submitted January 30, 2006; Revised February 13, 2006; Accepted March 6, 2006
Monitoring Editor: Reid Gilmore

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITP localization in mammalian cells. We demonstrate PITP localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITP. Domain mapping analyses show the targeting information within PITP consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W₂₀₂W₂₀₃ motif). Combination of the specificity elements with the W₂₀₂W₂₀₃ motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITP association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITP isoform with a naturally occurring S₂₆₂Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITP localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms or . These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser₂₆₂ is required for PITP targeting to Golgi membranes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Because the discovery that a phosphatidylinositol transfer protein (PITP) plays an essential role in regulating the interface between lipid metabolism and membrane trafficking from the yeast trans-Golgi network (TGN; Bankaitis et al., 1990; Cleves et al., 1991a, 1991b), it has become increasingly clear that lipid metabolism regulates many individual trafficking steps throughout the secretory pathway (Cleves et al., 1991a; DeCamilli et al., 1996; Simonsen et al., 2001). In vivo studies demonstrate PITPs either control the efficiency at which trafficking reactions occur (Bankaitis et al., 1989, 1990; Cleves et al., 1991b; Kearns et al., 1997) or impart spatial organization to these reactions (Carmen-Lopez et al., 1994; Nakase et al., 2001; Vincent et al., 2005). PITPs do so by coupling their ability to bind and/or transfer specific lipids to the coordination of lipid metabolic pathways with specific membrane trafficking steps (see Phillips et al., 2006). In vitro reconstitution of various membrane trafficking or receptor-coupled signaling reactions also identify involvements for PITPs in these events (Hay and Martin, 1993; Ohashi et al., 1995; Cunningham et al., 1996; Jones et al., 1998).

The available evidence indicates confident assignment of function for any individual PITP requires in vivo studies with model genetic systems. The reconstituted systems that show PITP dependence are remarkably promiscuous from the perspective of source of PITP. This is amply demonstrated by the stoichiometric interchangeability of yeast and mammalian PITPs in such reconstitutions (Ohashi et al., 1995; Cunningham et al., 1996; Jones et al., 1998), even though these PITPs exhibit unrelated structural folds (Sha et al., 1998; Yoder et al., 2001; Tilley et al., 2004). By contrast, in vivo studies show even very closely related PITPs play nonredundant functions in cells (Li et al., 2000; Alb et al., 2002, 2003; Routt and Bankaitis, 2004; Vincent et al., 2005).

Mammalian cells express three soluble PITPs. PITP and PITP share 77 and 95% primary sequence identity and similarity, respectively, and are encoded by distinct genes. The third, rdgB, is considerably more diverged and remains largely unstudied (Fullwood et al., 1999). The shared homologies notwithstanding, PITP and PITP are functionally distinct (Alb et al., 2002, 2003). In this regard, PITP binds PtdIns and PtdCho, whereas PITP binds both those phospholipids and, in addition, sphingomyelin (SM; De Vries et al., 1995). Moreover, recombinant PITP and PITP localize to distinct compartments, the former to the cytosol and nucleus and the latter to the cytosol and a perinuclear compartment that is likely the Golgi complex (De Vries et al., 1995, 1996; van Tiel et al., 2002). The relationship between the distinct biochemical properties of these two PITP isoforms and localization and function (if any) remain to be determined.

Herein, we report that endogenous PITP (and a novel spliceoform thereof) localizes predominantly to TGN membranes and that localization is specified by a functionally redundant set of three short C-terminal motifs. These motifs are collectively insufficient to target a naive reporter to Golgi membranes, but cooperate with a W₂₀₂W₂₀₃ motif to generate an efficient TGN-targeting module. We also show that the phospholipid-bound status of PITP does not contribute to its association with the TGN. Finally, in contrast to a previous claim (van Tiel et al., 2002), our data indicate that neither localization of PITP nor its novel spliceoform to Golgi membranes is obligately regulated by conventional protein kinase C (PKC)-mediated phosphorylation of residue serine 262.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mammalian Cell Culture and Transfections
Murine embryonic fibroblasts (MEFs) were derived from E16.5 wild-type and PITP^–/– embryos as previously described (Alb et al., 2003). The mammalian cell lines used in this study were cultured in DMEM containing 10% fetal bovine serum, 1 U/ml penicillin G, 100 µg/ml streptomycin, and 4.2 µl -mercaptoethanol (for 500 ml of complete medium). Cultures were incubated at 37°C and in 5% CO₂.

COS-7 cells were transfected using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). Briefly, 24 h before transfection the cells were plated at 50–60% confluency in six-well plates containing glass coverslips. DNA (1.5–2 µg) was reconstituted in 100 µl of OptiMEM (Invitrogen), mixed with 2 µl of Plus reagent, and incubated at room temperature for 15 min. In a separate microcentrifuge tube, 3 µl of Lipofectamine was diluted in 100 µl of OptiMEM for each transfection. After 15 min, the solutions were mixed and then incubated for 15 min at 25°C. Cells were washed twice with OptiMEM and incubated at 37°C with DNA mixture in 1 ml OptiMEM for 3 h. Subsequently, 4 ml of complete medium was added, and cells were cultured for 18–24 h before processing for immunocytochemistry. MEFs were transfected using the Amaxa (Cologne, Germany) nucleofector following the manufacturer's directions.

Antibody Reagents
PITP antibodies used in this study included: a PITP isoform–specific rabbit polyclonal antibody directed against the C-terminal 25 amino acid of PITP (generous gift from Bruce Hamilton), a PITP isoform–specific chicken polyclonal antibody directed against the last 15 amino acids of PITP (Alb et al., 2002), and the NT-PITP-antibody rabbit polyclonal immunoglobulin (Ig) raised against the N-terminus of PITP and that recognizes both PITP and PITP (generous gift of Prof. George Helmkamp, Jr.).

The following primary antibodies were used: a monoclonal antibody directed against actin (Chemicon, Temecula, CA), sheep polyclonal anti-TGN38 Ig (Serotec), monoclonal anti-GM130 antibodies (BD Bioscience, San Diego, CA), and murine monoclonal anti-giantin Ig (generous gift from Dr. Hans Peter Hauri, Switzerland). Secondary antibodies used included: Alexa fluorescein isothiocyanate 488 (Molecular Probes, Eugene, OR), Cy5-conjugated anti-mouse and fluorescein isothiocyanate–conjugated anti-mouse (Jackson ImmunoResearch, West Grove, PA), and goat anti-rabbit, goat anti-mouse, or goat anti-chicken horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch).

Immunocytochemistry
Cells were cultured on glass coverslips. Cells were fixed for 15 min with 3.7% formaldehyde in phosphate-buffered saline (PBS), permeabilized in 0.2% Triton X-100 in PBS for 4 min, rinsed once in PBS, and then preincubated for 30 min in blocking buffer (2% BSA in PBS). Permeabilized cells were subsequently incubated with suitable primary antibody appropriately diluted in blocking buffer for 1 h at room temperature, rinsed four times 5 min with PBS, and then incubated with the secondary antibodies appropriately diluted in blocking buffer for 1 h. Cells were rinsed four times in PBS, and coverslips were mounted onto glass slides and examined in a Leica SP2 Laser Scanning Confocal Microscope (Leica, Deerfield, IL). Images were processed with the use of Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA).

In classifying PITP localization profiles as "‘Golgi," two major criteria were applied. First, for to score a profile as Golgi the appropriate query profile (GFP or PITP) must exhibit obvious and predominant colocalization with a Golgi marker (TGN38 or GM130). Second, the Golgi component of the query profile must be the strongest signal recorded in the cell being scored. Failure to satisfy both these criteria resulted in a non-Golgi score. Fixed and stained samples were blinded before scoring to control for investigator bias.

Pharmacological Challenge
PITP^–/– MEFs were grown on glass coverslips to subconfluency and intoxicated with chelerethryne chloride (0.66 µM; Sigma, St. Louis, MO) or G109203X (10 nM; Sigma) for appropriate times. PKC activity in MEFs was also stimulated by exposure of cells grown on coverslips to PMA (100 nM, Sigma) for 15 min in serum-free medium. Cells were subsequently fixed for PITP immunostaining as described above. Cell-free extracts were prepared for parallel-treated cultures and processed for immunoblot analysis as described below.

SDS-PAGE and Immunoblotting
Cultures were rinsed with ice-cold PBS and scraped into lysis buffer (20 mM Tris-HCL, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 mM NaF, 1% Triton 1 mM orthovanadate supplemented with a cocktail of protease inhibitors (Complete; Roche, Indianapolis, IN). For preparation of cell-free extracts, cells (grown to confluency in a 100-mm dish) were incubated with 700 µl of lysis buffer at 4°C for 10 min and then scraped with a rubber policeman into microcentrifuge tubes. After centrifugation at 14,000 x g for 10 min, the supernatant was mixed in Laemmli sample buffer and heated for 5 min at 95°C. Samples were resolved by SDS-PAGE (10%) and transferred to nitrocellulose (Millipore, Billerica, MA). Membranes were blocked overnight at 4°C in TBST (5% dry nonfat milk in 0.05% Tween 20 in Tris-buffered saline) and then incubated for 3 h at room temperature with the appropriate primary antibodies diluted in TBST. Membranes were rinsed four times for 5 min each with TBST and then incubated with the appropriate HRP-conjugated secondary antibody for 1 h, and washed four times for 5 min each with TBST. Blots were developed on x-ray film (Eastman Kodak, Rochester, NY) using the enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham, Arlington Heights, IL).

Generation of PITP-GFP and PITP-GFP cDNAs
PCR primers for rat-PITP and rat-PITP cDNA sequences were flanked on the 5' end with the restriction enzyme site HindIII and on the 3' end with the restriction enzyme site BamHI. The HindIII-BamHI PCR fragments were cloned into the pEGFP-C1 plasmid (Clontech, Palo Alto, CA). Yeast plasmids harboring PITP and PITP cDNAs (Skinner et al., 1993) were used as templates in the PCR reactions used for generating the appropriate DNA fragments for cloning. The resulting plasmids were designated pRE772 (PITP-GFP) and pRE774 (PITP-GFP). Primer sequences used are available from the authors by request.

Yeast Complementation Assay
Wild-type and mutant PITP or PITP-GFP cDNAs, as appropriate, were cloned into the multicopy yeast URA3 vector YEplac195 such that the cDNA was expressed either under control of the powerful constitutive PGK promoter or the constitutively expressed but weaker SEC14 promoter. This expression vector was transformed into the sec14-1^ts yeast strain (CTY 1-1A, MATa ura3-52 his3200, lys2-810 sec14-1^ts; Cleves et al., 1991b) using the lithium acetate method of Ito et al. (1983). As matched controls, isogenic vectors with either no insert or with SEC14 or PITP cDNA inserts were also transformed into the sec14-1^ts yeast host strain. Transformants were selected and cultured in uracil-free glucose minimal medium (Sherman et al., 1983). Five OD₆₀₀ equivalents of each strain were resuspended in 200 µl Tris-EDTA buffer and serially diluted 10-fold in Tris-EDTA buffer. An aliquot (5 µl) of each dilution was spotted on duplicate YPD agar plates. One plate was incubated at the 30°C (a permissive temperature for sec14-1^ts mutants) to report unrestrained growth and viability. The companion plate was incubated at 37°C (normally a restrictive temperature for sec14-1^ts mutants) to assess phenotypic rescue of sec14-1^ts.

Phospholipid-Transfer Assays
Assays were performed using cytosol prepared from the sec14 cki1 host strain CTY303 expressing the desired PITP as described previously (Kearns et al., 1998; Phillips et al., 1999; Li et al., 2000; Vincent et al., 2005). Cytosol fractions generated from CTY303 variants expressing Sec14p (positive control) or no PITP (negative control) were generated and assayed in parallel with those fractions containing PITP, PITP, or PITP variants.

Site-directed Mutagenesis
The QuickChange kit (Stratagene, La Jolla, CA) was used. Sequences of the various mutagenic primers used are available from the authors by request. All mutant versions generated were verified by nucleotide sequence analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Endogenous PITP Localizes to the Mammalian Golgi Complex
Previous experiments suggesting a Golgi localization of PITP in mammalian cells relied on microinjection of purified fluorophore-modified protein into cells (De Vries et al., 1996) or creation of stable cell lines that overexpress PITP (van Tiel et al., 2002). As a result, several key questions regarding PITP localization remain. First, it remains to be demonstrated whether endogenous PITP is genuinely a Golgi membrane–associated protein. Second, the precise distribution of PITP within the Golgi stack also remains to be determined.

Specific localization of endogenous PITP was complicated by our observation that antibodies generated against the extreme C-terminal 15- and 25-residue peptides of these proteins, although facile for distinguishing PITP from PITP by immunoblotting, are not satisfactory for immunofluorescence experiments (unpublished data). To circumvent this issue, we used polyclonal antibodies raised against amino-terminal sequences conserved between PITP and PITP. These antibodies (NT-PITP-antibody) are suitable for immunofluorescence but are not specific reagents in that these recognize both PITP and PITP isoforms in immunoblotting experiments. The specificity issue notwithstanding, we inspected the endogenous PITP immunofluorescence staining profiles obtained with NT-PITP-antibody in an array of cell lines. Swiss 3T3 fibroblasts exhibited a strong perinuclear staining of what appears to be the Golgi apparatus and a diffuse signal in the cytoplasm and the nuclear matrix (Figure 1A). The PITP profiles obtained with Swiss 3T3 cells and NT-PITP-antibody as reporter were typical. Very similar results were also obtained with a variety of other cell lines including astrocytes, primary neurons, and COS-7, HeLa, and HEK293 cells. That the perinuclear PITP staining identifies the Golgi complex is indicated by the coincidence of this profile with that obtained for the cis-Golgi marker GM130 (Figure 1A). As the NT-PITP-antibody immunofluorescence profiles collected with immortalized cell lines represent the sum of endogenous PITP and PITP distribution, and previous studies indicate PITP localizes to the cytoplasm and nuclear matrix (De Vries et al., 1996), these various localization profiles suggest that endogenous PITP targets to Golgi membranes in a variety of cell types.

View larger version (48K): [in this window] [in a new window]   Figure 1. Endogenous PITP localization profiles. (A) Fixed and permeabilized cells of the indicated cell type were stained with a PITP antibody that detects PITP and PITP and antibodies directed against the Golgi marker GM130. The PITP (top panels) and GM130 profiles (bottom panels) are shown. Arrows indicate one example of the clear colocalization of an endogenous PITP with Golgi membranes for each cell type and orient the remaining Golgi profiles in the matched panels. (B) PITP–/– MEFs were fixed and decorated with primary antibodies directed against PITP antigen or the cis-Golgi marker GM130. Representative individual profiles for endogenous PITP and GM130 are shown in the left panels, as indicated, and the merged profile is depicted in the right panel. (C) PITP-GFP chimera is a functional protein. Serial 10-fold dilutions of isogenic sets of a sec14-1ts strain, derivatives of that strain carrying a high-copy plasmid (YEp) driving expression of either PITP, PITP-GFP, or a wild-type SEC14 gene (as indicated) were spotted onto YPD agar and incubated at 37°C for 48 h. The 37°C condition, although permissive for growth of wild-type yeast, is restrictive for growth of sec14-1ts yeast mutants. This sec14-1ts growth defect is rescued by expression of either PITP or the PITP-GFP chimera, indicative of preservation of PITP activity in the PITP-GFP chimera. Strains used: CTY1-1A (sec14-1ts), and CTY1-1A transformed with YEp(SEC14), YEp(PITP), and YEp(PITP-GFP), respectively. The respective PITP genes were driven by the strong and constitutively expressed yeast PGK promoter. (D) PITP-GFP faithfully targets to the Golgi complex. PITP nullizygous MEFs were transfected with a PITP-GFP expression plasmid, fixed, and decorated with primary antibodies directed against GFP antigen and antibodies directed against GM130, as indicated. Representative individual profiles for PITP-GFP and GM130 are shown in the left panels, and the merged profile is depicted in the right panel.

To confirm localization of the known PITP, we constructed a PITP-GFP chimera, where GFP was fused to the C-terminus of PITP. The activity of the PITP-GFP chimera was established with a yeast phenotypic rescue assay. This assay capitalizes on previous demonstrations that high-level expression of mammalian PITPs in yeast rescues the growth and secretory defects associated with inactivation of the essential yeast PITP Sec14p (Skinner et al., 1993; Tanaka and Hosaka, 1994). This rescue is dependent on robust PtdIns-binding/transfer by the heterologous mammalian PITP (Alb et al., 1995). As shown in Figure 1C, a sec14-1^ts yeast strain carrying an ectopic copy of the wild-type SEC14 gene grows robustly at 37°C. By contrast, the isogenic sec14-1^ts strain fails to grow at all at 37°C, i.e., the restrictive temperature at which the thermolabile sec14-1^ts gene product is inactive. Expression of PITP-GFP restored robust growth to the sec14-1^ts yeast mutant at the restrictive 37°C temperature.

The functional PITP-GFP was expressed in MEFs and the distribution of the chimera was monitored. These localization experiments confirm an unambiguous affinity of PITP-GFP for Golgi membranes in MEFs (Figure 1D) and also in COS-7 cells (see below).

PITP Selectively Associates with the TGN
Although both Golgi and ER membranes harbor pools of PITP, Golgi localization predominates and how PITP targets to the Golgi membrane system is the focus of this study. To more precisely assign the Golgi subcompartment of residence for endogenous PITP, we performed a series of double-label immunofluorescence experiments. In these experiments, NT-PITP-antibody was used in combination with compatible antibodies raised against markers for specific Golgi compartments. These markers included GM130 for cis-Golgi, giantin for cis- and medial-Golgi, and TGN38 for the TGN. PITP nullizygous MEFs were used to ensure specific detection of endogenous forms of PITP.

As shown in Figures 2, A and B, endogenous PITP exhibits little coincidence of staining with the cis-Golgi marker GM130, or the medial-Golgi marker giantin, even though the general profiles for PITP and these markers are very similar. Endogenous PITP species exhibit a higher degree of colocalization with the trans-Golgi membrane marker TGN38, however (Figure 2C). The predominant localization of PITP to TGN membranes is emphasized in a stereo reconstruction of the MEF Golgi apparatus generated from triple-label experiments monitoring PITP, giantin, and TGN38 (Supplemental Video, Figure S1). The rotating image distinguishes giantin staining from the yellow staining that reports colocalization of TGN38 and PITP. We infer from these experiments that PITP targets predominantly to the trans-aspect of the Golgi stack in MEFs.

View larger version (84K): [in this window] [in a new window]   Figure 2. PITP localizes specifically to TGN membranes. PITP nullizygous MEFs were fixed and decorated with primary antibodies directed against PITP antigen (rabbit polyclonal NT-PITP-antibody) and antibodies directed against either the cis-Golgi marker GM130 (A), the medial-Golgi marker giantin (B), or the trans-Golgi marker TGN38 (C). The individual and merged profiles are identified at the top. The respective insets represent a higher magnification of the boxed region of the corresponding merged profile for purposes of enhanced detail.

PITP C-terminal Motifs Necessary for TGN Targeting
The distinctive localization profiles for PITP and PITP are remarkable in light of the high degree of primary sequence identity shared by these PITPs. To map the determinants specifying targeting of PITP to the mammalian TGN in an unbiased manner, we constructed a reciprocal series of PITP/PITP hybrid proteins in the context of a functional PITP-GFP chimera. The functional status of key chimeras was confirmed in the heterologous yeast sec14-1^ts phenotypic rescue assay (Skinner et al., 1993; described above and in Figure 1C). All chimeras generated were active in the yeast phenotypic rescue assay and were expressed both in PITP^–/– MEFs and in COS-7 cells. The respective intracellular distributions were imaged and quantified for both cell types. In describing the results of the mapping experiments, we present data obtained with MEFs and report the COS-7 data in Supplemental Materials.

The C-terminal 28 PITP residues are both necessary for PITP targeting to Golgi membranes and are sufficient to efficiently redirect PITP to that location (Figure 3A). The results were robust because the incidence of Golgi targeting in cells was >90% for PITP and the PITP/ chimera and <5% for PITP and the PITP / chimera. Representative images for each chimera are shown in Figure 3B. In the imaging experiments reported herein, we typically identify the Golgi region by surveying the cis-Golgi marker GM130 but confirmed that assignment by costaining with the pan-Golgi marker wheat germ agglutinin and, for key reporter/mutant constructs, by costaining for TGN38 (see below).

View larger version (32K): [in this window] [in a new window]   Figure 3. C-terminal PITP localization elements necessary for TGN association. (A) Alignment of the C-terminal 28 residues of PITP with the corresponding region of PITP is given. Schematic illustrations of PITP, PITP, and each of the reciprocal C-terminal swaps are depicted at bottom. At right, for each corresponding PITP version is given the number of imaged cells that exhibited a Golgi (G) or non-Golgi (N) immunofluorescence profile when that construct was expressed in MEFs as a PITP-GFP chimera and visualized along with the GM130 marker. The percentage of imaged cells with Golgi profiles is also given. (B) Imaging of PITPs with exchanged C-terminal regions. Representative localization profiles for PITP/-GFP and PITP/-GFP when expressed in PITP–/– MEFs are shown. Individual PITP-GFP and GM130 profiles are presented in the bottom panels underneath the corresponding merged profile. (C) Swap of divergent BOX motifs from PITP into the context of PITP. The BOX motifs are defined at top, and the most divergent residues within each are highlighted (). The series of hybrid PITPs analyzed is illustrated and each swap is further defined at left by identification of which PITP residues were introduced to generate the swap. Quantification of PITP–/– MEFs expressing each individual hybrid with respect to number of cells displaying Golgi (G) or non-Golgi (N) localization profile, along with percentages of cells displaying Golgi localization, is also given. (D) Representative images of PITP–/– MEFs individually expressing each of the three PITP-GFP chimeras where two of the three BOX motifs were mutagenized to PITP versions. The identities of the swaps are indicated at top. Individual PITP-GFP and GM130 profiles are presented in the bottom panels underneath the corresponding merged profile.

PITP C-terminal Motifs Sufficient for Targeting PITP to Golgi Membranes
To address the dual criteria of necessity and sufficiency, we tested whether any BOX residues sufficient for PITP localization to the TGN were capable of redirecting PITP to the same. To this end, PITP BOX1 or BOX3 residues were incorporated into the context of an otherwise wild-type PITP. The localization profiles of both constructs (PITP^QET and PITP^TSA) fully recapitulated the nuclear and cytoplasmic distribution of the PITP control (Figure 4A). Thus, neither BOX1 nor BOX3 has an assignable targeting function on its own in the context of PITP. However, BOX2 residues, although dispensable for PITP targeting to the Golgi complex, increased the efficiency with which an otherwise wild-type PITP reporter associates with Golgi membranes. That construct (PITP^KGSR) was scored as targeting to Golgi membranes in 51% of the transfected cells analyzed. Although this level of targeting is not as robust as that observed with the PITP positive control (>90%), it is substantial when compared with the basal association of the PITP control with the Golgi complex (ca. 5%; Figure 4A).

View larger version (60K): [in this window] [in a new window]   Figure 4 (facing page). C-terminal PITP localization elements sufficient for redirecting PITP to TGN membranes. (A) Swap of divergent BOX motifs from PITP into the context of PITP. The BOX motifs are defined at the top, and the most divergent residues within each are highlighted (). The series of hybrid PITPs is illustrated and each swap is further defined at left by identification of which PITP residues were introduced to generate the swap. Quantification of PITP–/– MEFs expressing each individual hybrid with respect to number of cells displaying Golgi (G) or non-Golgi (N) localization profile, along with percentages of cells displaying Golgi localization, is also given. Representative images of PITP–/– MEFs individually expressing: (B) each of the three PITP-GFP chimeras where two of the three BOX motifs were mutagenized to PITP versions. The identities of the swaps are indicated at top. Individual PITP-GFP and GM130 profiles are presented in the bottom panels underneath the corresponding merged profile. (C) Each of the three PITP-GFP chimeras where residue S262 is mutagenized to A, D, E, or P as indicated. Individual PITPS262-GFP and GM130 profiles are presented in the bottom panels underneath the corresponding merged profile. (D) Each of the three PITP-GFP chimeras where residue P263 is mutagenized to S (the corresponding PITP residue) or the phosphomimetic residues D or E as indicated at top. Individual PITPP263-GFP and GM130 profiles are presented in the bottom panels underneath the corresponding merged profile. (E) The C-terminal 28 residues of PITP and the novel PITPQGQR spliceoform are aligned at top, and the BOX motifs are identified. Differences in primary sequence are highlighted in red. The position of the S259A mutation in PITPQGQR is also indicated. Representative profiles for the corresponding GFP chimeras and GM130 are shown, as are the merged profiles. In B–E quantification of number of cells displaying Golgi (G) or non-Golgi (N) localization profile, along with percentages of cells displaying Golgi localization, is given.

PITP Ser₂₆₂ Is Nonessential for Golgi Localization
The dispensability of BOX2 for PITP localization to TGN membranes was counter to the findings of van Tiel et al. (2002), who reported that phosphorylation of a BOX2 residue (S₂₆₂) is essential for PITP targeting to Golgi membranes. Yet, our demonstration that swap of PITP BOX2 residues significantly improved PITP targeting to Golgi membranes (i.e., the PITP^KGSR construct; Figure 4A) is consistent with a more substantial role for BOX2 in Golgi targeting. To investigate these paradoxical findings in more detail, we analyzed the involvement of S₂₆₂ itself in PITP localization. Consistent with the results of the BOX2 chimera experiments, PITP^S262A, PITP^S262D, PITP^S262E, and PITP^S262P all targeted to Golgi membranes as efficiently as the PITP control (Figure 4C). These results were recapitulated in the context of COS-7 cells (Supplemental Figures and Supplemental Table S1).

To determine whether an analogous phosphorylation may be sufficient to redirect PITP to Golgi membranes, we incorporated phosphomimetic amino acids at the corresponding P₂₆₃ residue of PITP to generate the PITP^P263D and PITP^P263E mutants. The ability of each to associate with MEF TGN membranes was then assessed. Neither PITP^P263D nor PITP^P263E targeted to Golgi membranes any more efficiently than the PITP control (Figure 4D). We repeated these analyses in COS-7 cells. Again, neither incorporation of the P₂₆₃S missense substitution, nor P₂₆₃E, into the context of PITP-GFP had any major effect on the intracellular distribution of the chimera (Supplemental Materials, Supplemental Table S1).

A Novel PITP Isoform with Altered BOX2 Residues Targets to the TGN
All of the experiments described above rely on mutagenesis of canonical PITP. The novel murine PITP spliceoform described above (PITP^QGQR) differs from canonical PITP predominantly in BOX2 (Figure 4E). Interestingly, S₂₆₂ of canonical PITP is Q₂₆₂ in PITP^QGQR. PCR assays indicate both PITP and PITP^QGQR are expressed in PITP^–/– MEFs at approximately equal levels.

Localization experiments using GFP-tagged forms show PITP^QGQR, like PITP, associates with MEF Golgi membranes (Figure 4E). These results are consistent with data indicating S₂₆₂ is nonessential for efficient targeting of PITP species to that compartment. PITP^QGQR displays a single nonconserved serine residue (S₂₅₉) in the region of divergence, but the S₂₅₉A mutation has no effect on targeting of a PITP^QGQR-GFP chimera to TGN membranes (Figure 4E). These data lead us to form two conclusions in addition to S₂₆₂ dispensability for PITP targeting to Golgi. First, S₂₅₉ does not offer an alternative phosphorylation site required for PITP^QGQR association with Golgi membranes. Second, mammalian cells can express more than one PITP species in cells but, in this case, both PITP and PITP^QGQR isoforms home to Golgi membranes. Comparison of the localization profiles of PITP-GFP and PITP^QGQR-GFP chimeras indicates both target to the TGN, although PITP^QGQR-GFP also exhibits partial colocalization with the medial-Golgi marker mannosidase II (Supplemental Materials, Figure S4, A and B). PITP^QGQR-GFP also appears to target more efficiently to cis-Golgi membranes than does PITP-GFP (Supplemental Materials, Figure S4C). Thus, PITP^QGQR may represent more of a pan-Golgi PITP than the canonical PITP.

PITP Targeting to Golgi Membranes Is Independent of PtdIns- or SM-Transfer Activity
As described in detail below, the C-terminal 28 PITP residues are sufficient to redirect PITP to Golgi membranes but are insufficient to target a naive protein to this intracellular location. These data suggest that multiple localization signals may be involved in localizing PITP to the TGN and that a subset of these determinants likely resides in the PITP domain itself. As PITP domains represent specific lipid-binding modules, PITP lipid-binding properties themselves could potentially define components of a combinatorial targeting signal.

To test this possibility, we took advantage of mutant PITP derivatives with selective defects in the loading/transfer of defined phospholipid substrates. Given that PITP is distinguished from PITP in its ability to bind SM in addition to PtdIns and PtdCho, one attractive possibility is that SM loading contributes to the affinity of PITP for Golgi membranes. Data obtained from three independent lines of experimentation demonstrate that SM-loading and Golgi targeting are not strictly coupled. First, wholesale swap of the PITP C-terminal 28 residues into the context of PITP leads to a PITP chimera that fails to associate with the Golgi complex (see Figure 3, A and B above). Yet, this PITP/ chimera exhibits robust SM transfer in vitro (Figure 5A). Second, reciprocal swap of the PITP C-terminal 28 residues into the context of PITP results in a hybrid PITP/ that efficiently targets to the Golgi complex (see Figure 3, A and B above). This PITP/ chimera, while elaborating both PtdIns- and PtdCho- transfer activity, exhibits no detectable SM-loading/transfer activity in vitro (Figure 5A). Third, substitution of only two amino acids in PITP to the cognate PITP residues is sufficient to confer robust SM-transfer activity to PITP (PITP^LF221,225IL; Figure 5A). Reciprocally, conversion of those cognate PITP residues to the corresponding PITP residues strongly and specifically compromises the SM-transfer activity of PITP (PITP^IL220,224LF; Figure 5A). In neither case does modulation of SM-loading/transfer affect PtdIns- or PtdCh-transfer activity or PITP localization. PITP^IL220,224LF fully retains the ability to target efficiently to Golgi membranes, whereas PITP^LF221,225IL does not (Figure 5B).

View larger version (87K): [in this window] [in a new window]   Figure 5. PITP localization to TGN membranes is independent of phospholipid loading. (A) Phospholipid-transfer properties of select PITP chimeras. Abilities of each individual PITP or PITP chimera to transfer [14C]-SM, [3H]PtdIns, or [14C]PtdCho (as indicated) was determined in cytosol fractions prepared from yeast strain CTY303 (sec14 cki1) expressing recombinant versions of the respective PITPs (Phillips et al., 1999; Li et al., 2000). CTY303/YEp(URA3) cytosol was prepared and used as negative control. Activity is represented as the percentage of total input radiolabeled phospholipid transferred from donor membranes to unlabeled acceptor membranes during the course of the experiment. Assay blanks represented addition of buffer alone to the transfer assay reactions, and these background values were subtracted from the other measurements. Values represent the averages of triplicate determinations from a representative experiment, and at least three independent experiments were performed. In the experiment shown, input phospholipid-transfer substrate was 19,850 cpm [14C]SM; 21,050 cpm [3H]PtdIns; 21,250 cpm [14C]PtdCho. Background values for these respective transfer assays were 700, 315, and 820 cpm. A representative image of PITP–/– MEFs expressing a (B) PITPIL220,224LF-GFP or a PITPLF221,225IL-GFP chimera. Cells were imaged for GFP and the pan-Golgi marker wheat germ agglutinin (WGA), as indicated. Corresponding merged profiles are shown. (C) PITPT58D-GFP chimera. PITPT58D-GFP and GM130 profiles are presented in the bottom panels underneath the corresponding merged profile. (D) PITPS165A-GFP or a PITPS165,262A-GFP chimera. Cells were imaged for GFP and GM130, as indicated. Corresponding merged profiles are shown. For C and D, quantification of number of cells displaying Golgi (G) or non-Golgi (N) localization profiles, along with the percentages of cells displaying Golgi localization, are given for each construct at the bottom of the corresponding panel set.

Uncoupling of Phospholipid-Transfer Activity from PITP Targeting to Golgi Membranes
Although neither PtdIns- or SM-loading/transfer activity are required for PITP association with the TGN, a combination of phospholipid-loading/transfer activities could contribute to such targeting. We therefore tested whether residue Ser₁₆₅ is required for targeting of PITP to the Golgi complex. This residue lies in the PITP regulatory loop (Yoder et al., 2001). The side-chain status of this residue is functionally important because incorporation of either alanine or glutamate at this position abolishes all PITP phospholipid-transfer activities (van Tiel et al., 2000).

The PITP^S165A mutant was generated and its biochemical properties were assayed in vitro. As reported by van Tiel et al. (2000) for PITP^S166A, we too find PITP^S165A exhibited no measurable PtdIns-, PtdCho-, or SM-transfer activity, even though full-length PITP was readily detected in the cytosolic fractions by immunoblot (unpublished data). The S₁₆₅A missense substitution had no effect on PITP localization when assayed in PITP^–/– MEFs. PITP^S165A-GFP was targeted as efficiently to Golgi membranes as the PITP-GFP control (Figure 5D). Because PITP^S165A exhibits no detectable phospholipid-transfer activity, PITP^S165A-GFP association with Golgi membranes is independent of phospholipid-transfer activity.

We also expressed PITP^S165,262A-GFP in PITP^–/– MEFs and assessed the ability of this double mutant to target to TGN membranes. This experiment was motivated by the demonstration that PITP residue S₁₆₆ and PITP residue S₁₆₅ are minor PKC phosphorylation sites (van Tiel et al., 2000, 2002). PITP^S165,262A is therefore devoid of the PKC phosphorylation sites for which there is any evidence of use. Yet, PITP^S165,262A-GFP homes to TGN membranes in PITP^–/– MEFs (Figure 5D). These data provide further support for our conclusion that PKC-mediated phosphorylation of PITP (at least on the presently known S₁₆₅ and S₂₆₂ sites) does not play an essential role in localization of this protein to mammalian TGN membranes.

A WW Motif Common to PITP and PITP Contributes to Association of PITP with TGN Membranes
PITP occupied with either PtdCho or PtdIns crystallizes as a dimer, and the dimerization interface is defined by two small hydrophobic motifs displayed on exposed loops of the PITP fold (Figure 6A; Yoder et al., 2001; Tilley et al., 2004). These two motifs are represented by ₇₂FVRML₇₆ and W₂₀₃W₂₀₄ of PITP and ₇₁FVRMI₇₅ and W₂₀₂W₂₀₃ of PITP, respectively, and both are suggested to play critical roles in mediating membrane binding by PITP (Schouten et al., 2002; Tilley et al., 2004). To test whether ₇₁FVRMI₇₅ or W₂₀₂W₂₀₃ contribute to localization of PITP to the murine TGN, we mutagenized these motifs in the context of a PITP-GFP reporter and analyzed localization of the corresponding reporters in PITP^–/– MEFs. The comprehensive data are quantified in Figure 6B, and representative imaging profiles for the corresponding mutant PITP forms are given in Figure 6C.

View larger version (55K): [in this window] [in a new window]   Figure 6. General PITP elements required for PITP localization to TGN membranes. (A) Ribbon diagram of the PtdIns-bound PITP crystal structure with space-fill renditions of the M74I75 and W202W203 side-chains, as indicated. (B) Quantification of percentage of transfected PITP–/– MEFs displaying Golgi localization profiles for each PITP-GFP construct (identified at bottom). The ratio of number of cells imaged with clear Golgi profiles (G) for the indicated PITP-GFP chimera to the number of cells imaged for that chimera that show a non-Golgi profile (N) is given above each corresponding bar. (C) Representative images of PITP–/– MEFs expressing the indicated PITP-GFP chimeras. The localization profiles for the indicated PITP-GFP (bottom left panels), corresponding TGN38 (bottom right panels), and merged profiles (top panels) are presented.

Previous data obtained from PtdIns loading assays performed with permeabilized cells indicated PITP^WW202,203AA is strongly defective in PtdIns loading and is incompetent for the membrane interaction step of a phospholipid-transfer reaction (Tilley et al., 2004). We obtained two lines of evidence that are not congruent with this conclusion, at least in the PITP context. First, biochemical assays for phospholipid-transfer activity demonstrate PITP^MI74,75AA and PITP^WW202,203AA exhibit significant levels of PtdIns-, PtdCho-, and SM-transfer activity in vitro (Figure 7A). Second, we again took advantage of the yeast phenotypic rescue assay described above to independently assess whether the phospholipid-binding/transfer activities of the double mutant PITPs were strongly compromised. The results from that rescue assay also support the conclusion that both PITP^MI74,75AA and PITP^WW202,203AA are substantially functional proteins. As shown in Figure 7B, a wild-type yeast strain grows robustly at 30 and 37°C. By contrast, an isogenic sec14-1^ts strain grows only at the permissive temperature of 30°C and not at all at the restrictive temperature of 37°C, i.e., the temperature at which the thermolabile sec14-1^ts gene product is inactive. PITP expression from either a strong constitutive promoter (P_PGK) or a weaker constitutive promoter (P_SEC14) restored essentially wild-type growth properties to the sec14-1^ts yeast mutant. Similarly, expression of either PITP^MI74,75AA or PITP^WW202,203AA from the P_PGK driver also supported efficient rescue of sec14-1^ts-associated growth defects at 37°C (Figure 7B). Rescue mediated by both mutant PITP forms was also recorded when the mutant proteins were expressed from the weaker P_SEC14 promoter, although quality of rescue was diminished slightly under those conditions for PITP^WW202,203AA (Figure 7B).

View larger version (65K): [in this window] [in a new window]   Figure 7. Properties of PITPW202W203 interaction with membranes. (A) Phospholipid-transfer assays. Abilities of each individual PITP to transfer [3H]PtdIns, [14C]PtdCho, or [14C]-SM, (indicated at top) was determined in cytosol fractions prepared from yeast strain CTY303 (sec14 cki1) expressing the negative control gene URA3 (black bars), or recombinant versions of the respective PITPs (PITP, white bars; PITPMI74,75AA, hatched bars; PITPWW202,203AA, stippled bars). Activity is represented as the percentage of total input radiolabeled phospholipid transferred from donor membranes to unlabeled acceptor membranes during the course of the experiment. Values represent the averages of triplicate determinations from a representative experiment, and at least three independent experiments were performed. Assay blanks represented addition of buffer alone to the transfer assay reactions, and corresponding background values were subtracted from the other measurements. In this set of assays, input substrate was 14,792 cpm [3H]PtdIns; 27,940 cpm [14C]PtdCho; 22,216 cpm [14C]SM, respectively. Background values were 295, 485, and 236 cpm for each respective assay. (B) PITP WW202,203AA mutants preserve function as assayed in yeast. Serial 10-fold dilutions of isogenic sets of a sec14-1ts strain, derivatives of that strain carrying a high-copy plasmid (YEp) driving expression of either PITP, PITP-WW202,203AA, PITP-MI74,75AA, or a wild-type SEC14 gene (as indicated) were spotted onto YPD agar and incubated at 37°C for 48 h. Strains used were CTY1-1A (sec14-1ts), and CTY1-1A transformed with YEp(SEC14), YEp(PITP), YEp(PITP-MI74,75AA), and YEp(PITPWW202,203AA), respectively. The PITP genes were driven by the strong and constitutively expressed yeast PGK promoter (PPGK) or the weaker constitutively expressed SEC14 promoter (PSEC14), as indicated.

PITP Motifs Sufficient for Redirection of GFP to the TGN
The collective data suggest it is the combination of weak membrane targeting/association signals defined by the C-terminal BOX residues and the W₂₀₂W₂₀₃ motif that specifies PITP association with TGN membranes. To test this prediction we fused the C-terminal 35 and 71 residues of PITP to the GFP C-terminus. The former chimera (GFP-PITP^237–271) elaborates all three of the C-terminal BOX motifs, is predicted to preserve the C-terminal PITP helix, but lacks both the W₂₀₂W₂₀₃ motif and obviously lacks an intact PITP fold. The latter chimera (GFP-PITP^201–271) elaborates both W₂₀₂W₂₀₃ and the three BOX motifs, is predicted to maintain the ultimate two PITP helices, but lacks an intact PITP fold. The chimeras were expressed in PITP^–/– MEFs and their respective intracellular distributions were determined.

As expected, the GFP control distributes to the cytoplasm and nuclear matrix and fails to associate with Golgi membranes as evidenced by its lack of colocalization with the TGN marker TGN38 (Figure 8). This profile was recapitulated for the GFP-PITP^237–271 chimera that harbors all three of the C-terminal BOX motifs but no W₂₀₂W₂₀₃ motif. By contrast, GFP-PITP^201–271 targeted efficiently to PITP^–/– MEF TGN as demonstrated by its colocalization with TGN38-positive structures (Figure 8). Some 85% of the cells showed coincident localization of GFP-PITP^201–271 with the TGN. Thus, linking the PITP W₂₀₂W₂₀₃ motif with the three BOX motifs generated a targeting module that satisfies the dual criteria of necessity and sufficiency for specific association with TGN membranes.

View larger version (74K): [in this window] [in a new window]   Figure 8. W202W203 and C-terminal BOX motifs in TGN targeting. Representative profiles for a GFP control, the GFP-PITP237–271 chimera, and the GFP-PITP201–271 chimera are shown. Quantification of transfected PITP–/– MEFs displaying Golgi localization profiles for each GFP-PITP construct is given at bottom as the ratio of number of cells imaged with clear Golgi profiles for the indicated GFP-PITP chimera to the total number of cells imaged for that chimera.

PITP Association with TGN Membranes and Action of PKCs

View larger version (54K): [in this window] [in a new window]   Figure 9. PITP localization and protein kinases C. (A) Profiles (individual and merged) for endogenous PITP and TGN38 in PITP–/– MEFs challenged with no inhibitor (MOCK), GF109203X (10 nM), or chelerythrine chloride (CHEL., 660 nM). The profiles shown at 6 h after challenge but are representative for what was observed at 3 and 16 h postchallenge as well. (B) Quantification of the imaging data presented in A. Number of cells imaged with clear Golgi profiles as a function of total number of cells imaged for each condition are indicated above each corresponding bar. (C) PITP–/– MEFs were challenged with no inhibitor (MOCK) or GF109203X (10 nM) or chelerythrine chloride (CHEL., 660 nM) for the indicated times. Cell-free lysates were prepared, resolved by SDS-PAGE and blotted to nitrocellulose, and blots were decorated with antibodies specific for phospho-MARCKs (a PKC substrate) and actin (loading control). Antibodies used detect MARCKS phosphorylated at Ser159Ser163 (Santa Cruz).