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Vol. 19, Issue 3, 1046-1061, March 2008
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Max Planck Institute of Molecular Cell Biology and Genetics, Dresden D-01307, Germany; Whitehead Institute for Biomedical Research, Cambridge, MA 02142; ||Mass Spectrometry Group, Leibniz Institute of Molecular Pharmacology, Berlin D-13125, Germany; and ¶A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia
Submitted February 16, 2007;
Revised December 13, 2007;
Accepted December 20, 2007
Monitoring Editor: Benjamin Glick
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The phosphatidylinositol 4-kinases (PI 4-kinase) Pik1p in yeast and PI 4-kinase IIIβ in vertebrates are found at the trans-Golgi network (TGN) (Godi et al., 1999
; Walch-Solimena and Novick, 1999; Sciorra et al., 2005; Strahl et al., 2005), and they are key regulators of membrane transport and Golgi homeostasis. Mutants with defects in PI(4)P production by these lipid kinases showed reduced secretion, delayed vacuolar transport, defective protein retrieval from endosomes to the Golgi, and aberrant Golgi morphology (Godi et al., 1999; Hama et al., 1999; Walch-Solimena and Novick, 1999; Audhya et al., 2000). Recent work, mainly in mammalian cells, has revealed effectors of phosphatidylinositol 4-phosphate [PI(4)P] such as the PI(4)P adaptor proteins FAPP1 and FAPP2, which are involved in the generation of transport carriers for exocytosis (Godi et al., 2004; Vieira et al., 2005) and the clathrin adaptor AP-1 (Wang et al., 2003; Heldwein et al., 2004) that participates in the formation of clathrin-coated vesicles. In the mammalian system, Golgi PI(4)P is not only generated by the type IIIβ PI 4-kinase (Wong et al., 1997; Godi et al., 1999, 2004) but also by type II
(Wei et al., 2002; Weixel et al., 2005).
In addition to their TGN localization, both Pik1p and PI 4-kinase IIIβ also localize to the nucleus (Garcia-Bustos et al., 1994; Walch-Solimena and Novick, 1999; de Graaf et al., 2002; Strahl et al., 2005). Although the nuclear function of the two PI 4-kinases remains unknown, it has been demonstrated that they undergo nucleocytoplasmic shuttling (de Graaf et al., 2002; Strahl et al., 2005). Reversible relocation of Pik1p from the nucleus to the cytoplasm has been observed upon block of secretion in several sec (secretion) mutants as part of the so-called "arrest of secretion response" (ASR) (Walch-Solimena and Novick, 1999; Nanduri and Tartakoff, 2001). However, the physiological significance and regulation of this process is not understood.
Nucleocytoplasmic shuttling is a behavior of many proteins, such as kinases, transcription factors, and cell cycle regulators (for review, see Nigg, 1997). There are several examples in which this shuttling process is regulated by 14-3-3 proteins, a family of phosphorylation-dependent chaperones. 14-3-3 proteins are ubiquitous and highly conserved proteins, which alter protein localization or activity, modify protein–protein interactions, and induce conformational changes of their targets (for reviews, see Muslin and Xing, 2000; Tzivion and Avruch, 2002; Yaffe, 2002; Dougherty and Morrison, 2004; van Heusden and Steensma, 2006). The 14-3-3 proteins play a role in cellular processes such as cell cycle regulation, apoptosis, and cell proliferation (for review, see Dougherty and Morrison, 2004). In yeast, the two 14-3-3 proteins Bmh1p and Bmh2p are redundant and in combination essential proteins. They have been shown to play a role in the polarization of the actin cytoskeleton, cell cycle regulation at the G1/S transition, and regulation of transcription factors involved in nutrient signaling (Lottersberger et al., 2006; van Heusden and Steensma, 2006).
The only currently known binding partner of Pik1p is the calmodulin-like protein frequenin-1 (Frq1p), which functions in binding of Pik1p to the Golgi (Hendricks et al., 1999; Ames et al., 2000; Strahl et al., 2005). We now report that Pik1p binds to the yeast 14-3-3 proteins Bmh1p and Bmh2p. The Pik1p–14-3-3 interaction requires phosphorylation, occurs primarily in the cytoplasm, and controls nucleocytoplasmic shuttling and availability of Pik1p for Golgi recruitment. This interaction interferes with secretion, because overexpression of BMH2 results in a phenocopy of pik1-101 mutant transport phenotypes in secretion. We further show that under nutrient deprivation the PI 4-kinase relocates from the TGN to the cytoplasm and into the nucleus. This relocation is rapidly reversed upon restoration of nutrient supply. Therefore, the nucleocytoplasmic shuttling of Pik1p and 14-3-3 interaction might be part of a regulatory circuit that coordinates cell growth cues with PI(4)P production at the TGN, possibly to adjust membrane transport to the growth requirements of the cell.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Yeast strain L40 was used for the two-hybrid system (MATa, trp1-, ade2-, leu2-, his3-, LYS::LexA-HIS3, URA::LexA-LacZ). Complete protease inhibitors were from Roche Diagnostics (Mannheim, Germany) and Zymolase 100-T was from MP Biomedicals (Irvine, CA). The anti-hemagglutinin (HA) antibody Y11 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-green fluorescent protein (GFP) antibody (goat) was from the protein expression facility of the MPI-CBG (Dresden, Germany). Monoclonal anti-GFP antibody (clones 7.1 and 13.1) was from Roche Diagnostics.
Plasmid and Strain Constructions
Constructs used in this study are listed in Supplemental Table 3. General molecular biological methods were as described previously (Sambrook and Russel, 2001). Plasmid pMBL31 (LexA yeast two-hybrid vector) was constructed by replacing the GAL4 DNA-binding domain of vector pGBKT7 (Clontech, Mountain View, CA) by the coding region of LexA flanked by ADH promoter and terminator as described for pMBL33 (Brickley et al., 2005). Fusion constructs with the GAL4 DNA-binding domain and with LexA were obtained by in frame ligation of polymerase chain reaction (PCR) products. The pik1S396A and pik1S396D mutations have been generated by site-directed mutagenesis (QuikChange Multi kit; Stratagene, La Jolla, CA), and they were introduced into pRS415 containing the ADH promoter fused to GFP for N-terminal tagging. The pik1-101 allele has been described previously (Walch-Solimena and Novick, 1999). The alleles pik1-145-148 were generated by site-directed mutagenesis (QuikChange Multi kit; Stratagene) and contain S396A, S605A (pik1-145); S605A, S780A (pik1-146); S396A, S780A (pik1-147); and all three mutations (pik1-148). Allele pik1-130 (H35R, T105M, D147G, M367T, N372S, R462G and F530Y) was obtained by error-prone PCR. Yeast strains genomically tagged with 3xHA, 13xMyc, or tandem affinity purification (TAP) tag were constructed as described previously (Longtine et al., 1998; Puig et al., 2001). In Pik1p-TAP (CSY370, CSY513, and YMB119), a SGPGS linker (TCTGGTCCATCTGGT) was inserted between the Pik1p and the TAP tag. The Frq1p-TAP strain was from Open Biosystems (Huntsville, AL). A GGSGG peptide linker (GGTGGTTCTGGTGGT) has been inserted between Bmh1p or Bmh2p and 13xMyc in the case of YMB054, YMB058, or YMB119. To create a Bmh1p-3xha fusion (YMB148), a SGPGS linker (TCTGGTCCATCTGGT) was inserted between the Bmh1p and the HA tag.
Interaction Assays Using the Yeast Two-Hybrid System
For two-hybrid interaction studies the GAL4 based system Matchmaker 3' (Clontech) and a LexA-system with a modified LexA vector were used as described previously (Brickley et al., 2005). For measurement of β-galactosidase activity in liquid culture, an O-nitrophenyl β-D-galactopyranoside (ONPG) solution assay was performed as described previously (Miller, 1972; Sambrook and Russel, 2001).
Glutathione S-transferase (GST) Pull-Down Assays
GST and GST-Bmh1/2p-fusions were expressed in Escherichia coli BL21. Cells were homogenized in phosphate-buffered saline (PBS) with protease inhibitors (Complete and phenylmethylsulfonyl fluoride [PMSF]) by sonication. Lysates were incubated with 1% Triton X-100 for 10 min at 4°C, cleared, and mixed with glutathione Sepharose 4B slurry (GE Healthcare, Chalfont St. Giles, United Kingdom). After binding for 10 min at room temperature, beads were harvested and washed twice with PBS (with protease inhibitors) and once with yeast lysis buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 100 µM Na3VO4, 25 mM NaF, 1 mM PMSF, and Complete). Aliquots were used to pull down Pik1p from yeast lysate (corresponding to 5 OD units of cells) overnight at 4°C. Beads were washed with yeast lysis buffer; three times with 20 mM Tris-HCl, pH 7.5, 350 mM NaCl, 2 mM EDTA, and 0.1% NP-40; and once with PBS. Thirty microliters of supernatant and the resuspended beads were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by immunoblotting. In some experiments, the effects of adding phosphatase inhibitors (100 µM Na3VO4 and 25 mM NaF) and/or 
-PPase to yeast lysates were examined.
In vitro transcription/translation of Pik1p was performed using the TNT T7 Quick-Coupled Transcription/Translation System (Promega, Madison, WI), including [35S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) according to the manufacturer's instructions from plasmid pLD103. GST and GST-Bmh1/2p were expressed and solubilized as described above. Five hundred microliters of cleared E. coli lysate was incubated with 150 µl of prewashed 50% glutathione-Sepharose 4B slurry on a rotator for 30 min at room temperature.
To precipitate Pik1p, 40 µl of the TNT reaction mix was incubated overnight at 4°C with GST only beads or beads loaded with GST-Bmh1p or GST-Bmh2p, respectively. Samples were washed once with PBS, with washing buffer (10 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 0.5% NP-40) containing 150 mM NaCl or 500 mM NaCl and once with 10 mM Tris-HCl, pH 8.0. Beads were resuspended in SDS-sample buffer and proteins were analyzed by SDS-PAGE followed by autoradiography.
TAP and Protein Identification
The strain CSY 370 was used for purification of Pik1p complexes and the Frq1p-TAP strain for Frq1p. TAP from 12 l of YPD culture was performed as described previously (Rigaut et al., 1999; Puig et al., 2001) with the modification that Complete protease inhibitors were used throughout the procedure. When indicated, extractions were performed in the presence of phosphatase inhibitors (25 mM NaF and 100 µM Na3VO4). Proteins were separated by SDS-PAGE (4–16% gradient) and visualized with Coomassie, and bands were excised and in-gel digested with trypsin and analyzed by a combination of matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) peptide mapping and nanoelectrospray (nanoES) tandem mass spectrometry (MS/MS) as described previously (Shevchenko et al., 1996) by using, respectively, REFLEX IV MALDI time of flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) and QSTAR Pulsar i hybrid quadrupole time-of-flight mass spectrometer (MDS Sciex, Concord, ON, Canada). Database searches were performed by Mascot, version 2.1 software (Matrix Science, London, United Kingdom) installed on a local server against the subset of S. cerevisiae proteins in MSDB protein sequence database.
Gap1p Activity Assay
The Gap1p activity was determined by measuring the uptake rate of [14C]citrulline as described previously (Roberg et al., 1997).
Identification of Phosphorylation Sites by Mass Spectrometry
Phosphorylated Pik1p bands were excised. Disulfide bond reduction/alkylation, tryptic in-gel digestion, and sample preparation before analysis by MS were performed as described previously (Bauer and Krause, 2005). Separation of phosphorylated peptides was performed by selective binding on titanium dioxide (Pinkse et al., 2004). Peptide samples (5 µl in 30% acetonitrile and 2% formic acid) were loaded on a TiO2 column (5 µm, 100 Å; 5 mm x 300 µm i.d.) at a flow rate of 1.5 µl/min. After washing, the phosphopeptides were eluted with 0.1 M ammonium bicarbonate. The eluate was dried in a vacuum centrifuge and redissolved in 6 µl of 0.1% trifluoroacetic acid in acetonitrile:water (1:9, vol/vol). Liquid chromatography (LC)-MS and MS/MS experiments were performed on a quadrupole orthogonal acceleration time-of-flight mass spectrometer Q-TOF Ultima (Micromass, Manchester, United Kingdom). A CapLC liquid chromatography system (Waters, Milford, MA) was used to deliver the peptide solution to the nanoelectrospray source. Peptides were separated using a capillary column (PepMap C18; 3 µm, 100 Å; 150 mm x 75 µm i.d.; Dionex, Idstein, Germany) and an eluent flow rate of 200 nl/min. Mobile phase A was 0.1% formic acid in acetonitrile:water (3:97, vol/vol), and mobile phase B was 0.1% formic acid in acetonitrile:water (8:2, vol/vol). Runs were performed using a gradient of 4–65% B in 60 min. To perform MS/MS experiments, automatic function switching (survey scanning) was used. The processed MS/MS spectra (MassLynx version 4.0 software) were compared with the theoretical fragment ions of enzymatic fragments of Pik1p.
Live Cell Imaging
For the visual Gap1p trafficking assay, yeast strains transformed with TPQ99 [encoding Gap1p(K9A,K16A)-GFP fusion protein, called Gap1*p-GFP in the text, mutations according to Soetens et al. (2001)] were grown to early log phase in YP, containing 2% raffinose, and then reporter expression was induced by adding galactose to 3% for 3 h. For GFP–Pik1p microscopy, late log phase cultures were grown in SC-URA medium for 48 h. Early log phase cultures were grown to an OD600 of 0.3. Cells using glycerol as a carbon source were cultured for 3 d in YPG medium. To deplete glucose or glycerol from the medium, cells from early log phase cultures were harvested from SC-URA or YPG medium, respectively. Cells were washed twice with medium lacking carbon sources, and then they were resuspended in glucose or glycerol-free medium for 45 min. Cells were depleted of glucose (45 min) and at indicated times, glucose (2% final concentration) was added. Cells were then harvested and directly observed without fixation under a fluorescent microscope (Axioplan 2 MOT; Carl Zeiss, Jena, Germany).
Image Quantification
The images were processed with MotionTracking program: The background window size was chosen so that the fluorescence of cytosolic GFP pool was treated as a background. The background was calculated by modification of "dynamic" algorithm. The mean value and SD was calculated inside the square window. Then the mean value was recalculated but all intensities above mean + 2 were excluded from consideration. The last procedure was repeated four times. The mean values after this procedure well correspond to "background intensity," because the small bright structures were excluded from the averaging procedure. The intensities of small structures were fitted by hat-like analytical function:
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I is angle between main axes and the axes X/Y of image, pi is power (sharpness) of function, the index i refers to ith particle. For details of the fitting procedure see Rink et al. (2005). The number of dotted structures was calculated inside every cell. Because the program could not well distinguish cells that are touching each other, the cell boundaries were drawn manually. The integral intensity of structures was calculated by analytical integration of fitting functions.
Immunofluorescence Microscopy
Immunofluorescence experiments were performed as described in Walch-Solimena et al. (1997). Images were acquired using a microscope (Zeiss Axiovert 200) equipped with a Plan-Apo 100x/1.4 oil objective or Zeiss Axioplan 2 MOT with a Plan-Neofluar 100x/1.3 oil objective (SPOT camera; Diagnostic Instruments, Sterling Heights, MI).
Cell Fractionation and Immunoprecipitation
Cell fractionation was performed as described previously (Du and Novick, 2001), with protease inhibitors (Complete) used throughout the procedure unless otherwise indicated. Equal volumes were analyzed by SDS-PAGE and immunoblotting. For immunoprecipitations from cellular fractions, membrane-bound and soluble fractions were prepared from yeast cells (YMB148) by 100,000 x g centrifugation for 45 min. All samples were then adjusted to 2 ml, Tween 20 was added to 0.5% final concentration, and samples incubated on ice for 30 min. After preadsorption with protein A-Sepharose, precipitation was carried out using anti-myc antibody (9E10), followed by protein A-Sepharose incubation for 2 h at 4°C. Beads were harvested, washed six times with cold 20 mM HEPES, pH 7.4, containing 200 mM KCl, 0.5 mM dithiothreitol (DTT), 2 mM EDTA, 10 mM NaF, 100 µM Na3VO4, and 0.5% Triton X-100 followed by two washes without detergent.
For glucose depletion experiments, yeast cultures (YMB148) were grown overnight in YPD to mid-log phase (OD600 = 0.8–1.0). Glucose depletion was performed as described above. Cells were then harvested, washed with washing buffer 1 (50 mM Tris-HCl, pH 7.5, and 10 mM NaN3) and washing buffer 2 (20 mM Tris-HCl, pH 7.5), and resuspended in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1% Tween 20, and Complete). Cells were lysed using 2 g of Zirconia beads and a Mini-BeadBeater-8 (Biospec Products, Bartlesville, OK). The lysate was cleared by centrifugation (15 min; 14,000 x g; 4°C). Equal amounts of protein were used for the immunoprecipitation. Immunoprecipitation was performed as described above, and beads washed four times with lysis buffer. Samples were analyzed by SDS-PAGE and immunoblotting.
PI 4-Kinase Activity Assay
PI 4-kinase activity was measured essentially as described previously (Flanagan and Thorner, 1992; Walch-Solimena and Novick, 1999). Immunoprecipitations from soluble and membrane fractions were performed as described above. Immunocomplexes were washed, resuspended in PI-kinase buffer (50 mM Tris-HCl, 10 mM MgCl2, and 0.25% Triton X-100), and aliquots were used for control Western blots and phosphoinositide analysis by thin TLC. Quantification of radioactive PI(4)P was performed by autoradiography of TLC plates by using the PhosphorImager BAS-1800II (Fuji, Tokyo, Japan).
Phosphorylation-dependent Gel Shift of Pik1p
Yeast cultures were grown to mid-log phase at 25°C, and if indicated, shifted to 37°C for 30 min. Cells were washed with ice-cold PBS, and the cultures were resuspended in lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, and Complete) with or without phosphatase inhibitors (25 mM NaF and 100 µM Na3VO4). All samples were homogenized by bead beating, and lysates were cleared by centrifugation (5 min; 2500 x g). Seventy-five micrograms of total protein of the sample without phosphatase inhibitors was treated with 5000 U of -PPase (New England Biolabs, Ipswich, MA) at 30°C for 30 min. Equal protein amounts were analyzed by 10% SDS-PAGE (500:1, acrylamide:bisacrylamide ratio) and immunoblotting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Besides Pik1p, the Bmh2p complex contained Bmh1p, the second 14-3-3 protein found in yeast, the neutral trehalase proteins Nth1/2p, the transcription regulator Rtg2p, and the serine/threonine protein kinase Fun31p (Gavin et al., 2002). Using a yeast two-hybrid approach, we confirmed the interaction between Pik1p (amino acids [aa] 2–1066) and Bmh2p (Figure 1A, middle). Pik1p did not interact with other components of the Bmh2p complex in this assay (data not shown). The Pik1p–Bmh2p interaction was 10 times weaker than the interaction of Pik1p with Frq1p (Figure 1B). This interaction was lost upon deletion of the C-terminal catalytic domain of Pik1p (aa 770-1066) (Figure 1A, left). Bmh1p, which is highly similar to Bmh2p, also bound Pik1p in the yeast two-hybrid assay (Figure 1, A and B).
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97% identity between Bmh1p and Bmh2p (GST-Bmh11-198) interacted with Pik1p from yeast lysates (Figure 1C). We then asked whether the Pik1p interaction with 14-3-3 is direct. Full-length Pik1p was synthesized by in vitro transcription and translation in the presence of [35S]methionine. Radiolabeled Pik1p was incubated with GST-Bmh1p or GST-Bmh2p. As shown in Figure 1D, either GST fusion protein was able to pull down Pik1p, suggesting a direct interaction between the tested partners.
To test whether this interaction occurs in vivo, we performed a TAP experiment. The chromosomal copy of PIK1 was fused to the TAP tag. The fusion protein of the essential PI 4-kinase was functional only when we inserted a six-amino acid linker (SGPGS) at the C terminus of Pik1p as suggested by normal growth of the resulting strain (data not shown). The identification of coisolated proteins by mass spectrometry revealed interaction of Pik1p, Bmh1p/Bmh2p, and Frq1p (Figure 1E), whereas other proteins represented common contaminants of the TAP procedure (Shevchenko et al., 2002). We conclude that the Pik1p–14-3-3 complex occurs in vivo and includes both Bmh1p and Bmh2p, which often function as a heterodimer (Jones et al., 1995).
Pik1p, Bmh1/2p, and Frq1p Occur in a Common Complex
The interaction of Pik1p with both Bmh1/2p and Frq1p opens the question whether these binding partners can occur in a common complex or bind in a mutually exclusive manner. We performed glycerol velocity gradient centrifugation to determine size and composition of Pik1p–14-3-3–containing protein complexes providing support for the existence of a heterotetrameric complex (Pik1p, Frq1p, and Bmh1/2p). As shown in Supplemental Figure 1, Pik1p and Frq1p were mainly present in fractions 3–10, with a peak in fractions 4–5 corresponding to 230 kDa. The major pool of Bmh1p seems to be present in oligomeric complexes, because this protein is only weakly detectable in the monomer and dimer fractions 1–3. An antibody detecting both Bmh1p and Bmh2p revealed an identical behavior in the gradients, supporting involvement of heterodimers in the complex with Pik1p. This is in agreement with published data reporting that Bmh1/2p mainly occur as heterodimers (Chaudhri et al., 2003). All four proteins together were found in a peak in fractions 5–7 that runs between 230 and 670 kDa, suggesting the presence of a heterotetrameric complex involving Pik1p, Frq1p, Bmh1p, and Bmh2p (Supplemental Figure 1).
For further confirmation of this hypothesis, we then isolated Frq1p–TAP-containing protein complexes using the TAP purification protocol (Figure 2). Western blotting of purified Frq1p–TAP complexes (Figure 2A) and mass spectrometry (Figure 2B and Supplemental Table 4) provided evidence for copurification of Bmh1/2p and thus for the presence of these proteins in a common protein complex.
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or bmh2 with an allele of pik1-101 impaired in catalytic activity (data not shown), we did observe enhancement of the growth defect of pik1-101 by overproduction of Bmh proteins (2µ BMH1 or BMH2) (Figure 3A). We further tested the effect of the overproduction of 14-3-3 in mutants along the secretory pathway (Supplementary Table 1). Synthetic enhancement of the growth defect by 14-3-3 overproduction was found in mutants of genes involved in Golgi vesicle formation (arf1, vps1, ypt31 ypt32ts, and sec14-3) and in mutants of genes required for TGN-to-plasma membrane transport (sec1-1, sec2-41, sec2-59, sec3-2, sec4-8, and sec15-1). This type of interaction was also found in mutants of cytoskeleton genes required for polarized transport of secretory vesicles (act1-2 and myo2-66). No or a very weak genetic interaction was detected upon overexpression of BMH1 or BMH2 in mutants of genes in the general secretion gene SEC19 (sec19-1) or genes of endoplasmic reticulum-to-Golgi transport (sec21-1, sec23-1). Only the N-ethylmaleimide sensitive factor homologue SEC18 (sec18-1) showed suppression of the growth phenotype upon 14-3-3 overexpression. In most cases, the synthetic genetic interaction of BMH1/2 was found with mutant alleles of those sec mutants, which show synthetic sickness or lethality when combined with pik1-101 (Walch-Solimena and Novick, 1999). These data suggested that both Pik1p and 14-3-3 might be involved in regulation of post-Golgi trafficking and that 14-3-3 overproduction counteracts Pik1p function.
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). In this assay, Gap1*p-GFP activity was fivefold reduced upon increased Bmh2p level (Figure 3D). Thus, in agreement with the described genetic interaction, Bmh2p overexpression seems to inhibit secretion, and likely negatively regulates Pik1p in this process as it shows similarities to loss of Pik1p activity. To understand how 14-3-3 proteins regulate Pik1p function, we first asked about the localization of the interaction.
Pik1p–14-3-3 Interaction Occurs in the Cytoplasm
Pik1p has been found at the TGN and in the nucleus (Walch-Solimena and Novick, 1999; Sciorra et al., 2005; Strahl et al., 2005). 14-3-3 proteins have a more widespread distribution. Fluorescence microscopy revealed a general cytoplasmic distribution of Bmh1p-myc with nuclear exclusion (Figure 4A). Bmh2p-myc was found in a punctate pattern throughout the entire cell. There was no obvious colocalization of Bmh proteins with Pik1p at the TGN (Figure 4A, insets). Because Pik1p immunofluorescence did not perfectly overlap with DAPI staining of DNA, we asked whether Pik1p could be concentrated in the nucleolus. As shown in Figure 4B, Pik1p colocalizes with a nucleolar marker (detected by monoclonal antibody [mAb] 2.3b; Yang et al., 1989). Due to the general cytoplasmic labeling for Bmh1p-myc and Bmh2p-myc, we could not exclude that there could be a pool of these proteins that localizes to this compartment. Therefore, we further investigated the subcellular localization by differential fractionation.
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Pik1p Is Primarily Nuclear upon Loss of 14-3-3 Function
To address a possible role of 14-3-3 in regulation of Pik1p distribution between cytosol and nucleus, we studied the localization of GFP-Pik1p in the background of bmh mutant alleles. Although GFP-Pik1p localized to the TGN and to the nucleus in wild-type cells (Figure 5, A–C), we observed an increased number of cells with nuclear Pik1p and decreased Pik1p levels at the TGN in bmh1 or bmh1 bmh2ts mutants at 25°C, an effect more pronounced at 37°C (Figure 5, A–C). Whole cell Pik1p levels were unchanged in bmh mutants at 25°C and in bmh1 at 37°C. There was a slight decrease at the restrictive temperature in the bmh1 bmh2ts double mutant (Supplemental Figure 2). Thus, the microscopy data indicate a true redistribution of Pik1p, suggesting that 14-3-3 interaction is required for maintenance of a pool of Pik1p in the cytoplasm and thus might make it accessible to the TGN. This idea is supported by our finding that in bmh mutants, GFP-Pik1p still localizes to the Golgi; however, it does so with strongly decreased fluorescence intensity (Figure 5D and Supplemental Figure 3A). For quantification of the fluorescence intensity, we measured only structures smaller than 0.95 µm, thus excluding nuclei (Supplemental Figure 3B).
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; Yaffe, 2002; Dougherty and Morrison, 2004), we decided to investigate a possible phosphorylation dependence of the Pik1p–14-3-3 interaction.
GST pull-down experiments (GST-14-3-3) of genomically tagged Pik1p-TAP from yeast lysates were performed in the presence or absence of -PPase or phosphatase inhibitors. As shown in Figure 6A, Pik1p-TAP only bound to either GST-Bmh1p or GST-Bmh2p in the presence of phosphatase inhibitors, thus, Pik1p–14-3-3 interaction does require phosphorylation. This finding is consistent with experiments in Figure 1, C and E, which were performed in the presence of phosphatase inhibitors. In vitro-translated Pik1p used for GST pull-down experiments shown in Figure 1D was partially phosphorylated (data not shown).
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; Rittinger et al., 1999). Among the identified sites for phosphorylation, two (S396 and S605) are part of a predicted 14-3-3 target interaction motif according to Motif Scan (http://scansite.mit.edu). A third putative 14-3-3 binding motif was found at S780, a site that has not been identified by our mass spectrometry analysis. We generated mutants in these sites, tested for interaction of these mutated Pik1p proteins with Bmh1p and Bmh2p in the yeast two-hybrid assay and found that S396 is required for 14-3-3 interaction (Figure 6B). Together with the observed loss of interaction between Pik1p and 14-3-3 upon deletion of the catalytic domain of the PI 4-kinase (Figure 1A), we suggest that S396 is necessary but not sufficient for this interaction.
Pik1p Phosphorylation Correlates with Nucleocytoplasmic Relocalization
We next asked whether phosphorylation of Pik1p shows any correlation with its subcellular localization. We have previously described that Pik1p rapidly and reversibly relocalizes from the nucleus to the cytoplasm upon block of secretion, e.g., in a sec6-4 mutant. The ASR occurs in sec mutants along the entire secretory pathway and also results in relocation of other nuclear proteins (Walch-Solimena and Novick, 1999; Nanduri and Tartakoff, 2001), probably by nuclear import inhibition (Nanduri et al., 1999). We took advantage of the ASR and analyzed Pik1p phosphorylation in wild-type and sec6-4 mutant cells after a 30-min shift to the restrictive temperature (Figure 6C). Under these conditions, a massive relocalization of nuclear Pik1p to the cytoplasm has been observed previously (Walch-Solimena and Novick, 1999).
To resolve the phosphorylated and nonphosphorylated form of Pik1p by SDS-PAGE, we empirically determined an acrylamide:bisacrylamide ratio of 500:1 as the best composition. On ASR in sec6-4, we detected a shift of Pik1p to a higher molecular weight form in lysates from sec6-4 cells at the restrictive temperature compared with wild type. This shift was reversed upon treatment with -PPase, indicating that the higher molecular weight form is phosphorylated (Figure 6C). Together, the increase in phosphorylation and shuttling out of the nucleus of Pik1p during the ASR suggested that this modification might play a role in regulation of Pik1p distribution.
To test this further, we investigated the phosphorylation status of Pik1p in a mutant version of the protein showing a localization defect on its own. For this, we focused on the N-terminal regulatory domain of Pik1p, and we generated temperature-sensitive pik1 mutant alleles by error-prone PCR. Among the mutants we identified a new allele, pik1-130, which had a defect in nuclear but not the cytoplasmic punctate labeling for Pik1p (Figure 6D). Like Pik1p in Figure 4B, at the permissive temperature, mutant Pik1-130p localized immediately adjacent to DAPI, suggesting nucleolar localization (Figure 6D). At the restrictive temperature, when Pik1-130p no longer is nuclear, the protein was in part shifted to a higher molecular weight, phosphorylated form (Figure 6C) that could be detected even though the stability of the mutant protein was decreased. We conclude that the distribution of Pik1p correlates with its phosphorylation status. Together with the finding that Pik1p-14-3-3 interaction occurs in the cytoplasm and requires phosphorylation, we propose a model in which 14-3-3 proteins modulate nucleocytoplasmic shuttling of Pik1p. What could be the physiological significance of such a shuttling mechanism?
Pik1p Relocates from the TGN to the Nucleus under Conditions of Nutrient Limitation
We have shown that Pik1p redistributes from the nucleus to the cytosol during the ASR (Walch-Solimena and Novick, 1999), a condition where a rapid adjustment of the protein synthesis machinery occurs in response to a block in the secretory pathway (Mizuta and Warner, 1994; Nanduri and Tartakoff, 2001). Therefore, we asked whether a change in localization of Pik1p could also be observed under different growth conditions. For this, we observed GFP–Pik1p localization first in either early or late log phase growth. In early log phase cultures, only a small percentage of cells (9.0 ± 9.1%) showed nuclear GFP-Pik1p (Figure 7, A and E). Instead, all cells exhibited GFP-Pik1p labeling at the TGN (Figure 7, A and G). There was no obvious change in localization during the cell cycle (as judged by the size of the bud, data not shown). In late log phase, GFP-Pik1p became mostly cytoplasmic (Figure 7, A and G) without major change in nuclear (8.1 ± 9.1%) labeling (Figure 7, A and E). The TGN integrity was unchanged under these conditions since labeling with the TGN marker Sec7p-GFP was unaffected in late log phase cells (Figure 7D).
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The relocalization of Pik1p from the TGN to the nucleus might represent a mechanism by which cells can quickly adjust both PI(4)P production and vesicle formation from the late Golgi to growth conditions. If such a signaling pathway existed, one would expect rapid restoration of the TGN localization of Pik1p if the appropriate carbon source (glucose, galactose or raffinose) is added back to the cells. As shown in Figure 7C, we did observe such a restoration of TGN localization of Pik1p within minutes after glucose addition. We obtained similar results using galactose (Supplemental Figure 5).
Pik1p Relocation under Nutrient Deprivation Involves Phosphorylation and 14-3-3 Binding
We next asked whether the observed relocation of Pik1p from the TGN to the cytoplasm and into the nucleus during nutrient deprivation might be mediated by Pik1p phosphorylation and 14-3-3 binding. For this, we performed a glucose deprivation experiment as shown in Figure 7, but this time we lysed cells after 45 min without glucose and 20 min after glucose readdition. As shown in Figure 8, more phosphorylated Pik1p was recovered after glucose deprivation than in control cells or after glucose readdition. Also, glucose deprivation resulted in an increase of Bmh1p in complex with Pik1p (Figure 8). Phosphorylation and 14-3-3 complex formation thus correlates with the observed loss of Pik1p from the TGN that results in relocation to the cytoplasm and to the nucleus.
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We further took advantage of the S396 mutants to ask whether phosphorylation at this site regulates Pik1p–Frq1p interaction. In the yeast two-hybrid assay, we found no differences in the interaction of mutant versus wild-type Pik1p (Supplemental Figure 6). Together with our velocity gradient (Supplemental Figure 1) and Frq1p–TAP experiments (Figure 2), this suggests that S396 phosphorylation and 14-3-3 interaction do not function to regulate the Pik1p–Frq1p complex.
We have already shown that BMH overexpression can interfere with trafficking (Figure 3) and with nucleocytoplasmic shuttling (Figure 9). To investigate more directly the role of Pik1–14-3-3 interaction through phosphorylated S396 and trafficking, we measured endogenous Gap1p permease activity with the [14C]citrulline uptake assay in yeast strains carrying pik1S396A or pik1S396D, respectively, as the only PIK1 copy of the cell compared with cells carrying PIK1 on the same plasmid (Supplemental Figure 7). We found a decrease in surface transport for S396A. These data further support the hypothesis that regulated nucleocytoplasmic shuttling and Pik1p function during trafficking are closely connected.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Walch-Solimena and Novick, 1999; Sciorra et al., 2005; Strahl et al., 2005). A similar dual localization has been found for PI 4-kinase IIIβ (Godi et al., 1999; de Graaf et al., 2002). Pik1p shuttles constitutively between nucleus and cytoplasm (Strahl et al., 2005). We provide evidence that 14-3-3 proteins regulate nucleocytoplasmic shuttling of this enzyme.
Bmh1p and Bmh2p bind to Pik1p in vitro and in vivo depending on phosphorylation, a modification involved in 14-3-3 target binding (Tzivion and Avruch, 2002; Yaffe, 2002; Dougherty and Morrison, 2004). During the ASR, Pik1p rapidly relocates from the nucleus to the cytoplasm and undergoes a shift to the phosphorylated form, suggesting that Pik1p needs phosphorylation for cytoplasmic localization. Consistent with this model, we found an increase in phosphorylated Pik1p and Pik1p–14-3-3 complex during nutrient deprivation, when Pik1p is no longer TGN-bound but becomes cytoplasmic, before it accumulates in the nucleus. Membrane-associated Pik1p binds to 14-3-3 only to a small extent.
We were unable to disrupt Pik1p–14-3-3 complexes from cell lysates either with phosphatases or by competition with the R18 peptide, a peptide which competes with 14-3-3 interactors (Wang et al., 1999; data not shown), suggesting that dissociation of 14-3-3 from Pik1p is regulated and might precede dephosphorylation. This interaction might thus protect cytoplasmic Pik1p from dephosphorylation, possibly to offer phosphorylated Pik1p to the TGN. According to our data, membrane binding of Pik1p and Pik1p–Frq1p interaction occurs by a mechanism not requiring 14-3-3 function (see model in Figure 11). Instead, Pik1p binding to the Golgi does require Frq1p (Strahl et al., 2005). Such a model would be consistent with our finding that 14-3-3 overproduction interferes with function of the late secretory pathway.
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An important role of phosphorylation in PI 4-kinase regulation was also described for PI 4-kinase IIIβ (Hausser et al., 2005). Protein kinase D, which mediates fission of transport carriers from the TGN (Liljedahl et al., 2001), phosphorylates PI 4-kinase IIIβ at a site (S294) within a region of homology between class III PI 4-kinases (Hausser et al., 2005). Interestingly, one of the phospho-serines identified in our study (S396) is located within this conserved region and the phosphorylation site S294 of PI 4-kinase IIIβ also lies within a 14-3-3 consensus binding motif (Hausser et al., 2006). Even though phosphorylated PI 4-kinase IIIβ localizes to the TGN, phosphorylation (in position S294) is not required for this localization but rather for enzymatic activity (Hausser et al., 2005; Szivak et al., 2006). We did not find differences in Pik1p activity immunoprecipitated from cytosol or membrane fractions or in mutants of S396, suggesting that control of Pik1p enzymatic activity is not a major function of S396 phosphorylation and binding of 14-3-3 proteins.
PI 4-kinase IIIβ accumulated in the nucleus of cells treated with leptomycin B (de Graaf et al., 2002). However, the mammalian PI 4-kinase was not detected in the nucleus under steady-state conditions, making it difficult to compare the nucleocytoplasmic shuttling process with the yeast system. Therefore, it will be of interest to address whether control of nucleocytoplasmic shuttling of type III PI 4-kinases by 14-3-3 proteins is conserved.
Regulation of the localization of enzymes of PI metabolism has been documented in other studies, e.g., for the PI(4)P 5-kinase Mss4p (Audhya and Emr, 2003) and the lipid phosphatase Sac1p (Faulhammer et al., 2005; Faulhammer et al., 2007). Together with our data showing nucleocytoplasmic shuttling of Pik1p, we propose that relocation of PI-metabolizing enzymes between different compartments might be a general mechanism for the rapid adjustment of PI levels on organelles. Our work revealed one mechanism by which such shuttling of a PI kinase might be controlled: formation of a complex with 14-3-3 proteins.
Do 14-3-3 Proteins Coordinate Golgi Function with Nutrient Signaling?
Use of a phosphorylation-dependent and 14-3-3–dependent mechanism for regulated subcellular distribution of Pik1p provides in addition to rapid and local control of PI(4)P production the possibility of coupling Pik1p function to signaling processes, which might need to be coordinated with membrane transport. We did indeed find that 14-3-3 proteins affect secretion, and that Pik1p–14-3-3 interaction mediates relocation of Pik1p in response to nutrient limitation. We propose that 14-3-3 proteins act as a gatekeeper for Golgi Pik1p through control of cytoplasmic Pik1p levels and nucleocytoplasmic shuttling.
14-3-3 proteins have previously been implicated in membrane transport regulation (Gelperin et al., 1995; Vasara et al., 2002) and cytoskeleton organization (Roth et al., 1999), but so far little is known about the targets of these proteins. We found here that overexpression of BMH1 or BMH2 was lethal in the actin mutant act1-2 and a mutant of the myosin motor of secretory vesicles (myo2-66), together suggesting that 14-3-3 proteins, on their own or with Pik1p, could regulate the actin cytoskeleton. This regulation is likely to contribute to the effects of 14-3-3 on post-Golgi transport. Besides a defect in actin polarization, bmh and pik1 mutants both exhibit defects in budding and cell wall integrity (Lottersberger et al., 2006; data not shown), which could be related to membrane transport but could also point toward additional roles of both proteins.
We found that phosphorylation of Pik1p and Pik1p–14-3-3 complex formation is subject to regulation both in the ASR and under nutrient deprivation conditions. Therefore, we propose that this interaction could be part of a signaling pathway that coordinates growth conditions with Golgi function. 14-3-3 proteins have been implicated in many cellular processes. In yeast, they participate in the Ras/mitogen-activated protein kinase cascade of pseudohyphal development (Roberts et al., 1997), the target of rapamycin (TOR) pathway (Gelperin et al., 1995; Bertram et al., 1998; Beck and Hall, 1999), the RAS–cAMP pathway (Gelperin et al., 1995), and possibly in the cell wall integrity pathway (Lottersberger et al., 2006). Because we found that deprivation of fermentable carbon sources results in rapid and reversible relocation of Pik1p and increased Pik1p–14-3-3 interaction, the TOR and the Ras pathways are possible candidates for upstream signaling. It will now be important to identify the kinases for Pik1p phosphorylation.
Is there a coordination of growth with secretion via PI(4)P signaling? Interestingly, we found Pik1p loss from the TGN under similar conditions that caused relocation of the lipid phosphatase Sac1p from the ER to the Golgi (Faulhammer et al., 2005, 2007). Because Pik1p generates PI(4)P and Sac1p dephosphorylates this lipid, the coordinated relocation of both enzymes would result in a drastic and rapid reduction of PI(4)P on the TGN membrane. This in turn would reduce PI(4)P-dependent membrane transport and coordinate SV production with growth conditions. Indeed, it has been reported that a fluorescent PI(4)P-binding PH domain no longer localized to the Golgi under conditions of glucose deprivation (Faulhammer et al., 2005) similar to the conditions used in our study. It is now of great interest whether this signaling pathway is conserved in vertebrates. Interestingly, Frq1p, a positive regulator of function of the fission yeast Pik1p homologue, participates in nutrient-regulated signaling pathways (Hamasaki-Katagiri et al., 2004).
The Golgi apparatus is not only a major hub for transport in the secretory pathway but also an important signaling platform. Further work on the role of the Pik1p–14-3-3 interaction will provide new insights of how membrane transport at the Golgi is coordinated with growth.
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ACKNOWLEDGMENTS |
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