Conserved role for Gga proteins in phosphatidylinositol 4-kinase localization to the trans-Golgi network

Abstract

Phosphoinositides serve as key membrane determinants for assembly of clathrin coat proteins that drive formation of clathrin-coated vesicles. At the trans-Golgi network (TGN), phosphatidylinositol 4-phosphate (PtdIns4P) plays important roles in recruitment of two major clathrin adaptors, Gga (Golgi-localized, gamma-adaptin ear homology, Arf-binding) proteins and the AP-1 (assembly protein-1) complex. The molecular mechanisms that mediate localization of phosphatidylinositol kinases responsible for synthesis of PtdIns4P at the TGN are not well characterized. We identify two motifs in the yeast phosphatidylinositol 4-kinase, Pik1, which are required for binding to the VHS domain of Gga2. Mutations in these motifs that inhibit Gga2-VHS binding resulted in reduced Pik1 localization and delayed accumulation of PtdIns4P and recruitment of AP-1 to the TGN. The Pik1 homolog in mammals, PI4KIIIβ, interacted preferentially with the VHS domain of GGA2 compared with VHS domains of GGA1 and GGA3. Depletion of GGA2, but not GGA1 or GGA3, specifically affected PI4KIIIβ localization. These results reveal a conserved role for Gga proteins in regulating phosphatidylinositol 4-kinase function at the TGN.

Keywords: Gga adaptors; clathrin; phosphatidylinositol 4-kinase; phosphatidylinositol 4-phosphate; trans-Golgi network.

Fig. 1.

Identification of a Gga2–VHS binding site in Pik1. (A) Pik1(283–425) directly binds Gga2–VHS. Purified fragments of Pik1-His6 were incubated with GST (G) or GST-Gga2–VHS (V) immobilized on glutathione-Sepharose. Bound proteins were eluted and analyzed by SDS/PAGE and immunoblotting with His tag antibody. Input (I) represents 8% of the sample used for binding. (B) Residues in Pik1(283–425) required for Gga2–VHS binding. Pik1(283–425) mutants were tested for binding to Gga2–VHS as described in A. Bound proteins were detected by staining with Coomassie blue (Upper) or immunoblotting with His tag antibody (Lower). Input (I) = 2%. (C) Mutation of residues 286–291 in Pik1(283–425) eliminates binding to Gga2–VHS. Pik1(283–425) mutants were tested for binding to Gga2–VHS as described in B. (D) Residues 286–291 are not required for binding of full-length Pik1 to Gga2–VHS. Lysates from strains overexpressing HA-tagged WT Pik1 (HA-Pik1, GPY4966) or Pik1 containing (286–291)^6A mutations (HA-pik1^6A, GPY5063) were incubated with GST (G) or GST–VHS (V) on glutathione-Sepharase and bound proteins were analyzed by SDS/PAGE and staining with Coomassie blue (Top), immunoblotting with antibody to the HA tag (Middle) or clathrin heavy chain (Chc1p; Bottom). Input (I) = 2%.

Fig. S1.

Pik1 contains two Gga binding sites. (A) Schematic representation of Pik1 domains: LKU (lipid kinase unique amino acids 35–110), Frq (Frequenin binding site, amino acids125–169) (28), VBS1 and VBS2 (VHS binding sites 1 and 2), PI4K-homology (amino acids 437–528), and catalytic domain (amino acids 777–1066) (42). (B) VBS1 and VBS2 (underlined) and selected mutations described in the text. Underlined are residues critical for binding to Gga2–VHS domain.

Fig. S2.

Gga2–VHS binding is sensitive to single amino acid changes in VBS1 but not VBS2. (A) The indicated VBS2 single amino acid mutations in Pik1(283–425) were analyzed for binding to GST (G) or GST-Gga2–VHS (V). Bound proteins were eluted, separated by SDS/PAGE and detected by staining with Coomassie blue. Input (I) represents 8% of the samples used for binding. (B) Lysates from strains expressing untagged Pik1 (WT, GPY404.2), or strains overexpressing HA-tagged WT Pik1 (HA-Pik1, GPY4966) or Pik1 containing (286–291)^6D/E (HA-pik1^6D/E, GPY5064) under control of the glyceraldehyde-3-phosphate dehydrogenase promoter (GPD) were tested for binding to Gga2–VHS, as described in the legend to Fig. 1D. An asterisk denotes nonspecific protein band. Input = 8%. (C) The indicated VBS2 single amino acid mutations in Pik1(215–325)^6D/E were analyzed for binding to GST (G) or GST-Gga2–VHS (V) as in A. Input (I) represents 8% of the samples used for binding.

Fig. 2.

VBS1 and VBS2 are major determinants of Pik1 binding to Gga2–VHS. (A) Identification of a second Gga2–VHS binding site in Pik1. Purified Pik1-His6 fragments, all bearing (286–291)^6D/E mutations, were tested for binding to Gga2–VHS as in Fig. 1A. (B) Residues in Pik1(215–325)^6D/E required for Gga2–VHS binding. Pik1(215–325)^6D/E fragments carrying the indicated mutations were tested for binding to Gga2–VHS as in Fig. 1B; the KR→A mutant carries mutations of all positive residues in amino acids 218–225 to alanines. (C) The two Gga2–VHS binding sites in Pik1(215–325) are redundant. Pik1(215–325) mutants were tested for binding to Gga2–VHS as described in Fig. 1B. (D) Gga2–VHS binding to full-length Pik1 is reduced by mutation of both VBS1 and VBS2. Lysates from strains overexpressing HA-tagged WT Pik1 (HA-Pik1, GPY4966), HA-Pik1 containing (218–220)^EEA and (286–291)^6D/E mutations (HA-pik1^gga, GPY5062), or HA-Pik1 containing (218)^A and (286–288)^3A mutations (HA-pik1^A-3A, GPY5061) were tested for binding to Gga2–VHS as in Fig. 1D.

Fig. S3.

PIK1 mutant allele expression and effects of pik1^A-3A on PtdIns(4)P accumulation and AP-1 recruitment. (A) Lysates (5 × 10⁶ cells) from PIK1 (GPY404.2), 9xMyc-PIK1 (GPY5067), 9xMyc-pik1^gga (GPY5069), or 9xMyc-pik1^A-3A (GPY5068) strains were analyzed by SDS/PAGE and immunoblotting using c-Myc (Upper) or Gga2 (Lower) antibodies. (B and C) Representative images of cells acquired at 100× magnification by spinning disk confocal microscopy. White arrowheads show puncta used for peak-to-peak analysis summarized in Fig. 3C. The kymograph below each image depicts the selected puncta in every third frame; the time to acquire one image pair varied between 1 and 1.2 s, depending on the image. Graphs to the right of each image show the change in normalized fluorescence intensity for the selected puncta in the GFP (green) and mRFP (red) channels.

Fig. 3.

Gga-binding mutations in Pik1 impair Pik1 localization, PtdIns4P accumulation, and adaptor recruitment. (A) Defective localization of pik1^A-3A. Diagram at top shows mutations in pik1 alleles; Lower panels show representative images of strains (GPY5065, GPY5066) expressing the indicated proteins, acquired at 100× magnification by spinning disk confocal microscopy of live cells. (B) Localization of pik1^A-3A is reduced. Bar and whisker plots of the indicated strains from A and isogenic strains, depicting fluorescence intensity ratios of GFP-Pik1 puncta to whole cells (Left) or Sec7-mRFP puncta to whole cells (Right). *P < 0.05 compared with WT by Student t test. Each point represents the average relative intensity of puncta to the whole cell in a still image of a field of cells; n = 40 images for WT, n = 33 images for pik1^A-3A. “x” represents mean. (C) Accumulation of PtdIns4P and recruitment of AP-1 are delayed in pik1 mutant cells. Bar and whisker plots depicting times between peaks of fluorescence intensity (peak-to-peak fluorescence) of Sec7-mRFP and GFP-PH^OSH1 (tan bars) or AP-1 β1-GFP (blue bars) in cells carrying PIK1 (WT) or the indicated mutant pik1 alleles. *P < 0.05 compared with WT by Student t test. Each point represents a puncta; n = 41 for SEC7-mRFP GFP-PH^OSH1 WT (GPY4938), n = 18 for pik1^A-3A (GPY5070), n = 51 for pik1^gga (GPY5071), n = 27 for SEC7-mRFP APL2-GFP WT (GPY4934), n = 19 for pik1^A-3A (GPY5072), n = 47 for pik1^gga (GPY5073). “x” represents the mean. (D) Localization of Ent5-mRFP in pik1 mutants is reduced. Representative images of Ent5-mRFP and Ent3-GFP, acquired at 100× magnification by spinning disk confocal microscopy of live WT (PIK1, GPY3912) or pik1 mutant cells (GPY5074, GPY5075). (E) Bar and whisker plots depicting fluorescence intensity ratios of Ent3-GFP or Ent5-mRFP puncta to whole cells, displayed as in A. *P < 0.00001 compared with WT by Student t test. n = 30 images for WT (GPY3912 and isogenic strains), n = 20 images for pik1^A-3A (GPY5074 and isogenic strains), n = 20 images for pik1^gga (GPY5075 and isogenic strains).

Fig. 4.

Functional interaction of mammalian PI4KIIIβ and GGA2. (A) PI4KIIIβ interacts selectively with GGA2–VHS. HeLa cell extract (+) or buffer (–) was incubated with GST (G) or GST fused to VHS domains of GGA1 (V1), GGA2 (V2) or GGA3 (V3). Bound proteins were eluted and analyzed by SDS/PAGE and immunoblotting with anti-PI4KIIIβ (Top), anti-PI4KIIα (Middle), or staining by Coomassie blue (Bottom). Input (I) = 0.2%. (B) Purified PI4KIIIβ (190–308) was tested for binding to GST (G) or the indicated GST–VHS fusions (V1, V2, V3). Bound proteins were eluted and analyzed by SDS/PAGE and immunoblotting with anti-PI4KIIIβ. Input (I) = 3.3%. (C) GGA2 is required for perinuclear PI4KIIIβ localization. HeLa cells treated with siRNA targeting luciferase (siLuciferase) or the indicated GGA genes were fixed and immunostained with antibodies against PI4KIIIβ (red) and either GGA2 or GGA3 (green). An asterisk indicates a cell that retains perinuclear labeling of Gga2 and the corresponding PI4KIIIβ localization. (D) AP-1 is not required for PI4KIIIβ localization. (Left) HeLa cells treated with siLuciferase or siRNA targeting AP-1 γ1 and σ1 subunits were fixed and immunostained with antibodies against PI4KIIIβ (red) or AP-1 γ1 (green). (Right) Effects of siRNA targeting GGA2 or AP-1 were quantified as the percentage of cells with perinuclear PI4KIIIβ staining. n = number of cells. Error bars indicate SE. (E) PI4KIIIβ localization defect in siGGA2 cells is rescued by GGA2 expression. HeLa cells were treated with the indicated siRNA followed by transfection with vector alone (pCDNA3) or vector expressing siGGA2-resistant Flag-tagged GGA2. Cells were analyzed by immunofluorescence and PI4KIIIβ localization was quantified as in D. An asterisk highlights perinuclear labeling of Gga2-Flag with corresponding localization of PI4KIIIβ. All microscopy images acquired at 40× magnification.

Fig. S4.

PI4KIIIβ sequence and siRNA effects on protein expression and localization. (A) Comparison of regions in Pik1 (amino acids 215–325) and PI4KIIIβ that directly interact with VHS domains. Basic residues are highlighted in red. Pik1 VBS1 and VBS2 are boxed. (B and C) Lysates from HeLa cells treated with the indicated siRNA (Top) were analyzed by SDS/PAGE and immunoblotting for the indicated proteins (Right). (D) HeLa cells treated with the indicated siRNA (Left side) were fixed and immunostained for PI4KIIIβ (red) and either AP-1(γ1) or GM130 (green). Images acquired at 40× magnification.