PtdIns4P recognition by Vps74/GOLPH3 links PtdIns 4-kinase signaling to retrograde Golgi trafficking

Abstract

Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting. In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor). We show that targeting of Vps74 and GOLPH3 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol (PtdIns) 4-phosphate (PtdIns4P) by the Pik1 PtdIns 4-kinase and that modulation of the levels and cellular location of PtdIns4P leads to mislocalization of these proteins. Vps74 and GOLPH3 bind specifically to PtdIns4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74. Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo. These results implicate Pik1 signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi PtdIns 4-kinases.

Figure 1.

Intracellular targeting of yeast Vps74 and human GOLPH3 is determined by Pik1 PtdIns 4-kinase and Sac1 lipid phosphatase. (A) GFP-Vps74 or GFP-GOLPH3 was expressed in the indicated strains and visualized by fluorescence microscopy. Cultures were incubated at 26 or 37°C for 30 min before microscopy. Grayscale micrographs are not scaled equivalently so that cellular features can be better visualized. False-color images of the same micrographs are scaled equivalently so that the intensity of punctate Golgi compartments can be directly compared (from blue [lowest intensity] to yellow [highest intensity]). (B) GFP-Vps74 was expressed in sac1Δ vps74Δ cells that also express a resident ER protein, Sec63, fused to mCherry (Sec63-mCh). Colocalization of GFP-Vps74 and Sec63-mCherry is compared in a merged image. Localization of GFP-GOLPH3 in sac1Δ vps74Δ cells is shown to the right. Bar, 3 µm.

Figure 2.

Specific recognition of PtdIns4P by GOLPH3 and Vps74. (A) Radiolabeled in vitro–translated Vps74 and GOLPH3 were incubated with nitrocellulose membranes spotted with the indicated phosphoinositides (4, 2, 1, and 0.5 µg). Bound proteins were visualized using a phosphorimager. Unprgrm, unprogramed. (B and C) SPR of GOLPH3 (B) and Vps74 (C) binding to phosphoinositides. A series of samples at the indicated concentrations was passed over an L1 chip that had been prepared with vesicles of the indicated phosphoinositides at 3% (mol/mol) in a DOPC background. The steady-state SPR response for a representative dataset, corrected for background binding to a DOPC surface, is plotted against protein concentration. The inset shows data for 50 µM Vps74. The curves indicate the fit to a simple one-site binding equation. K_D values from at least three independent binding experiments are listed in Table I. RUs, relative units.

Figure 3.

Structure of human GOLPH3 and identification of a putative PtdIns4P-binding site. (A) Orthogonal cartoons of GOLPH3. Core α helices are shown in gold (labled in the right panel only), amphipathic α helices in red, and β strands, including the conserved β3/β4 hairpin, in blue. A well-ordered and partially buried sulfate ion is shown in stick representation. (B) The β hairpin–mediated crystallographic dimer of GOLPH3 (left) is almost identical to one of the intersubunit interfaces in the Vps74 tetramer (right). The other subunits of the Vps74 tetramer are shown in blue and cyan. (C) A magnified view of the sulfate-binding pocket (boxed regions in B) from GOLPH3, showing the sulfate and amino acids lining this pocket in stick representation. Interactions with the sulfate ion are shown with dashed lines. The same region of Vps74 is shown on the right. A water molecule occupies approximately the same position as the sulfate. The K178 side chain is disordered in the Vps74 structure. Underlined amino acids were substituted with alanine (see Results and discussion).

Figure 4.

PtdIns4P binding is essential for Golgi localization of Vps74 and GOLPH3 and for Vps74-mediated retention of the Kre2 Golgi mannosyltransferase. (A) GFP-tagged forms of Vps74 were expressed in vps74Δ and vps74Δ sac1Δ cells and visualized by fluorescence microscopy. (B) SDS-PAGE autoradiography was used to compare the relative mobilities of radiolabeled proteins that are secreted into the medium by cells that express wild-type VPS74, an empty vector, or the indicated Vps74 point mutants. The migration of mass standards (kD) is indicated. (C) Localization of Kre2-GFP, a medial Golgi mannosyltransferase that requires functional Vps74 to be retained in the Golgi, is shown in vps74Δ cells that express wild-type or the indicated untagged Vps74 point mutants. (D) Kre2-GFP localization is shown in pik1-83 and frq1-1 cells grown at 26°C (permissive temperature) or incubated for 60 min at 37°C (restrictive temperature). Maximum intensity projections of z stacks are shown. Each pair of micrographs (isogenic wild type and mutant) was identically scaled so that the intensity of Kre2-GFP signals can be directly compared. Bar, 3 µm.