An in vitro Lipid Binding Screen Identifies GOLPH3 as a PtdIns(4)P Binding Protein
To identify phosphoinositide binding proteins we devised a high throughput proteomic screen based on the lipid blot assay of Dowler et al., 2000
. This assay involves spotting phosphoinositides on a membrane, blotting with a protein of interest, washing, and detecting bound protein. We optimized the assay for use with proteins produced by in vitro
transcription and translation (IVT) with 35
S-methionine to allow detection.
Critical to the reliability of the assay is the quality of the phosphoinositides. We screened individual lots of lipids from commercial suppliers by thin layer chromatography (TLC). We also validated each binding assay with a panel of positive control lipid binding proteins.
We screened the D. melanogaster
proteome, which is compact but contains examples of the known phosphoinositide-modifying enzymes and phosphoinositide-dependent signaling pathways found in higher organisms. The Drosophila
Gene Collection provides an arrayed set of 15,466 cDNAs of known sequence cloned behind T7 promoters allowing IVT (Stapleton et al., 2002
). To date, we have screened ~4000 unique cDNAs from this collection scoring positive hits for many previously identified PH, PX, FYVE, Tub, and Proppin family proteins, validating the method (). The screen has also identified unique phosphoinositide binding proteins. Here we describe one of these, a PtdIns(4)P binding protein (clone ID LD23816, FBgn0010704) which lacks homology to known phosphoinositide binding proteins. The mammalian homolog has been named GOLPH3 (Genbank), GMx33 (Wu et al., 2000
), GPP34 (Bell et al., 2001
), or MIDAS (Nakashima-Kamimura et al., 2005
), and the yeast homolog is Vps74p (Bonangelino et al., 2002
Proteomic screening identifies GOLPH3 as a PtdIns(4)P binding protein that requires PtdIns(4)P for Golgi localization
GOLPH3 is conserved from yeast to humans. It was originally identified in a proteomic characterization of rat Golgi and later suggested to be a Golgi matrix protein (Wu et al., 2000
; Bell et al., 2001
; Snyder et al., 2006
), although its mechanism of interaction with the Golgi and function were not determined. Deletion of VPS74
in S. cerevisiae
results in defective trafficking of vacuolar proteases (Bonangelino et al., 2002
). More recently Vps74p was shown to be Golgi-localized and necessary for localization of glycosyltransferases, glycosylation, and secretory function of the Golgi in yeast (Schmitz et al., 2008
; Tu et al., 2008
To validate our screen, we sought to confirm that GOLPH3 binds PtdIns(4)P and found that not only the D. melanogaster
, but also the H. sapiens
and S. cerevisiae
orthologs expressed in rabbit reticulocyte lysates all bind tightly and specifically to PtdIns(4)P in our lipid blot assay (). The binding to PtdIns(4)P is direct, as shown by using E. coli
expressed and purified GOLPH3 (Figure S1
). Binding of GOLPH3 to small unilamellar vesicles further demonstrates specific binding to PtdIns(4)P ().
Identification of PtdIns(4)P binding domain and pocket required for Golgi localization of GOLPH3
PtdIns(4)P is Required for GOLPH3 Localization to the Golgi
If GOLPH3 binding to PtdIns(4)P is functionally important in vivo
, then it should localize to the Golgi, as other groups have previously reported (Wu et al., 2000
; Bell et al., 2001
; Snyder et al., 2006
; Schmitz et al., 2008
). Specifically, GOLPH3 was shown by immunofluorescence microscopy (IF) to colocalize with trans
Golgi markers and found by immuno-electron microscopy at the rim of the trans
Golgi (Wu et al., 2000
) and enriched in budding vesicles and tubules (Snyder et al., 2006
). With our antibodies to GOLPH3 we too see that endogenous GOLPH3 colocalizes with the endogenous trans
Golgi network (TGN) markers TGN46 and p230 (Figure S2A
), but somewhat less with the cis
Golgi marker GM130, in several mammalian cell lines (data not shown). In yeast, EGFP-Vps74p colocalizes with the late Golgi marker Sec7p-dsRed (Figure S2B
If GOLPH3 binds PtdIns(4)P in vivo
, then the subcellular localization of the two should coincide. By coexpressing the PtdIns(4)P reporter EYFP-FAPP1-PH (Godi et al., 2004
) with mCherry-GOLPH3 in HeLa cells, time-lapse confocal fluorescence microscopy shows that the two proteins fully colocalize over space and time (Movie S1
). This colocalization is tighter than any other pair of Golgi markers we examined (e.g., Figure S7
), consistent with GOLPH3 binding to PtdIns(4)P in vivo
. Both EYFP-FAPP1-PH and mCherry-GOLPH3 were enriched on tubules and vesicles leaving the Golgi, similar to previous results localizing PtdIns(4)P (Godi et al., 2004
) and GOLPH3 (Snyder et al., 2006
If GOLPH3 binds PtdIns(4)P in vivo
, then reduction of PtdIns(4)P at the Golgi should result in GOLPH3 dissociation from the Golgi. We depleted PtdIns(4)P by expressing a mutant of the lipid phosphatase Sac1 (Sac1-K2A) that constitutively localizes to the Golgi, dephosphorylating PtdIns(4)P without affecting other phosphoinositide pools (Rohde et al., 2003
and Figure S3
). In HeLa cells, expression of EGFP-Sac1-K2A resulted in loss of GOLPH3 from the Golgi (), although the Golgi remained intact and other markers remained at the Golgi including p230, TGN46, β-1,4-galactosyltransferase-EYFP, and α-mannosidase II-EGFP ( and data not shown).
In S. cerevisiae
the PI-4-kinase Pik1p produces PtdIns(4)P at the Golgi. Using a temperature sensitive mutant allele of PIK1
(Audhya et al., 2000
), we show that EGFP-Vps74p dissociates from the Golgi within five minutes after shift to the non-permissive temperature ( and Movie S2
Finally, highly overexpressed EYFP-FAPP1-PH displaces endogenous GOLPH3 from the Golgi (). We conclude that PtdIns(4)P must be present and available for interaction for GOLPH3 to localize to the Golgi.
GOLPH3 binds PtdIns(4)P via a positively charged binding pocket on the hydrophobic face of the protein
Because GOLPH3 contains no domains found in previously characterized phosphoinositide binding proteins, we mapped the domain required for PtdIns(4)P binding. A series of truncations of Drosophila GOLPH3 tested for binding to PtdIns(4)P by lipid blot assay showed that binding to PtdIns(4)P requires amino acids 30–293 (). This corresponds to the most evolutionarily conserved region, and has been termed the GPP34 domain by PFAM. This series of truncations, fused to EGFP and expressed in HEK 293 cells (), shows that the GPP34 domain is also required for localization to the Golgi ().
Using the crystal structure of yeast Vps74p (Schmitz et al., 2008
), we identified a positively charged pocket on the hydrophobic face of the GPP34 domain (Figure S4
). To test the role of this pocket we made mutations in GOLPH3 designed to neutralize its charge yet retain proper folding. Two independent mutations of the pocket (R90L and R171A/R174L) each impaired binding to PtdIns(4)P in the lipid blot assay (data not shown) and the vesicle binding assay (). IF of 3xHA-tagged wild type and mutant GOLPH3 expressed in HeLa cells shows binding pocket mutants lose Golgi localization (). This further demonstrates the requirement for PtdIns(4)P binding for Golgi localization.
Our data show that GOLPH3 binds tightly, specifically, and directly to PtdIns(4)P in vitro in two assays, binding is evolutionarily conserved from yeast to humans, depletion or blocking of PtdIns(4)P place GOLPH3 downstream of PtdIns(4)P in vivo, and mutational analysis reveals that GOLPH3 requires the ability to bind PtdIns(4)P to localize to the Golgi. Taken together we conclude that GOLPH3 and Vps74p localize to the Golgi by binding PtdIns(4)P.
GOLPH3 is an Abundant Protein
To determine a role for GOLPH3 in Golgi function, we began by quantifying its abundance using quantitative Western blotting of HeLa whole cell lysates. We compared signal intensity from lysates of known numbers of HeLa cells to known quantities of purified bacterial expressed GOLPH3. We estimate that each HeLa cell expresses 25+/−5 fg (mean+/−SEM, n=4) or 500,000+/−100,000 molecules of GOLPH3, making it a highly abundant protein (Figure S5
). This abundance suggests that GOLPH3 is a major target of PtdIns(4)P, likely to be of general importance in Golgi function.
GOLPH3 is Required for Trafficking from Golgi to PM
To determine the requirement for GOLPH3 in Golgi function, we examined the effect of reducing its levels by siRNA knockdown. By Western blot, three different GOLPH3-specific siRNA oligos reduced GOLPH3 levels by at least 70–90% in HeLa or HEK 293 cells ( and data not shown).
GOLPH3 is required for trafficking and extended ribbon morphology of the Golgi
Using the anterograde cargo ts045-VSVG-EGFP (Hirschberg et al., 2000
), we examined trafficking in cells depleted of GOLPH3. Transport of ts045-VSVG-EGFP from ER to Golgi was unaltered by knockdown of GOLPH3 (data not shown). IF of unpermeabilized cells using an antibody specific to the extracellular domain of VSVG, allowing unambiguous detection at the PM (Bossard et al., 2007
), showed that knockdown of GOLPH3 impaired trafficking from the Golgi to PM (). Delivery to the PM was quantified by summing exofacial fluorescence over the volume of each cell and normalizing to total ts045-VSVG-EGFP fluorescence. We observed a highly significant (p<10−7
, t-test) defect in delivery of ts045-VSVG-EGFP to the PM in GOLPH3 knockdown cells, [control=6.5+/−0.3 (n=13), GOLPH3 knockdown=3.5+/−0.2 (n=12), arbitrary normalized fluorescence units, mean+/−SEM]. Thus, GOLPH3 is required for anterograde trafficking from the Golgi to the PM.
GOLPH3 is Required for the Normal Extended Golgi Ribbon
We next examined Golgi morphology upon GOLPH3 knockdown. Depletion of GOLPH3 altered the Golgi ribbon, changing its normal appearance of extending partially around the nucleus, to condensing at one end of the nucleus (). Quantification of Golgi extent, measured as the fraction of nuclear perimeter encompassed by the Golgi, demonstrated a highly significant condensation of the Golgi upon knockdown of GOLPH3 by each of three specific siRNA oligos (p<10−12
for each versus control, t-test, ). We examined several peripheral membrane and transmembrane Golgi markers, and all revealed condensation of cis
, and trans
Golgi as well as the TGN upon knockdown of GOLPH3 in HeLa, HEK 293, and NIH 3T3 cells (Figures S6, S7, S8, S9, S10, and S11
). We conclude that the entire Golgi condenses upon knockdown of GOLPH3.
Recently published data indicate that Vps74p is responsible for the stable cis
Golgi localization of glycosyltransferases involved in yeast cell wall biosynthesis (Schmitz et al., 2007; Tu et al., 2007). We examined the localization of three mammalian glycosyltransferases in GOLPH3 knockdown cells and found that their localization indicated condensation of the Golgi, but they still colocalized with other Golgi markers, indicating that GOLPH3 is not necessary for localization of these glycosyltransferases to the Golgi in mammalian cells (Figures S8, S9, S10, and S11
Rescue of GOLPH3 Knockdown Requires the Ability to Bind PtdIns(4)P
To further confirm specificity of the siRNA knockdown phenotype, we made silent mutations in GOLPH3 allowing expression resistant to our strongest siRNA oligo. Expression of siRNA-resistant wild type GOLPH3 completely rescued the normal extended Golgi ribbon morphology (). We also produced an siRNA-resistant construct with the R90L PtdIns(4)P binding mutation. This mutant, as expected did not localize to the Golgi, and was incapable of rescuing Golgi morphology. These results validate that the effects we see on the Golgi upon knockdown of GOLPH3 are specific to the function of GOLPH3 and further demonstrate the necessity of PtdIns(4)P binding in GOLPH3 function.
GOLPH3 Links the Golgi to the Actin Cytoskeleton
The condensation of the Golgi observed upon knockdown of GOLPH3 raised the suggestion that GOLPH3 may serve to mediate the application of a tensile force to the Golgi, pulling the ribbon around the nucleus. We figured a cytoskeletal motor would likely be needed to apply such a force. We considered the dyneins and kinesins acting on microtubules or the myosins acting on F-actin. The effect of using nocodazole to depolymerize microtubules results in dispersal of the Golgi (Rogalski and Singer, 1984
), quite different from the effect of GOLPH3 knockdown.
The role of actin at the Golgi is poorly understood. To examine the effect of actin depolymerization on Golgi morphology, HEK 293 cells expressing a Golgi marker were treated with Latrunculin B (LatB) while performing live cell imaging. Treatment with LatB led to rapid condensation of the Golgi (Movie S3
), recapitulating a published report (Lázaro-Diéguez et al., 2006
) and the effect of knockdown of GOLPH3.
We compared the effects on the Golgi of LatB treatment or GOLPH3 knockdown. Knockdown of GOLPH3 results in compaction of the Golgi without altering F-actin (). Depolymerization of actin by LatB leads to compaction of the Golgi without affecting the Golgi association of GOLPH3. Simultaneous knockdown of GOLPH3 and treatment with LatB produced a similar compaction of the Golgi, but without an additive effect (). These results raised the possibility that GOLPH3 may in some way link the Golgi to the actin cytoskeleton, exerting a tensile force on the Golgi.
GOLPH3 links the Golgi to F-actin via MYO18A
GOLPH3 Binds MYO18A
To determine how GOLPH3 might link the Golgi to actin and affect Golgi structure, we took an open-ended approach performing large-scale immunoprecipitation (IP) of GOLPH3 from HeLa whole-cell lysates. Analysis by mass spectrometry of prominent bands near 250 kDa identified the unconventional myosin, MYO18A as a major GOLPH3 interacting protein (Figure S12
The interaction between GOLPH3 and a Golgi-localized myosin suggested a model to explain a role for GOLPH3 in linking the Golgi to the actin cytoskeleton (). Our model builds upon GOLPH3 binding to PtdIns(4)P at the Golgi and suggests that GOLPH3 further interacts with MYO18A, bringing it to the Golgi and thus linking to the actin cytoskeleton.
To confirm interaction between GOLPH3 and MYO18A, we IP’d GOLPH3 from whole cell lysates and Western blotted with a MYO18A-specific antibody. MYO18A coIP’d specifically with GOLPH3 (). The interaction is not mediated by actin, as it was not disrupted by LatB ( and Figure S13
). The Golgi-localized myosins MYO2B or MYO6 did not coIP with GOLPH3 (), indicating that GOLPH3 interacts uniquely with MYO18A. The GOLPH3/MYO18A complex is likely distinct from the MYO18A/LRAP35a/MRCK complex (Tan et al., 2008
), as we did not detect MRCKβ in GOLPH3 IP’s and only a fraction of MYO18A coIP’d with GOLPH3 (data not shown).
We next tested if GOLPH3 and MYO18A interact directly. We tagged N-terminal, Middle, and C-terminal segments of MYO18A with 6xHis-SUMO, expressed them in bacteria, and purified on Ni beads. Incubation of each MYO18A fragment with purified bacterial expressed GOLPH3 and GST (negative control) showed that GOLPH3 was specifically pulled down by the N-terminal and Middle fragments of MYO18A, but not by the C-terminal fragment or 6xHis-SUMO alone (). This pull-down of GOLPH3 with two fragments of MYO18A indicates a bipartite interaction, suggesting the interaction with intact MYO18A may be particularly strong.
We next examined subcellular localization of MYO18A by IF. Endogenous MYO18A was observed at the Golgi, colocalizing with endogenous GOLPH3 and p230 in cells treated with control siRNA (). Localization of GOLPH3 at the Golgi is independent of MYO18A, as shown by knockdown of MYO18A. However, MYO18A no longer localized to the Golgi upon knockdown of GOLPH3, showing that MYO18A depends on GOLPH3 for Golgi localization, providing genetic confirmation of the biochemical interaction.
Depletion of PtdIns(4)P Phenocopies Knockdown of GOLPH3
Our model predicts that depletion of PtdIns(4)P should also result in a condensed Golgi ribbon. In , depletion of PtdIns(4)P by expression of EGFP-Sac1-K2A caused dissociation of GOLPH3 from the Golgi. p230 IF revealed that depletion of PtdIns(4)P also caused the Golgi ribbon to condense into a compact ball ( and data not shown). Quantification of Golgi extent around the nuclear perimeter confirmed that the Golgi compaction was highly significant (p<10−8
, t-test, Figure S14
). Similar results were observed with the Golgi reporter α-mannosidase II-EGFP (data not shown) and indicate that depletion of PtdIns(4)P phenocopies knockdown of GOLPH3 and both are essential for an extended Golgi ribbon.
Knockdown of MYO18A Phenocopies Knockdown of GOLPH3
To determine if knockdown of MYO18A causes the same condensed Golgi phenotype, we used siRNA to knock down MYO18A expression in HeLa cells (). IF to endogenous GOLPH3 and p230 showed that each of three MYO18A-specific siRNAs produced the condensed Golgi phenotype (). Quantification of Golgi extent confirmed that the Golgi compaction in MYO18A knockdown cells was highly significant and similar to GOLPH3 knockdown (p<10−10, t-test, ). Similar results were observed in HEK 293 cells (data not shown).
MYO18A knockdown phenocopies GOLPH3 compact Golgi phenotype
Rescue of MYO18A Knockdown Requires an Intact MYO18A ATPase Pocket
To confirm the specificity of the MYO18A siRNA phenotype, we expressed GFP-tagged wild type mouse MYO18A, predicted to be resistant to our human siRNA oligos, to rescue the compact Golgi phenotype. We found that overexpressed mouse MYO18A-EGFP was diffuse in the cell, but fully rescued Golgi morphology resulting from knockdown of human MYO18A ().
Mutation of the MYO18A ATPase pocket was recently demonstrated to result in a dominant negative effect on actomyosin retrograde flow (Tan et al., 2008
). We used our ability to rescue human MYO18A knockdown with mouse MYO18A-EGFP to test the function of a mutation in conserved residues in the ATPase pocket. The G520S/K521A ATPase mutant failed to rescue the condensed Golgi phenotype (). MYO18A’s ability to generate force on actin has not been formally demonstrated, however since MYO18A is homologous to other myosins, especially in important functional regions, and fails to function when the ATPase pocket is mutated ( and Tan et al., 2008
), we favor that MYO18A functions, like other myosins, to generate force on F-actin.
trans Golgi Cisternae are Dilated in Cells Depleted of GOLPH3 or MYO18A
Our data demonstrate that GOLPH3 functions to bind specifically to Golgi membranes by binding to PtdIns(4)P and also to MYO18A, which we propose generates a pulling force, one consequence of which is to stretch the Golgi ribbon around the nucleus. We considered that interfering with this apparatus may also produce ultrastructural changes. Depolymerization of actin has been shown to cause rounding of the normally flat Golgi cisternae (Lázaro-Diéguez et al., 2006
). We examined Golgi ultrastructure in control, GOLPH3, or MYO18A knockdown cells and found that knockdown of GOLPH3 or MYO18A produced dilated Golgi cisternae, frequently dramatically so (). Cells appeared similar to those observed upon depolymerization of actin (Lázaro-Diéguez et al., 2006
). The dilation persists despite cycloheximide treatment, suggesting it is not due to filling of cisternae with protein (data not shown). We also observed that cisternal dilation was asymmetrically localized to one side of the Golgi (). Since PtdIns(4)P (Godi et al., 2004
) and GOLPH3 (Wu et al., 2000
) localize to the trans
Golgi, we infer that these dilated cisternae represent the trans
Golgi. The difference in cisternal thickness at the trans
Golgi was highly statistically significant (p<10−4
, unpaired t-test, ), so we conclude that GOLPH3, MYO18A, and F-actin are required to maintain the familiar flattened appearance of the trans
Knockdown of GOLPH3 or MYO18A produces dilated Golgi cisternae
PtdIns(4)P/GOLPH3/MYO18A/F-actin are Required for Efficient Vesicle Budding
Our data argue that PtdIns(4)P/GOLPH3/MYO18A/F-actin form a molecular apparatus to pull on trans Golgi membranes. We considered the purpose of this apparatus, and noted that it may contribute to the efficient extraction of tubules or vesicles from the Golgi, and thus may be required for normal Golgi vesicle trafficking. If true, we predicted that interfering with any component of the apparatus depicted in would impair trafficking. Indeed, knockdown of GOLPH3 significantly impaired trafficking of ts045-VSVG-EGFP from the Golgi to the PM ().
We next examined the effect of actin depolymerization, GOLPH3 knockdown, or MYO18A knockdown on the rate of tubule or vesicle exit from the Golgi by measuring the effect on vesicles bearing PtdIns(4)P. We performed fluorescence microscopy of cells expressing low levels of EYFP-FAPP1-PH. Live time-lapse images were obtained and tubules or vesicles emanating from the Golgi identified (Movie S4
). The number of vesicles or tubules leaving the Golgi was counted from the same cells before and after treatment with LatB. Depolymerization of actin caused a dramatic reduction in the frequency of Golgi exit events (), consistent with previously reported results (Lázaro-Diéguez et al., 2007
). Furthermore, knockdown of GOLPH3 or MYO18A each caused a similar reduction in the frequency of tubule and vesicle exit from the Golgi. We further validated these results by measuring the rate of exit of tubules and vesicles marked with the cargo ts045-VSVG-EGFP (Figure S15
). Thus, GOLPH3, MYO18A, and F-actin are required for efficient exit from the Golgi.
GOLPH3/MYO18A/F-actin are necessary for Golgi vesiculation
We tested the requirement for GOLPH3 binding to PtdIns(4)P for efficient Golgi trafficking. We attempted rescue of siRNA knockdown of GOLPH3 with siRNA-resistant wild type or R90L mutant GOLPH3 and observed trafficking of EYFP-FAPP1-PH. Wild type GOLPH3 rescued efficient trafficking, but the R90L PtdIns(4)P binding mutant did not (). We thus conclude that efficient exit from the Golgi depends on the ability of GOLPH3 to bind PtdIns(4)P.
Our model argues that the extension of the Golgi ribbon around the nucleus is a consequence of a tensile force applied via GOLPH3, and that this force is necessary for extracting tubules and vesicles from the Golgi. If true, the model predicts that tubules and vesicles exit the Golgi on a trajectory parallel to the path of the Golgi ribbon around the nucleus. As above, we expressed low levels of EYFP-FAPP1-PH to mark PtdIns(4)P-bearing vesicles and tubules and measured the angle between the trajectory of exit from the Golgi and a tangent to the Golgi ribbon at the point of exit ( and Movie S4
). The trajectory angles were graphed for 99 exit events observed in eight cells (). These angles clustered highly significantly near 0° and 180°, or parallel to the Golgi (p<0.01, Kolmogorov-Smirnov). We also observed trafficking of ts045-VSVG-EGFP, and the angles again clustered around 0° and 180°, as predicted (p<0.01, Kolmogorov-Smirnov, ).
Taken together, our data indicate that exit of traffic from the Golgi depends on interaction of PtdIns(4)P, GOLPH3, MYO18A, and F-actin. The initial trajectory of vesicle exit follows the orientation of the tensile force that extends the Golgi ribbon and flattens Golgi cisternae. We argue that this force is produced by MYO18A and F-actin and transmitted to the Golgi by GOLPH3 and PtdIns(4)P. We propose that the molecular apparatus diagrammed in and functions to attach the Golgi to the actin cytoskeleton and is necessary to pull tubules and vesicles from it.