Recycling of Golgi glycosyltransferases requires direct binding to coatomer

Lin Liua, Balraj Doraya, and Stuart Kornfelda,1

aDepartment of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110

Contributed by Stuart Kornfeld, July 20, 2018 (sent for review June 14, 2018; reviewed by Juan S. Bonifacino and Suzanne R. Pfeffer) The glycosyltransferases of the mammalian Golgi complex must Results recycle between the stacked cisternae of that organelle to Specific Binding of Ptase N-Tail to Coatomer. Ptase is a type III maintain their proper steady-state localization. This trafficking transmembrane protein with a 17-amino acid N-tail and a 21- is mediated by COPI-coated vesicles, but how the glycosyltrans- amino acid C-tail, both of which are cytoplasmically oriented. ferases are incorporated into these transport vesicles is poorly Previously we showed that two mutations in the N-tail, Lys4Gln understood. Here we show that the N-terminal cytoplasmic tails (K4Q) and Ser15Tyr (S15Y), identified in patients with the ly- cis (N-tails) of a number of Golgi glycosyltransferases which sosomal storage disorder mucolipidosis III, resulted in the ϕ δ share a -(K/R)-X-L-X-(K/R) sequence bind directly to the -and being mislocalized to punctae tentatively identified as ζ -subunits of COPI. Mutations of this N-tail motif impair binding the endolysosomal compartment (15). We now document that to the COPI subunits, leading to mislocalization of the transfer- these mutant proteins, along with a protein containing a third ases to lysosomes. The physiological importance of these inter- patient mutation, Arg8Gly (R8G) (16), colocalize with the ly- actions is illustrated by mucolipidosis III patients with missense sosomal marker cathepsin D, establishing that the punctae rep- mutations in the N-tail of GlcNAc-1-phosphotransferase that resent mostly lysosomes (Fig. 1A and SI Appendix, Fig. S1 A–C). cause the transferase to be rapidly degraded in lysosomes. These We hypothesized that these mutations impaired binding to a studies establish that direct binding of the N-tails of mammalian protein(s) required for the retrograde transport of the trans- cis Golgi glycosyltransferases with COPI subunits is essential for ferase from the trans cisternae of the Golgi to the cis cisternae recycling within the Golgi. where the functions. To identify proteins that interact with the N-tail of Ptase in cells, we utilized the BioID2 system to COPI | coatomer | glycosyltransferase | Golgi biotinylate proteins in the immediate proximity of the transferase (17). Taking advantage of the fact that Ptase is a type III he Golgi complex contains numerous glycosyltransferases transmembrane protein, we attached the BioID2 biotin ligase to Tthat process the glycans present on newly synthesized glyco- the C-tail of the transferase (Fig. 1B), something not possible proteins as they move through this organelle (1). These en- with other glycosyltransferases. We first established that the at- zymes are arrayed in the specific order in which they act along tachment of the BioID2 protein did not alter the Golgi locali- the stacks of the cisternae. To maintain their steady-state lo- zation of WT Ptase nor impact the mislocalization of the mutants calization in the Golgi, glycosyltransferases undergo continuous (SI Appendix, Fig. S1D). Using these constructs, we set out to rounds of retrograde transport from late cisternae (trans)to cis identify proteins that bound to the WT tail substantially better earlier cisternae ( ) mediated by COPI vesicles (2, 3). Glyco- than to the patient mutants. Transiently transfected HEK 293 syltransferases are type II transmembrane proteins whose lo- calization in the Golgi has been shown to be impacted by several Significance of their domains, including the transmembrane segment, the luminal region, and the amino-terminal cytoplasmic tail (N-tail) (4, 5). In yeast it has been documented that the steady-state The mammalian Golgi contains numerous glycosyltransferases localization of a number of Golgi glycosyltransferases is medi- that continuously recycle from late cisternae to earlier cisternae ated by binding of the peripheral membrane protein Vps74 to a in COPI vesicles to maintain their steady-state localization in this conserved motif (F/L-L/I/V-X-X-R/K) present in the N-tails of organelle. How the glycosyltransferases are incorporated into these glycosyltransferases (6, 7). The Vps74, in turn, binds to these vesicular carriers is poorly understood. Here, we show that COPI coatomer, promoting the incorporation of the the N-cytoplasmic tails (N-tails) of a subset of these type II into COPI-coated vesicles to mediate their retrograde transport. transmembrane proteins bind directly to two of the seven sub- While mammalian cells contain an equivalent of Vps74 known as units of COPI coatomer. These glycosyltransferases share a com- GOLPH3, its potential role in recycling has only been analyzed mon amino acid motif in their N-tails. The importance of these in a few instances with variable results (8–13). To date no evi- interactions is illustrated by mucolipidosis III patients with missense dence has been presented for the direct binding of glycosyl- mutations within the N-tail motif of GlcNAc-1-phosphotransferase transferase N-tails to COPI subunits, in contrast to the situation that impair binding to the COPI subunits, resulting in the mis- with several type I endoplasmic reticulum (ER) proteins that get localization of this transferase to lysosomes. incorporated into COPI-coated vesicles through their carboxyl- Author contributions: L.L., B.D., and S.K. designed research; L.L. and B.D. performed re- terminal cytoplasmic tails (C-tails) which contain di-lysine (KK search; L.L. and B.D. contributed new reagents/analytic tools; L.L., B.D., and S.K. analyzed or KXKXX)-, arginine (RxR)- or FFXXBB (B is basic amino data; and L.L., B.D., and S.K. wrote the paper. acid)-based motifs (14). We now demonstrate that a number of Reviewers: J.S.B., Eunice Kennedy Shriver National Institute of Child Health and cis Golgi glycosyltransferases bind directly through their N-tails Development, National Institutes of Health; and S.R.P., Stanford University School to the δ- and ζ-subunits of COPI and that this interaction is of Medicine. essential to maintain the Golgi localization of these proteins. The authors declare no conflict of interest. GlcNAc-1-phosphotransferase (Ptase), the Golgi enzyme that Published under the PNAS license. catalyzes the first step in the synthesis of the mannose 6- 1To whom correspondence should be addressed. Email: [email protected]. phosphate recognition marker on lysosomal acid hydrolases, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. serves as a paradigm for what can go wrong at the cellular 1073/pnas.1810291115/-/DCSupplemental. level when this binding is impaired. Published online August 20, 2018.

8984–8989 | PNAS | September 4, 2018 | vol. 115 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1810291115 Downloaded by guest on October 3, 2021 − − Fig. 1. Identification of coatomer subunits as N-tail binding proteins using the BioID2 method. (A) Confocal immunofluorescence images of GNPTAB / HeLa cells transfected with either WT Ptase or the N-tail mutant (K4Q) cDNA and colocalized with the lysosomal enzyme cathepsin D. (Magnification: 1,260×.) (B) Schematic of Ptase with BioID2 biotin ligase fused to the C terminus with the HA epitope. (C) High-confidence N-terminal cytoplasmic tail interactors identified using the BioID procedure. (D) Ptase N-tail peptide pulldown of endogenous coatomer, probed with antibodies against β-COP and δ-COP. AP-1 was detected within an antibody against the μ1-subunit. Unless indicated otherwise, 5% of input and 50% of pellet fraction were loaded for all binding assays. (E) Schematic of the subunit arrangement of COPI subunits within the F subcomplex and the analogous clathrin adaptor AP-1. (F) Binding of Ptase WT N-tail immobilized on streptavidin–agarose beads to the indicated proteins expressed in HEK 293 cells.

cells were incubated with biotin for 16 h to allow for interacting Binding to δ-COP μ-Homology Domain and ζ-COP Is Direct. The hu- proteins to be biotinylated followed by capture of these proteins man δ-COP subunit contains a μ-homology domain (hMHD) on streptavidin beads and identification by mass spectrometry with a longin domain and helix region (Fig. 2A). To localize the analysis. This analysis identified a small number of proteins that Ptase binding site, we expressed separately the N- and C- interacted with WT Ptase two- to threefold greater in terms of terminal portions of δ-COP in HEK 293 cells and found bind- proximal biotinylation than with the mutant N-tails (Fig. 1C and ing only to the latter (Fig. 2B), implicating the hMHD as the SI Appendix, Fig. S2). Among the top hits were the δ-COP and binding domain. Compared with WT N-tail peptide, the binding β-COP subunits of COPI coatomer along with ARFGAP2, a of peptides with the patient mutations to bacterially expressed protein known to be associated with COPI (18). Of note, and purified δ-COP hMHD and ζ1-COP (SI Appendix, Fig. S4) GOLPH3 was not detected as a biotinylated protein in this sys- was substantially decreased (Fig. 2C). Similarly, binding of the tem. To confirm that the Ptase N-tail binds coatomer, we per- mutant peptides to COPI F subcomplex purified from SF9 insect formed pulldown assays using HEK 293 lysates and a peptide cells was decreased compared with WT (SI Appendix, Fig. S5). corresponding to the N-tail with a biotin moiety at the C ter- Consistent with these observations, bio-layer interferometry – minus, allowing immobilization on streptavidin agarose beads. (BLI) analysis revealed that the K4Q and R8G mutations im- The peptide bound coatomer as detected with antibodies against paired binding to both the hMHD of δ-COP and ζ1-COP relative β-COP and δ-COP, but not adaptor protein 1 (AP-1) or actin to the WT value, whereas the S15Y mutation impaired binding (Fig. 1D). only to the ζ1-COP subunit (Fig. 2D and SI Appendix, Fig. S6). Ptase N-Tail Binds to the δ- and ζ-Subunits of Coatomer. Coatomer Localization of Binding Site Within δ-COP μ-Homology Domain. Of consists of two subcomplexes: the cage-like B subcomplex, com- note, the WT N-tail peptide failed to bind to the yeast δ-COP posed of α-, β′-, and e-subunits, and the adaptor-like F sub- MHD (yMHD) (Fig. 2C) even though it is structurally quite complex, composed of β-, γ-, δ-, and ζ-subunits, the latter being similar in subunit organization to AP-1 (Fig. 1E). We next in- similar to its human ortholog. This provided an opportunity to dividually expressed each of the subunits of COPI, and also the localize the peptide-binding site within the hMHD by preparing – three additional proteins identified in the BioID2 screen (Fig. 1C), a series of yeast human chimeric proteins and determining the in HEK 293 cells and determined their binding to the Ptase N-tail minimum amount of human sequence required for the yeast peptide. As shown in Fig. 1F,theδ-COP and ζ1-COP subunits protein to acquire the ability to bind the N-tail peptide. As shown E F bound well while trace binding was detected with β-COP and γ1- in Fig. 2 and , the substitution of yeast MHD amino acid COP. Binding of the other COPI subunits was extremely weak or residues 252–255 (residues 532–535 in full-length yeast δ-COP) undetectable, as was that of ARFGAP2, GOLGA5, and TMF1. with the equivalent human MHD residues (residues 495–500 in Coexpression of ζ1-COP with its binding partner γ-COP in SF9 red in Fig. 2G of human δ-COP) was sufficient to confer peptide insect cells, or the complete F subcomplex, did not enhance ζ1- binding. Interestingly, these residues are located in the analo- binding (SI Appendix,Fig.S3, Upper). Similarly, coexpression of gous region serving as the binding site for YXXϕ (ϕ designates

δ-COP with β-COP or the F subcomplex had little effect on its bulky hydrophobic residue) on the μ2-subunit of adaptor protein CELL BIOLOGY binding (SI Appendix,Fig.S3, Lower). 2 (Fig. 2H) (19).

Liu et al. PNAS | September 4, 2018 | vol. 115 | no. 36 | 8985 Downloaded by guest on October 3, 2021 Fig. 2. Ptase N-tail binds directly to δ-COP and ζ1-COP. (A) Schematic representation of full-length δ-COP and its N-terminal longin/helix domain and its C-terminal hMHD. (B) Ptase N-tail peptide pulldown with HEK 293 cell-expressed full-length δ-COP, or the N- or C-terminal domains. (C) Binding of Ptase N-tail peptides (WT and mutants) immobilized on streptavidin–agarose beads to purified ζ1-COP, hMHD, or yMHD. (D) Binding constants for Ptase N-tail peptides (WT vs. mutants) with purified ζ1-COP and hMHD, as determined by BLI. (E) Schematic representation of the various yeast–human δ-COP chimeric cDNA constructs. (F) Ptase N-tail peptide pulldown of bacterially expressed and purified yeast–human MHD chimeric proteins. (G) Sequence of δ-COP N-tail binding site of human is identical to mouse and bovine species, but distinct from Saccharomyces cerevisiae (yeast). Amino acids in red correspond to residues 252–258 of the MHD, as shown in E.(H) Structure of bovine δ-COP MHD [Protein Data Bank (PDB) ID code 4O8Q] and adaptor protein AP-2 μ2-subunit (PDB ID code 1BXX). Arrowhead indicates site on the MHD localized to bind the Ptase N-tail, while the arrow is the site on AP-2 μ2 known to bind YXXϕ motif.

δ- and ζ-COP Bind to a Number of Glycosyltransferase N-Tails. Since of δ-COP (Fig. 3C, Upper) but binding to the purified ζ1-COP our results demonstrated that δ- and ζ-COP bound directly to the was variable (Fig. 3C, Lower). In contrast, the N-tail peptides of N-tail of Ptase, it was of interest to determine whether the N-tails six other glycosyltransferases failed to bind endogenous coat- of other glycosyltransferases also bound these two COPI sub- omer or its purified subunits in these assays (Fig. 3 A–C). units. Toward this end, we synthesized 12 different N-tails cor- responding to type II transmembrane proteins, including 11 Identification of Binding Motifs Within the N-Tails of Glycosyltransferases. glycosyltransferases and a control protein, sucrase–isomaltase Examination of the N-tail sequences of the glycosyltransferases that (SI), a plasma membrane protein (Fig. 3A). Remarkably, the N- bound COPI revealed the following common features lacking in the tails of the glycosyltransferases C2GNT1, GALNT3, GALNT4, nonbinders, ϕ-(K/R)-X-L-X-(K/R), that could potentially serve as GALNT6, and GALNT8 pulled down endogenous coatomer binding motifs for δ-COP and/or ζ-COP. Both K4 and R8 of Ptase (Fig. 3B). These peptides also bound well to the purified hMHD are part of this motif and mutation of either residue causes

8986 | www.pnas.org/cgi/doi/10.1073/pnas.1810291115 Liu et al. Downloaded by guest on October 3, 2021 Fig. 3. Binding of δ-COP and ζ1-COP to the N-tails of a number of glycosyltransferases dictates their Golgi localization. (A) The N-tail sequence of 12 Golgi glycosyltransferases is shown along with that of the plasma membrane protein sucrase–isomaltase. Amino acids (shown in red) of the binders conform to the consensus sequence ϕ-(K/R)-X-L-X-(K/R). (B and C) Pulldown assays of cytosolic coatomer (B), and purified δ-COP MHD and ζ1-COP (C) with the indicated N-tail peptides shown in A.(D) Binding of C2GNT1 N-tail peptides (WT and mutant) to purified δ-COP MHD and ζ1-COP. (E) Cellular distribution of WT C2GNT1 and the N-tail mutant, colocalized with GOLPH4, a specific Golgi marker not related to GOLPH3. (F) Cellular distribution of luminal GFP chimeras containing the transmembrane domain of the plasma membrane protein sucrase–isomaltase and the indicated N-tails. (Magnification: E–F, 1,260×.)

mislocalization of Ptase and a disease phenotype. Further, al- One of the glycosyltransferases, GALNT4, bound hMHD tering the spacing between K4 and R8 of Ptase resulted in quite well, although it lacked the consensus sequence present in mislocalization to the endosome/lysosome compartment (SI the N-tail of the other transferases (Fig. 3 A–C). Examination of Appendix,Fig.S7). It should be noted that one of the mucoli- its N-tail revealed a WTW motif known to bind the MHD (20). pidosis III patient mutations (S15Y) which only impairs binding In agreement with the previous study, a GST–WXW fusion peptide to ζ1-COP (Fig. 2 C and D), is outside the common motif, in- efficiently pulled down WT but not the mutant hMHD (H330A, dicating additional residues may be involved in COP1 binding. K363S) (SI Appendix,Fig.S9, Upper). This double mutation also Similarly, in the case of C2GNT1, mutation of the two Arg abolished binding of the hMHD to the GALNT4 N-tail peptide (SI Appendix Middle residues (R8 and R9) just outside of the putative motif was ,Fig.S9, ) but, as expected, did not affect binding to SI Appendix Lower required along with mutation of one of the Arg residues (R3 or the Ptase N-tail peptide ( ,Fig.S9, ) since this F R7) within the motif to cause mislocalization of the transferase binding occurs at a different site on the MHD (Fig. 2 ). (Fig. 3D and SI Appendix,Fig.S8A and B). Taken together, GOLPH3 Is Not Required for Golgi Localization of Ptase, C2GNT1, and these findings indicate that, in addition to the core motif, res- MGAT1 in HeLa Cells. To test the effect of GOLPH3 depletion on idues outside the defined sequence may impact binding. the Golgi localization of a number of glycosyltransferases, we Our findings raised the question of whether the N-tail of a utilized the CRISPR-Cas9 system to inactive the GOLPH3 glycosyltransferase with the consensus sequence that bound in HeLa cells (Fig. 4A). We found that the absence of GOLPH3 coatomer is sufficient in itself to direct Golgi localization of a did not alter the Golgi localization of Ptase, C2GNT1, and reporter protein. To test this possibility, a number of chimeric MGAT1, nor did it impair the ability of the Ptase and C2GNT1 constructs were made containing the N-tail of a glycosyltransferase peptides to pull down cytosolic COPI (Fig. 4 B–E). In addition, – with the transmembrane segment of sucrase isomaltase and a siRNA knockdown of GOLPH3 did not impact the binding of luminal GFP. We found that N-tails containing the consensus the Ptase N-tail to coatomer (SI Appendix, Fig. S10). Thus, in the sequence (Ptase, C2GNT1, GALNT3, and -8) were competent subset of Golgi glycosyltransferases we tested, GOLPH3 is not in directing Golgi localization of the chimeric reporters, whereas required for maintaining their Golgi localization. N-tails lacking the sequence (SI, MGAT1) failed to do so (Fig. 3F and SI Appendix,Fig.S8C). These results demonstrate that N-tails Discussion

harboring the consensus sequence are necessary and sufficient for Taken together, these findings indicate that the retrograde CELL BIOLOGY Golgi targeting. trafficking of Ptase and a number of other glycosyltransferases

Liu et al. PNAS | September 4, 2018 | vol. 115 | no. 36 | 8987 Downloaded by guest on October 3, 2021 this protein to the plasma membrane (8). Similarly, knockdown of GOLPH3 in CHO cells results in partial redistribution of α-2,6 SialT to the plasma membrane (8), while knockdown of GOLPH3 in either HeLa or HEK 293 cells had no effect on the localization of α-2,6 SialT; β-1,4 GalT; or α-mannosidase (10, 12). Moreover, depletion of GOLPH3 leads to accumulation of POMGnT1 in the endoplasmic reticulum (13). These differences may reflect vari- ability in the regulation of specific glycosyltransferase localization among different cell types (8). While our data clearly demonstrate that the N-tails of a number of glycosytransferases interact directly with δ-COP and/ or ζ1-COP, which is essential for their localization in the Golgi, this is not the case with every N-tail we have examined (Fig. 3 B and C). In examining the Golgi localization of the various gly- cosyltransferases analyzed in this study, it is evident that all of the transferases that were shown to bind directly to coatomer are either established cis Golgi residents or likely to be so, based on their role in oligosaccharide assembly. With the exception of GALNT2, which has a very short N-tail, the glycosyltransferases that fail to bind coatomer are localized to the medial–trans re- gions of the Golgi. This raises the possibility that this mechanism for incorporation into COPI vesicles is primarily used by cis Golgi glycosyltransferases. It also underscores that resident Golgi proteins utilize a variety of mechanisms, including their Fig. 4. GOLPH3 is not required for the Golgi localization of Ptase and transmembrane and luminal elements, in addition to their N- C2GNT1. (A) Two HeLa CRISPR knockout (k/o) cell lines show complete absence of GOLPH3, while the levels of GOLPH3-like (GOLPH3L) are unaffected, as tails, to maintain their steady-state localization in this highly determined with the pan-GOLPH3 antibody that detects both GOLPH3 and dynamic organelle. GOLPH3L (12). (B) N-tail peptide pulldown of cytosolic coatomer from normal HeLa cells and the two GOLPH3 k/o cell lines. Ten percent of input and 50% of Materials and Methods pellet fraction were loaded. (C–E) Cellular distribution of Ptase (C), C2GNT1 Reagents and Plasmids. All antibodies and cell lines used in this study are listed (D), and MGAT1 (E) colocalized with the Golgi marker GOLPH4, in normal in detail in SI Appendix, Materials and Methods. The human full-length HeLa cells and the two GOLPH3 k/o cell lines. (Magnification: C–E,1,260×.) Ptase-V5/His constructs in pcDNA6 have been described (22). Mutants in the N-terminal tail were generated by site-directed mutagenesis. The various cDNA constructs including the GOLPH3 CRISPR-Cas9 and GST fusion protein involves the direct interaction of their N-tails with the δ-COP plasmids are described in SI Appendix, Materials and Methods. and ζ1-COP subunits of coatomer. When this interaction is dis- rupted, as occurs in mucolipidosis III patients with missense Protein Expression and Purification. All GST-fusion proteins were expressed in mutations in the N-tail of Ptase, the result is mislocalization of the Escherichia coli strain BL-21 (RIL) (Stratagene) and purified essentially as described previously (23). For expression in Sf9 insect cells, the various the mutant to the lysosome where they are degraded pFastBac-Dual constructs were transformed into E. coli DH10Bac-competent (15) with a concomitant disease phenotype. As far as we are cells to generate recombinant bacmids as previously described (24). Bacmid aware, this is the first documentation of a direct interaction of DNAs were prepared and transfected using standard protocol into Sf9 cells Golgi glycosyltransferase N-tails with COPI coatomer. Further, it (Invitrogen) to produce recombinant baculoviruses that were amplified and is quite interesting that the binding occurs to two of the COPI used to express or coexpress the different coatomer subunits in Sf9 subunits, as these two proteins have very different structures. insect cells. Analysis of the N-tail peptide binding sites in these proteins should help to clarify this issue. BioID Screen for Identification of Interacting Proteins. To achieve proximity- Other than cytosolic proteins containing the WxW motif, dependent labeling of proteins interacting with the N-terminal cytoplas- δ mic tail of Ptase, Ptase-BioID2/HA pcDNA6 with cDNA encoding either the WT -COP MHD binding motifs have only been reported in the N-tail or point mutants (K4Q, R8G, or S15Y) was transfected into HEK 293 cytoplasmic domain of the ER-resident protein, Sec71, and in an cells. Forty-eight hours posttransfection, free biotin was added to the cell ER-retention motif in the C-tail of the CD36E chain of the media at a final concentration of 50 μM, and cells were incubated for a T-cell receptor, a type I transmembrane protein (21). In- further 16 h to allow for the biotinylation of interacting proteins. After terestingly, in these proteins the binding motifs contain a critical rinsing twice with PBS wash, cells were harvested and lysed by sonication in aromatic residue, whereas the essential residues of the binding lysis buffer (25 mM Tris, pH 7.2, 150 mM NaCl, 1% Triton X-100) containing motifs of the glycosyltransferase N-tails are basic residues. The protease inhibitor (Inhibitor Mixture, Sigma). Cell lysates were cleared by centrifugation at 20,000 × g for 10 min, and the supernatant was collected glycosyltransferase N-tails appear to be the first ligands identi- μ – ζ and incubated with 100 L streptavidin agarose beads (Thermo Fisher Sci- fied as binding partners with 1-COP. These COP binding motifs entific) overnight. Beads were collected after brief centrifugation at 2,000 × are highly conserved among species, consistent with having an g, washed once with PBS containing 1% SDS, twice with lysis buffer, and

important role in the function of the enzymes. once with wash buffer containing 50 mM Na2HPO4, pH 7.4, 500 mM NaCl In our experiments the inactivation of the gene encoding and 1% Triton X-100, before mass spectroscopy analysis. GOLPH3 in HeLa cells had no effect on the Golgi localiza- Mass spectrometry analysis was performed on the WT and mutant Ptase at tion of Ptase, C2GNT1, or MGAT1, nor did it inhibit the ability the Proteomics Core Laboratory at Washington University School of Medicine of Ptase and C2GNT1 N-tail peptides to pull down cytosolic in St. Louis. Isobaric labeling-based relative quantitation was used to score for COPI. Similar findings were obtained using siRNA knockdown high-confidence proximity interactors. of GOLPH3. These findings with C2GNT1 differ from reports Biotinylated Peptide and GST Pulldown Assays. Peptides corresponding to the documenting that knockdown of GOLPH3 in KG1 lymphoblasts N-terminal cytoplasmic tails (WT or mutants) of the various Golgi glycosyl- results in C2GNT1 redistributing to the endoplasmic reticulum transferases or the plasma membrane protein, sucrose–isomaltase, were (9), whereas in Chinese hamster ovary (CHO, Agilent Technolo- synthesized by Lifetein (LifeTein, LLC). All peptides were covalently linked to gies) cells, the loss of GOLPH3 leads to partial redistribution of a biotin moiety at the C terminus to mimic the orientation of the peptides in

8988 | www.pnas.org/cgi/doi/10.1073/pnas.1810291115 Liu et al. Downloaded by guest on October 3, 2021 the native state when immobilized on streptavidin beads. Pulldown assays siRNA Knockdown of GOLPH3. siGENOME human GOLPH3 siRNA (Cat. no. D- were performed as described in SI Appendix, Materials and Methods. 006414-01-002) and control nontargeting siRNA (Cat. no. D-001210-01-05) were purchased from Dharmacon (GE Healthcare). HeLa cells were seeded Direct Binding Using BLI. Direct binding of the N-terminal cytoplasmic tail with 30% confluence in six-well plates 1 d before siRNA transfection. The peptides to either the purified coatomer δ-subunit MHD or to the ζ1-subunit cells were transfected with 5 μM siRNA using DharmaFECT 4 transfection was determined by BLI using a ForteBio Octet. Streptavidin-coated biosen- reagent (Dharmacon) following the manufacturer’s protocol. Two days later, sors from ForteBio were used to capture the biotinylated peptides onto the the cells were analyzed by Western blotting. surface of the sensor. After reaching base line, sensors were moved to the association step containing serial dilutions of purified coatomer subunits for Immunofluorescence Microscopy. To visualize the subcellular localization of −/− 300 or 600 s and then dissociated for 300 s. A buffer-only reference was various constructs, the constructs were transfected into GNPTAB or pa- subtracted from all curves. Affinities were estimated from global kinetic rental HeLa cells using Lipofectamine 3000 (Life Technologies) according to ’ analysis of the five concentrations using Octet RED software version 5.2. R2 the manufacturer s protocol. Twenty-four hours posttransfection, the cells – (Fig. 2D) is the square of the sample correlation coefficient between the treated with or without 10 mM NH4Cl for 4 6 h were fixed with 4% form- outcomes and their predicted values. aldehyde (Sigma-Aldrich) and permeabilized in 0.1% (vol/vol) Triton X-100 in PBS. Cells were blocked for 1 h with 2% IgG-free BSA (Jackson Immuno- Research) and probed with the indicated combinations of antibodies as Generation of CRISPR Knockout GOLPH3 Cell Line. HeLa cells (described in SI described in SI Appendix, Materials and Methods. The images were acquired Appendix, Materials and Methods) were transfected with 500 ng pX330- with an LSM880 confocal microscope (Carl Zeiss, Inc.) in the Molecular Mi- GOLPH3 plasmid and 150 ng pEGFP-puro (Addgene no. 45561) in 12-well crobiology Imaging Facility at Washington University School of Medicine in plates with 50% confluence using Lipofectamine 3000 (Life Technologies) St. Louis. Images were analyzed by ImageJ software (Fiji). according to the manufacturer’s protocol. One day later, the cells were treated with 10 μg/mL puromycin in medium. Twenty-four hours later, the ACKNOWLEDGMENTS. We thank Reid Townsend and Qiang (Tim) Zhang cells were washed twice with PBS and trypsinized and diluted into two to from the Proteomics Core (Washington University School of Medicine in three 96-well plates with around one cell per well and cultured in 15% FBS St. Louis) for the mass spectrometry and data analysis. This work was supported for ∼10 d. Single colonies were expanded and screened by Western blotting by National Institutes of Health Grant CA-008759 and the Yash Gandhi using GOLPH3 antibody. Foundation.

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