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Recycling of Golgi Glycosyltransferases Requires Direct Binding to Coatomer

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Recycling of Golgi Glycosyltransferases Requires Direct Binding to Coatomer 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 transferase 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 enzyme 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 transferases 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 Human 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 cell 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.
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