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A BLOC-1–AP-3 Super-Complex Sorts a Cis-SNARE Complex Into

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A BLOC-1–AP-3 Super-Complex Sorts a Cis-SNARE Complex Into ARTICLE ABLOC-1–AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers Shanna L. Bowman1,2,3,4,LinhLe1,2,3,5, Yueyao Zhu1,2,3,6, Dawn C. Harper1,2,3, Anand Sitaram1,2,3, Alexander C. Theos7, Elena V. Sviderskaya8, Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202005173/1413924/jcb_202005173.pdf by St George's, University Of London user on 26 May 2021 Dorothy C. Bennett8, Graça Raposo-Benedetti9, David J. Owen10,MeganK.Dennis1,2,3,11,andMichaelS.Marks1,2,3 Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1–dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3–BLOC-1 super-complex. Introduction The endolysosomal system requires proper regulation of mem- and Südhof, 2012). In endosomal fusion, the four SNARE helices brane trafficking for maintenance of organelle identity, cargo can be differentially distributed among opposing membranes distribution, signaling, metabolic control, and organelle bio- (Zwilling et al., 2007). A key step in determining the fusion genesis (Huotari and Helenius, 2011). Membrane-bound vesic- capacity of a transport carrier with its target is the incorporation ular or tubular transport carriers bud from donor organelles and of the R-SNARE at the time of transport carrier formation. are transported to the appropriate target organelle, to which However, our understanding of the mechanisms by which they then dock and fuse to deliver their contents (Bonifacino and R-SNAREs are sorted into transport carriers is incomplete. Glick, 2004). Docking and fusion are primarily mediated by Lysosome-related organelles are specialized, tissue-specific specific interactions between SNARE proteins on the membrane structures that derive from the endolysosomal network. carrier and the target membrane (Jahn and Scheller, 2006; Yoon Lysosome-related organelle biogenesis and maturation via and Munson, 2018). Typically, a vesicular SNARE (or R-SNARE, membrane transport are well studied for melanosomes, organ- defined by the central R residue in the SNARE helix) on the elles within pigment cells of the skin, hair, and eyes that syn- vesicle membrane engages a three-helix target SNARE complex thesize and store melanin pigments (Bowman et al., 2019; (or Qa, Qb, and Qc helices, defined by the central Q residue in the Sitaram and Marks, 2012). Melanosome precursors segregate SNARE helix) on the target membrane to form a stable four- from early endosomes (Raposo et al., 2001) and then mature in a helix bundle “trans”-SNARE complex that drives fusion (Jahn process that requires delivery of melanogenic enzymes and and Scheller, 2006), often promoted by Sec1-Munc18 family transporters by at least three distinct membrane transport members and other regulators (Baker and Hughson, 2016; Rizo pathways, one from the Golgi and two from distinct domains of ............................................................................................................................................................................. 1Department of Pathology & Laboratory Medicine, Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA; 2Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 3Department of Physiology, University of Pennsylvania, Philadelphia, PA; 4Department of Biology, Linfield University, McMinnville, OR; 5Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA; 6Department of Biology, University of Pennsylvania, Philadelphia, PA; 7Department of Human Science, Georgetown University, Washington, DC; 8Cell Biology Research Centre, Molecular and Clinical Sciences Research Institute, St George’s, University of London, London, UK; 9Institut Curie, Paris Sciences et Lettres Research University, Centre National de la Recherche Scientifique Unite´ Mixte de Recherche 144, Compartiments de Structure et de Membrane, Paris, France; 10Cambridge Institute for Medical Research, Cambridge, UK; 11Department of Biology, Marist College, Poughkeepsie, NY. Correspondence to Michael S. Marks: [email protected]; A. Sitaram’s present address is Biology Department, Stonehill College, North Easton, MA. © 2021 Bowman et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press https://doi.org/10.1083/jcb.202005173 1of20 J. Cell Biol. 2021 Vol. 220 No. 7 e202005173 early endosomes (Bowman et al., 2019; Sitaram and Marks, Q-SNAREs that interact with VAMP7 is syntaxin 12/13 (STX13; 2012). The endosome-derived pathways are regulated by the Chung et al., 2013; Kuster et al., 2015), a Qa SNARE that is protein complexes adaptor protein-3 (AP-3) and biogenesis of present on BLOC-1–dependent transport carriers but is not itself lysosome-related organelles complex (BLOC)-1, -2, and -3, sub- incorporated into melanosomes (Delevoye et al., 2016; Dennis units of which are mutated in heritable disorders called the et al., 2016; Dennis et al., 2015) and thus is not likely a part of Hermansky-Pudlak syndromes (HPS; Bowman et al., 2019; Di the fusogenic complex for cargo delivery. STX13 binds in vitro to Pietro and Dell’Angelica, 2005). One function of AP-3 is to sort the pallidin (Pldn; also known as BLOC1S6) subunit of BLOC-1 the enzyme tyrosinase (TYR) from early endosomes into via its SNARE domain (Ghiani et al., 2010; Huang et al., 1999; clathrin-coated vesicles destined for maturing melanosomes Moriyama and Bonifacino, 2002), suggesting a potential direct (Huizing et al., 2001; Theos et al., 2005). BLOC-1, in collaboration mechanism for sorting STX13 into BLOC-1–dependent tubules. If with actin nucleators and the microtubule motor, KIF13A, is re- this were true, then VAMP7 could be sorted into these tubules by quired to generate tubular membrane transport carriers that association with STX13. Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202005173/1413924/jcb_202005173.pdf by St George's, University Of London user on 26 May 2021 emerge from vacuolar early endosomes, have features of re- Here, we tested both models for VAMP7 sorting into BLOC-1– cycling endosomes, and transiently fuse with melanosomes to dependent transport carriers in melanocytes by exploiting cell deliver essential melanosome cargoes such as TYR-related pro- lines lacking AP-3δ, the BLOC-1 Pldn subunit, or both and re- tein 1 (TYRP1; Delevoye et al., 2016; Delevoye et al., 2009; Dennis constituting these cells with variants of AP-3δ and/or Pldn that et al., 2016; Dennis et al., 2015; Setty et al., 2008; Setty et al., do or do not bind their target SNAREs. We show that binding of 2007). BLOC-2 helps target the tubular transport carriers to either AP-3 to VAMP7 or BLOC-1 to STX13 is both necessary and maturing melanosomes (Dennis et al., 2015). Fusion of these sufficient to support VAMP7 sorting and melanogenesis, doc- carriers and subsequent cargo delivery to melanosomes requires umenting an example of redundancy in a SNARE sorting step. the R-SNARE, vesicle-associated membrane protein 7 (VAMP7; This mechanism of SNARE sorting and regulation provides in- also known as tetanus neurotoxin insensitive VAMP, or TI- sight into how SNAREs that localize broadly within the endo- VAMP; Dennis et al., 2016; Tamura et al., 2011). VAMP7 is lysosomal system can maintain specificity in fusion reactions, subsequently recycled from melanosomes in distinct tubular and how recognition by multiple molecular components can carriers (Dennis et al., 2016; Ripoll et al., 2018). Sorting of VAMP7 ensure the sorting of an essential SNARE component. into melanosome-derived tubules likely requires binding to the scaffolding protein VAMP7- and Rab32/38-interacting protein (VARP; Dennis et al., 2016; Schafer¨ et al., 2012; Tamura et al., Results 2009), but how VAMP7 is sorted from endosomes into The BLOC-1–dependent cargo TYRP1 is partially missorted in melanosome-bound tubular carriers is unknown. AP-3–deficient melanocytes VAMP7 is one of several R-SNAREs that possess a longin In AP-3–deficient melanocytes from AP3B1−/− HPS type 2 pa- domain (Daste et al., 2015), which folds back onto the SNARE tients or Ap3b1−/− pearl mice, the BLOC-1–independent cargo TYR domain to impede SNARE complex formation (Kent et al., 2012; is largely depleted from melanosomes (Huizing et al., 2001; Martinez-Arca et al., 2003; Schafer¨ et al.,
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