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The Two-Pore Channel (TPC) Interactome Unmasks Isoform-Specific Roles for Tpcs in Endolysosomal Morphology and Cell Pigmentation

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The Two-Pore Channel (TPC) Interactome Unmasks Isoform-Specific Roles for Tpcs in Endolysosomal Morphology and Cell Pigmentation The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation Yaping Lin-Moshiera, Michael V. Keeblera, Robert Hooperb, Michael J. Boulwarea, Xiaolong Liua, Dev Churamanib, Mary E. Aboodc, Timothy F. Walsetha, Eugen Brailoiuc, Sandip Patelb, and Jonathan S. Marchanta,1 aDepartment of Pharmacology, University of Minnesota, Minneapolis, MN 55455; bDepartment of Cell and Developmental Biology, University College London, London WC1E 6BT, United Kingdom; and cDepartments of Anatomy, Cell Biology, and Pharmacology and the Center for Substance Abuse, Temple University School of Medicine, Philadelphia, PA 19140 Edited by Richard W. Aldrich, University of Texas at Austin, Austin, TX, and approved July 30, 2014 (received for review April 20, 2014) The two-pore channels (TPC1 and TPC2) belong to an ancient family and their regulation. The dataset reveals a predomination of links of intracellular ion channels expressed in the endolysosomal system. between TPCs and effectors controlling membrane organization Little is known about how regulatory inputs converge to modulate and trafficking, relevant for disease states involving lysosomal TPC activity, and proposed activation mechanisms are controversial. proliferation where TPC functionality may be altered (14). Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins Results involved in Ca2+ homeostasis, trafficking, and membrane organi- TPC Affinity Purification. For affinity purification, we used a method zation. Among these interactors, TPCs were resolved to scaffold based upon “One-Strep”-tagging (Fig. 1A). This method uses a Rab GTPases and regulate endomembrane dynamics in an isoform- short (∼2 kDa), stable tag exhibiting high affinity for an engineered specific manner. TPC2, but not TPC1, caused a proliferation of endo- streptavidin (Strep-Tactin) to enable one-step purification of TPC lysosomal structures, dysregulating intracellular trafficking, and cel- complexes. The utility of this approach for defining protein inter- lular pigmentation. These outcomes required both TPC2 and Rab actomes (including dynamic, transient interactions) has been activity, as well as their interactivity, because TPC2 mutants that shown in several applications (15) and is outlined schematically in were inactive, or rerouted away from their endogenous expression Fig. 1B. Potential TPC-interacting candidates, with matched sec- locale, or deficient in Rab binding, failed to replicate these out- tions from control lanes, were processed for identification by mass C comes. Nicotinic acid adenine dinucleotide phosphate (NAADP)- spectrometry (Fig. 1 ). Common hits, absent from controls, were evoked Ca2+ release was also impaired using either a Rab binding- then used to define the basal TPC interactome. Table 1 collates defective TPC2 mutant or a Rab inhibitor. These data suggest a fun- common TPC interactors prioritized by total peptide number damental role for the ancient TPC complex in trafficking that holds (greater than five peptides in experimental samples and absence relevance for lysosomal proliferative scenarios observed in disease. from controls). Unique TPC1/2 interactors (greater than five peptides in experimental samples and absence from the alternate + Ca2 signaling | lysosome | Xenopus isoform purification) are shown in Table S1. The quality of the proteomic dataset was interrogated in sev- eral ways. First, the predominance and coverage of bait (TPCs) wo-pore channels (TPCs) are an ancient family of intra- in dataset (and absence from controls) was assessed. TPCs were Tcellular ion channels and a likely ancestral stepping stone the predominant protein found in their respective purification + + in the evolution of voltage-gated Ca2 and Na channels (1). > 2+ + datasets (peptide coverage was 60% of the predicted non- Architecturally, TPCs resemble a halved voltage-gated Ca /Na transmembrane regions for both TPCs) (Fig. S1). Second, channel with cytosolic NH2 and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative Significance pore-forming domain between the fifth and sixth membrane- spanning regions. Since their discovery in vertebrate systems, many studies have investigated the properties of these channels Two-pore channels (TPCs) are a recently discovered family of (2–7) that may support such a lengthy evolutionary pedigree. endolysosomal ion channels, but their regulation is controver- In this context, demonstration that (i) the two human TPC sial. By defining the TPC interactome, we provide a community isoforms (TPC1 and TPC2) are uniquely distributed within the resource that illuminates TPC complex regulation and resolves endolysosomal system (2, 3) and that (ii) TPC channel activity is associations with novel partners and processes. Physical inter- + activated by the Ca2 mobilizing molecule nicotinic acid adenine actions with endolysosomal trafficking regulators predominate, dinucleotide phosphate (NAADP) (4–6) generated considerable and Rab GTPases impart isoform-specific roles for TPCs in excitement that TPCs function as effectors of this mercurial organelle proliferation and cellular pigmentation. These data + second messenger long known to trigger Ca2 release from imply a fundamental role for TPCs in trafficking that augurs “acidic stores.” The spectrum of physiological activities that have significance for disease states exhibiting lysosomal prolif- been linked to NAADP signaling over the last 25 years (8, 9) may eration where TPC dysregulation may drive pathogenesis. therefore be realized through regulation of TPC activity. How- ever, recent studies have questioned the idea that TPCs are Author contributions: Y.L.-M., S.P., and J.S.M. designed research; Y.L.-M., M.V.K., R.H., CELL BIOLOGY NAADP targets (10, 11), demonstrating instead that TPCs act as M.J.B., X.L., D.C., M.E.A., T.F.W., E.B., S.P., and J.S.M. performed research; Y.L.-M., + Na channels regulated by the endolysosomal phosphoinositide M.V.K., R.H., M.J.B., X.L., D.C., M.E.A., T.F.W., E.B., S.P., and J.S.M. analyzed data; and Y.L.-M. and J.S.M. wrote the paper. PI(3,5)P2. Such controversy (12, 13) underscores how little we know about TPC regulatory inputs and the dynamic composition The authors declare no conflict of interest. of TPC complexes within cells. This article is a PNAS Direct Submission. Here, to generate unbiased insight into the cell biology of the Freely available online through the PNAS open access option. TPC complex, we report a proteomic analysis of human TPCs. 1To whom correspondence should be addressed. Email: [email protected]. The TPC interactome establishes a useful community resource This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. as a “rosetta stone” for interrogating the cell biology of TPCs 1073/pnas.1407004111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1407004111 PNAS | September 9, 2014 | vol. 111 | no. 36 | 13087–13092 Downloaded by guest on October 1, 2021 annexins, and sigma receptors). Rab GTPases, small monomeric GTPases that control intracellular trafficking (21, 22), predomi- nated in terms of peptide number (Table 1) and interactivity Table 1. Common TPC interactors Peptide no. TPC1 No. Candidate description Gene name Control and -2 TPC2 TPCN2 5 282 TPC1 TPCN1 1 254 1 Na/K-transporting ATPase ATP1A1 0 132 subunit alpha-1 2 Rab GTPase family RAB 0 129 3 Transmembrane emp24 TMED1-5, 7, 9, 10 0 98 domain family 4 3-Hydroxyacyl-CoA PTPLAD1 0 65 dehydratase 3 5 Surfeit locus protein 4 SURF4 0 55 6 ADP/ATP translocase SLC25A5/6 0 54 7 Membrane-associated PGRMC1 0 49 progesterone receptor component 1 8 Isoform 2, 4F2 cell-surface SLC3A2 0 42 antigen heavy chain 9 Aldolase A ALDOA 0 40 10 Annexin family ANXA1-7,11 040 11 Transmembrane 9 superfamily TM9SF1-3 0 38 12 Peroxiredoxin Family PRDX1,4,6 0 38 13 Transmembrane protein 33 TMEM33 0 36 Fig. 1. Strategy for affinity purification and validation of TPC interactors. 14 Rab GDP dissociation GDI2 0 33 (A) Diagram of affinity purification method that illustrates TPC (open) and inhibitor beta TPC-associated protein complexes (shaded) binding to Strep-Tactin via the 15 VDAC family VDAC1-3 0 31 Strep-tag moiety. (B) TPC isolation followed the same protocol for TPC1/2 16 Myosin, heavy chain 9 MYH9 0 30 purifications with elution of TPC complexes tracked via an anti–Strep-tag 17 Prohibitin-2 PHB2 0 30 monoclonal. (Upper) Representative purification followed by Western blot 18 Transmembrane protein 165 TM165 0 26 detection (anti–Strep-tag) of TPC2 protein in specific eluates. (Lower) Sche- 19 Isoform 2, magnesium MRS2 0 23 matic showing eluate “3” was processed for SDS/PAGE and mass spectrom- transporter MRS2 homolog etry (“C”). Validation of interactors was performed by Western blotting of 20 Ceramide synthase 2 CERS2 0 22 TPC-enriched eluates (“D”) and coimmunoprecipitation (“E”). (C) Example 21 Sideroflexin family SFXN1,2,4 0 21 of silver-stained gel highlighting positions of excised bands in TPC1/2 puri- 22 Ancient ubiquitous protein AUP1 0 21 fications together with bands cut from equivalent positions in controls 23 Large neutral amino acids SLC7A5 0 20 (no Strep-tag). (D) Western blot for endogenous TMEM33, a representative transporter small subunit 1 interactor. (E) Interaction between endogenous TMEM33 and TPC confirmed 24 Ca-binding protein p22 CHP 0 18 by coimmunoprecipitation in HEK293 cells expressing TPC1-MYC. 25 Vesicular integral protein36 VIP36 0 17 26 Isoform ADelta10 of LMNA 0 16 prelamin-A/C candidate TPC interactors assigned by mass spectrometry were 27 Reticulocalbin-2 RCN2 0 16 validated by (i) screening for presence in the TPC-enriched eluate 28 Inorganic pyrophosphatase PPA1 0 15 (Fig. 1D)before(ii) interactivity was assessed by coimmunopreci- 29 Isoform 2, heat shock HSP90AA1 0 14 pitation (Fig. 1E). These steps are exemplified for TMEM33, one protein 90α candidate TPC interactor. Third, the entire purification dataset 30 Isoform 2, adenylyl CAP1 0 14 was cross-referenced with in silico datasets to ascertain potential cyclase-associated protein 1 interactivity.
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