© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs218610. doi:10.1242/jcs.218610

RESEARCH ARTICLE Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane Varsha Singh1,*, Jianbo Yang1, Jianyi Yin1, Robert Cole2, Ming Tse1, Diego E. Berman3, Scott A. Small3, Gregory Petsko4 and Mark Donowitz1,*

ABSTRACT intestinal epithelial barrier function via cyclic AMP (cAMP)-induced Cholera toxin (CT) causes severe diarrhea by increasing intracellular disruption of Rab11- and exocyst-dependent delivery of endocytic – cAMP leading to a PKA-dependent increase in Cl− secretion through recycling cargo to cell cell junctions (Guichard et al., 2013). CFTR and decreased Na+ absorption through inhibition of Na+/H+ CT is a classic AB toxin. CT translocates from the plasma exchanger 3 (NHE3; also known as SLC9A3). The mechanism(s) by membrane (PM) through the trans-Golgi network (TGN) into the which CT inhibits NHE3 is partially understood, although no drug endoplasmic reticulum (ER) in a retrograde fashion by binding the therapy has been successful at reversing this inhibition. We now ganglioside GM1 via the B subunit of the native holotoxin (Orlandi describe that CT phosphorylates an amino acid in the PDZ domain of and Fishman, 1998; Wernick et al., 2010). Once it is released from the SNX27, which inhibits SNX27-mediated trafficking of NHE3 from the ER lumen, the enzymatic moiety CT-A subunit causes a pathological early endosomes to the plasma membrane (PM), and contributes to increase in cellular cAMP levels and PKA activity via the induction reduced basal NHE3 activity through a mechanism that involves of host PM adenylyl cyclase (De Haan and Hirst, 2004; Sack et al., reduced PM expression and reduced endocytic recycling. Importantly, 2004; Wernick et al., 2010). PKA then activates CFTR and inhibits + + mutagenesis studies (Ser to Asp) showed that the effect of this the brush border (BB) Na /H antiporter NHE3 (also known as + phosphorylation of SNX27 phenocopies the effects seen upon loss of SLC9A3), which is a major contributor to small intestinal Na SNX27 function, affecting PM trafficking of cargo proteins that bind absorption. Inhibition of NHE3 by CT and in other cAMP-related SNX27–retromer. Additionally, CT destabilizes retromer function by diarrheal diseases involves increased endocytosis, which reduces BB decreasing the amount of core retromer proteins. These effects of CT NHE3 expression (Musch et al., 2007, 2010). However, it is not can be partially rescued by enhancing retromer stability by using known whether changes in NHE3 exocytosis are also caused by CT, ‘pharmacological chaperones’. Moreover, pharmacological chaperones particularly from the early endosome (EE) to the PM. canbeusedtoincreasebasalandcholeratoxin-inhibitedNHE3activity An early endosomal PDZ domain-containing protein, sorting and fluid absorption by intestinal epithelial cells. nexin 27 (SNX27) binds and regulates exocytosis of NHE3 from the EE to the PM (Singh et al., 2015). SNX27 has important regulatory This article has an associated First Person interview with the first roles in EE-to-PM trafficking of multiple classes of proteins; this author of the paper. occurs via interacting with the retromer complex. The retromer complex is composed of the vacuolar protein sorting (VPS) trimer KEY WORDS: SNX27, Retromer, NHE3, PDZ, Secretory diarrhea, core sub-complex (VPS26–VPS29–VPS35) and a membrane- Apical trafficking, Exocytosis, Cholera toxin, Early endosomes associated sorting nexin (SNX) dimer (SNX1–SNX2 or SNX5– SNX6) (Seaman, 2005). The retromer complex is important in INTRODUCTION regulating transmembrane receptor recycling from the EE either to the To infect human cells, pathogenic microorganisms have evolved TGN or to the PM (Belenkaya et al., 2008; Seaman, 2007; Yang et al., numerous strategies to suppress host defenses and exploit host cellular 2008), with SNX27 involved in the EE-to-PM pathway (Joubert signaling machinery. Specific pathogen virulence factors disable, et al., 2004; Lauffer et al., 2010). In this function, SNX27 directly subvert or even stimulate vesicular trafficking pathways to and from binds VPS26 via the SNX27 PDZ domain. In regulating this aspect of the host cell surface, which promotes pathogen entry, replication or trafficking, the mammalian retromer complex binds other protein escape. One prominent trafficking pathway that pathogens modulate or complexes including Wiskott–Aldrich protein and SCAR homolog exploit by multiple mechanisms is the final step of endocytic recycling, (WASH) complex, actin, ankrin-repeat 50 domain, VPS-ankrin repeat at which cargo-containing vesicles dock at the cell surface. As one domain protein and FAM21 proteins (Burd and Cullen, 2014). example, cholera toxin (CT) secreted by Vibrio cholerae compromises Retromer-mediated trafficking defects has been implicated in a growing number of neurological diseases (Follett et al., 2014; Small 1Departments of Medicine and Physiology, School of Medicine, Johns Hopkins et al., 2005; Zimprich et al., 2011). Until now, the effect of CT on University, Baltimore, MD 21205, USA. 2Department of Biological Chemistry, retromer-mediated movement of cargo proteins in the intestine has School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA. 3The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, not been described. In the present study, we demonstrate that CT Department of Pathology, Columbia University College of Physicians and increases phosphorylation of a serine residue in the PDZ domain of Surgeons, New York, NY 10032, USA. 4Helen and Robert Appel Alzheimer’s SNX27, which inhibits SNX27 binding to NHE3 and reduces NHE3 Disease Research Institute and Department of Neurology, Weill Cornell Medical College, New York, NY 10021, USA. exocytosis to the PM. Moreover, CT destabilizes retromer function by reducing the expression of two core components of the retromer *Authors for correspondence ([email protected]; [email protected]) complex – VPS35 and VPS26. This previously undescribed activity V.S., 0000-0003-0796-2619; M.D., 0000-0003-0477-8824 of CT identifies a site at which CT affects PM transporters, including NHE3, and identifies a step in trafficking for the potential targeting of

Received 2 April 2018; Accepted 22 June 2018 drug development to treat diarrheal diseases. Journal of Cell Science

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RESULTS the level of BB NHE3 was reduced by CT exposure; however, this CT inhibits exocytosis of NHE3 in intestinal epithelial occurred in a time-dependent manner. Cells treated with CT Caco-2/bbe cells for 8 h had more NHE3 on the membrane compared to cells Basal NHE3 activity is known to be inhibited after CT exposure, treated for 24 h or 48 h, and the percentage of total NHE3 on the with the effect involving reduced PM NHE3 expression and PM at all three time points of CT exposure was significantly stimulation of endocytosis (Subramanya et al., 2007). However, reduced compared to the untreated control (10, 7 and 5% characterization of the effects of CT on both rates of endocytosis respectively for 8, 24 and 48 h of CT exposure, respectively, and exocytosis of NHE3 in the same model has not been reported. versus 15% in untreated control cells) (Fig. 1B,C). To define the To extend this characterization, we initially tested the timecourse effect of CT on NHE3 trafficking, we measured the rate of of CT-mediated inhibition of NHE3 activity. Polarized endocytosis and exocytosis of NHE3 in CT-treated Caco-2/bbe- monolayers of Caco-2/bbe cells expressing HA–NHE3 (Caco-2/ HA-NHE3 cells. Endocytosis was determined using a biotinylation- bbe-HA-NHE3) were treated with CT for 8, 24 and 48 h (CT added based assay (Singh et al., 2015). This demonstrated similar increased every 12 h), and NHE3 activity was determined in the presence of rates of NHE3 endocytosis with 8 and 48 h of CT exposure (Fig. 1D) 50 µM HOE694 to inhibit endogenous NHE1 and NHE2 activity A cell surface biotinylation-based exocytosis assay showed that the (Ikuma et al., 1999). Consistent with previous studies, an 8 h CT rate of exocytosis of NHE3 in 8 and 48 h CT-treated cells was treatment significantly decreased basal NHE3 activity (∼40%), significantly less than that of control cells (Fig. 1E; Fig. S1). These and the effect became more pronounced at 24 h and 48 h, at which results indicate that CT, in addition to its known effect on stimulating times there was a similar extent of inhibition (Fig. 1A). To further NHE3 endocytosis, also inhibits NHE3 exocytosis, which delineate the mechanism of inhibited NHE3 activity in response to contributes to a lower abundance of NHE3 on the PM and to lower CT, we determined the surface abundance of NHE3. Biotinylation- NHE3 activity. Since there were slightly greater CT-mediated based surface abundance analysis showed that at all times studied, effects on NHE3 activity and PM expression at 48 h compared to

Fig. 1. CT inhibits NHE3 activity, stimulates endocytosis and inhibits exocytosis in intestinal epithelial cells. (A) Initial rates of Na+/H+ exchange were + measured in either untreated or CT-treated (100 ng/0.5 ml for 8 h, 24 h or 48 h) Caco-2/bbe-HA-NHE3 cells as the Na -dependent pHi recovery by using the pH-sensitive dye BCECF. Results are means±s.e.m. with results from individual experiments shown; n=4 separate experiments. *P<0.05, comparison between control and CT-treated cells; #P<0.05, comparisons between 8 h and 24 h or 48 h CT-treated cells. NS, not significant. (B) Caco-2/bbe-HA-NHE3 cells were treated with CT (8 h, 24 h or 48 h) and surface NHE3 levels were analyzed. A representative western blot (IB) analysis is shown illustrating changes in PM expression of NHE3 in response to CT. (C) The quantification from at least three independent experiments, as in B, is shown for individual experiments expressed asthe percentage of total along with the mean±s.e.m. *P<0.05, comparison between control and CT treatment; #P<0.05, comparison between 8 h and 24 h or 48 h CT-treated cells. (D) Rate of endocytosis of NHE3 in control (Ctrl) and CT-treated (100 ng/0.5 ml for 8 h or 48 h) confluent Caco-2/bbe-HA-NHE3 cells. Biotinylated cells were incubated at 37°C for 0, 30 and 60 min. The amount of endocytosis of NHE3 (internalization of surface NHE3) at the indicated time points was determined by a GSH-resistant endocytosis assay. A representative blot from three independent experiments is shown. Quantitative analysis of the amount of internalized/ endocytosed NHE3 at each time point was calculated as the percentage of surface NHE3 of the corresponding control groups, which were always kept at 4°C and never exposed to GSH. Results are means±s.d., n=3. *P<0.05, comparison between control and CT-treated cells. (E) Rate of exocytosis of NHE3 in control (Ctrl) and CT-treated (100 ng/0.5 ml for 48 h) Caco-2/bbe-HA-NHE3 cells. Cells were incubated with NHS-acetate and then incubated for 0, 15, 30, and 60 min at 37°C to allow exocytosis of NHE3. Cells were then chilled to 4°C, and newly inserted NHE3 (exocytosed NHE3) was biotinylated and subjected to quantitative western blotting. The plot represent the mean±s.d. of three independent experiments. *P<0.05, comparison between control and CT-treated cells. Journal of Cell Science

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24 h, further studies examined CT effects at early (8 h) and later times the entry to the ligand-binding site (Fig. 2B). Although basal of exposure (48 h). phosphorylation of SNX27-Ser49 had been previously identified in rat and human tissues (Lundby et al., 2012; Zhou et al., 2013), no CT increases phosphorylation of Ser49 in the PDZ domain of functional relevance of this phosphorylation has been described. We SNX27 hypothesized that the increase in phosphorylation of Ser49 by CT It has been previously shown that exocytosis of NHE3 from EEs to might play a role in the regulation of SNX27-NHE3 binding. the PM is regulated by SNX27, and that SNX27 directly binds NHE3 (Singh et al., 2015). Therefore, we next investigated whether CT Phosphorylation of SNX27 Ser49 regulates NHE3 basal modified SNX27 by using two mass spectroscopy (MS)-based activity and surface expression by inhibiting SNX27–NHE3 proteomic approaches, both based on immunoprecipitation (IP) of rat association SNX27 stably expressed in HEK293 cells that were either untreated Whether CT-induced phosphorylation of Ser49 affected NHE3 basal or exposed to holo-CT for 8 h. In the first approach, spectral counting activity and surface expression was determined. Mutants where Ser49 from liquid chromatography tandem MS (LC-MS/MS) analysis of was replaced with a non-phosphorylatable alanine residue (S49A) or a two experiments showed a 3.5× average increase (2.5×, 4.5×) in phosphomimetic aspartic acid reside (S49D) were generated in rat- phosphorylation of the peptide SES49GYGFNVR at Ser49. A similar derived SNX27b (a specific splice variant) and transiently expressed increase in phosphorylation on Ser49 was caused by forskolin (2.6×) in human HEK293A cells expressing HA–NH3 (HEK293A-HA- when studied in one of the experiments. No changes in NHE3) previously transduced with shRNA lentivirus to knockdown phosphorylation were detected on Ser47.Similarly,E. coli heat- (KD) SNX27. The SNX27 protein expression was reduced by stable enterotoxin (STa), which is also known to inhibit NHE3 ∼60% in SNX27 shRNA stably expressing cells. These cells were activity by altering its trafficking, did not increase phosphorylation at used to measure NHE3 surface expression and basal activity. Ser49 of SNX27 (data not shown), suggesting mechanistic differences Immunoblotting demonstrated expression of the rat SNX27-S49A between cAMP- and cGMP-mediated pathways. In the second and -S49D. Surface biotinylation assays showed that the cells approach, isobaric mass tags (tandem mass tags; TMT 10-plex) were containing the S49D mutant had the least NHE3 PM expression used to directly quantitatively compare the IPs from three separately (Fig. 3A). Furthermore, the cells expressing the SNX27-S49D prepared biological replicates of unstimulated and stimulated cells mutant had a similar reduction in basal NHE3 activity to that seen in (Fig. 2A). CT caused a 2.6±0.39× (mean±s.e.m.) increase in SNX27KD cells (Fig. 3B). Since NHE3 surface expression largely phosphorylation at Ser49 (2.3×, 2.5×, 2.9× for each replicate). A depends upon its binding with SNX27, we next examined whether TMT-labeled phosphorylated Ser47 peptide was not detected. Ser49 phosphorylation altered SNX27-PDZ binding to NHE3. This Computational modeling of the SNX27-PDZ domain showed was achieved by generating GST fusion proteins of the wild-type that Ser49 is immediately adjacent to the conserved GYGF motif at (WT) and S49A or S49D PDZ-PX domains of SNX27. An extended

Fig. 2. CT increases phosphorylation of Ser49 in the PDZ domain of SNX27. (A) Identification of SNX27 phosphorylation site Ser49 by isobaric mass tag (TMT 10-plex) LC-MS/MS analysis of GFP-tagged rat SNX27. GFP–SNX27 (rat) was immunoprecipitated from HEK293 control and CT-treated (100 ng/0.5 ml for 8 h) cells and proteolyzed for LC-MS/MS analysis. The increase in phosphorylation of Ser49 was identified within the PDZ domain of SNX27. The MS/MS spectrum (A) shows the phosphorylation at Ser49. Phosphorylation analysis using MS was repeated in three separate experiments. The maximum Mascot score for Ser49 was 82. Results are shown below as means±s.e.m. (B) Schematic representation of SNX27 domains. The expanded box shows a sequence alignment of SNX27–PDZ domain (residues 41–60 human; 39–58 mouse and rat) of different species. Sequences were obtained from the Swiss/UniProt database. The CT-induced phosphorylation site Ser49 is underlined and the ligand-binding site GYGF is shown in red. A 3D image of the rat SNX27-PDZ domain was prepared using PyMOL computer software. The predicted structure shows that Ser49 is adjacent to the highly conserved GYGF carboxylate-binding loop. Journal of Cell Science

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Fig. 3. Ser49 phosphorylation of SNX27 phenocopies loss of SNX27 function. (A) Cell surface proteins were biotinylated from HEK293-HA-NHE3 cells containing control shRNA or SNX27 shRNA (SNX27-KD) with or without replacement by co-transfection of rat GFP–SNX27-PDZ-S49A or -S49D mutants. Biotinylated proteins were isolated with streptavidin–Sepharose, and the amount of biotinylated NHE3 was quantified by western blotting and compared to the amount of total NHE3. NHE3 surface expression was quantified in three independent experiments (lower panel). Results are means±s.e.m. P<0.05 and NS, not significant versus control (unpaired t-test). (B) Na+/H+ exchange was measured in HEK293-HA-NHE3 cells expressing control shRNA or SNX27 shRNA with or without reconstitution with rat GFP–SNX27-PDZ-S49A or -S49D mutants. Results are means±s.e.m., n=4. P values are as indicated. (C) GST, GST–SNX27-FL, GST–SNX27-PDZ-PX, GST–SNX27-PDZ-PX-S49A or -S49D fusion proteins were mixed with HEK-HA-NHE3-SNX27KD cell lysate and then subjected to pulldown assays with GSH resin. Samples were analyzed by western blotting (IB) with antibodies against NHE3 and GST. The experiment was repeated three times with similar results, and one representative result is shown. (D) Colocalization analysis of endogenous SNX27 or GFP–SNX27 (Alexa Fluor 488) and EE marker EEA1 (Alexa Fluor 594) in HEK WT and HEK-SNX27 depleted (shRNA) cells transfected with rat GFP–SNX27-S49A or S49D mutants. Insets are magnified views of the endosomes in the dashed box. Colocalization between SNX27 and EEA1 was quantified as the Pearson’s correlation coefficient over 10 images per condition containing more than 50 cells acquired from three independent experiments. Mean±s.e.m. are shown. Scale bars: 10 μm. All results were not significantly (NS) different from control (Student’s t-tests).

PDZ domain was used for this binding assay because it was associated with the SNX27–retromer pathway: (1) Glut-1 (also previously shown that the extension of the SNX27-PDZ domain to known as SLC2A1; Steinberg et al., 2013), (ii) ZO-2 (also known as include most of the PX domain (PDZ-PX) significantly increased TJP2; Zimmerman et al., 2013) and (3) the copper ATPase ATP7A, SNX27 binding with NHE3 (Singh et al., 2015). Purified WT and mutations in which causes Menkes disease (Steinberg et al., 2013). mutant proteins were used to pulldown NHE3 from lysates of cells The interactions between SNX27–PDZ-PX and Glut-1, ZO-2 and stably overexpressing HA–NHE3. The pulldown assay showed ATP7A was first investigated using GST pulldown assays. GST that the phosphomimetic S49D mutant of SNX27 pulled down fusion proteins of WT and SNX27 mutants (S49A/S49D) were used less NHE3 compared to the S49A mutant or WT PDZ-PX domain to pull-down cargo proteins from cell lysate prepared from Caco-2/ of SNX27 (Fig. 3C). This indicates that the increase in bbe cells with SNX27KD cells. Similar to what occurred with phosphorylation on Ser49 decreases SNX27–NHE3 binding and NHE3, the unmodified SNX27 PDZ-PX domain pulled down more also reduces NHE3 surface expression and basal activity. ATP7A, ZO-2 and Glut-1 compared to SNX27-S49A and -S49D In immunolocalization studies, we found that SNX27–GFP modifications (Fig. 4A). Of note, for all three proteins, the extent of colocalizes prominently with EEA1-containing endosomes, as protein pulled down by WT SNX27 and SNX27-S49A were not shown previously (Singh et al., 2015; Temkin et al., 2011). Similar significantly different while the phosphomimetic (D) mutation to WT SNX27, the S49A and S49D mutants also localized to the caused significantly less binding to all three proteins. Consistent EEA1 compartment (Fig. 3D) demonstrating that Ser49 with the role of SNX27 in Glut-1 trafficking, depletion of SNX27 or phosphorylation does not appear to change the intracellular expression of SNX27-S49D in SNX27KD cells, led to significant localization of SNX27. This was further supported by quantitative mis-sorting of Glut-1 to the lysosomal compartment, whereas it is image analysis (Fig. 3D). normally localized to the cell surface at the steady-state (Fig. 4B). Similarly, cell surface biotinylation studies revealed that there was Increased Ser49 phosphorylation affects SNX27 binding and decreased Glut-1 surface expression in SNX27-S49D-containing trafficking of multiple cargoes cells, which was similar to what was seen with SNX27KD, while in Next, we sought to investigate whether the effect of increased the presence of the SNX27-S49A, expression was similar to SNX27-Ser49 phosphorylation is limited to NHE3. To do this, we control (Fig. S2). These results indicate that Ser49 is critical for the verified the binding of three other ligands previously shown to be interaction of multiple cargo proteins with the SNX27-PDZ domain Journal of Cell Science

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Fig. 4. SNX27-PDZ domain cargo binding is regulated by Ser49 phosphorylation. (A) Caco-2/bbe-SNX27KD cell extracts (1 mg) were incubated with GST or GST fused to PDZ+PX (P-PX, amino acids 1–266), or PDZ+PX-S49A or -S49D (P-PX-A and P-PX-D) (all 1 nmol). The presence of ATP7A, ZO-2 and Glut-1 in the GST pull downs were detected by western blotting (IB, top; representative results from six independent experiments). A quantification of cargo proteins in pull downs normalized to the amount of GST from the 6 experiments is shown below as means±s.e.m. *P<0.05, **P<0.01; ***P<0.001; NS, not significant (compared to WT control). (B) HeLa cells expressing control shRNA, SNX27 shRNA and SNX27 shRNA transfected with SNX27-S49A or -S49D mutant were stained for endogenous GLUT1 (Alexa Fluor 488) and endogenous LAMP1 (Alexa Fluor 594). A representative experiment is shown above with magnified views of regions in the dashed boxes shown. Scale bars: 10 μm. Quantification of colocalization was performed across four independent experiments and is shown below. Results are shown as means±s.e.m. *P<0.05 compared to shRNA control. and that CT/cAMP-mediated phosphorylation of SNX27-Ser49 while VPS29 was slightly, but not significantly, reduced (data not disrupts these interactions and thereby regulates the PM sorting of shown) in CT-treated Caco-2/bbe cells. This reduction was evident multiple membrane proteins, including NHE3. after 8 h of CT treatment (VPS35, 78.8±9.4%, VPS26, 66.7±2.9% versus untreated control; mean±s.e.m., P<0.05) and became more Cholera toxin destabilizes the retromer by reducing the pronounced when CT was present for 48 h (VPS35, 53.3±2.9%; amount of core VPS proteins VPS26: 49.3±1.2% versus control; P<0.05) (Fig. 5B). However, SNX27 is a component of a multi-protein assembly, the ‘retromer the mRNA levels for VPS35, VPS29 and VPS26 were not affected tubule complex’, which forms a major sorting platform on EEs by CT (Fig. S3). We also investigated the effects of CT (8 h) on (Tsvetanova et al., 2015). This complex includes the core retromer, colocalization of the retromer components in HEK293 cells via which is composed of the VPS35–VPS29–VPS26 ternary complex. immunofluorescence. CT treatment did not appreciably alter the Retromer serves as a multifunctional scaffold forming an interaction colocalization between VPS35 and VPS26; however, the number of hub for a wide array of endosome-associated proteins, collectively VPS35-containing and VPS26-containing intracellular vesicles was termed the retromer interactome (Fig. 5A). These interactions aid in significantly reduced with CT treatment compared to in untreated the formation of the cargo-containing tubulovesicular membrane control cells (Fig. 5C). These results indicate that CT destabilizes carriers destined for other compartments, including the Golgi and retromer by reducing the levels of the core retromer proteins VPS35 PM (Lin et al., 2015; Steinberg et al., 2013; Tsvetanova et al., 2015). and VPS26. Retromer-related defects have been shown to reduce trafficking of cargo out of endosomes (Bhalla et al., 2012; Vieira et al., 2010). A ‘pharmacological chaperone’ stimulates NHE3 activity in Therefore, we investigated the effect of CT on the retromer human enteroid monolayers trimeric core complex in intestinal epithelial cells and HEK293 Recently, small-molecule ‘pharmacological chaperones’ that are cells. At 14 days post plating, confluent Caco-2/bbe cells were thiophene thiourea derivatives, including R55 [thiophene-2,5- exposed to CT and the total protein levels of core retromer proteins diylbis(methylene) dicarbamimidothioate dihydrochloride] have was determined. Initially, effects on the core retromer protein been shown to improve thermal stability of the core retromer VPS35 were determined, demonstrating a significant reduction in proteins, and to increase the retromer-mediated trafficking of cargo cells treated with CT for 8 h (Fig. 5B). Reduction in amount of one proteins from EEs in cultured hippocampal neurons (Mecozzi et al., protein of the VPS35–VPS29–VPS26 trimeric core often leads to a 2014). The effect of these compounds on intestinal ion transport secondary reduction in other retromer proteins (Fuse et al., 2015). physiology has not been described. Since SNX27 is part of the Therefore, we also examined the levels of VPS26 and VPS29 in retromer tubule complex, we investigated the effect of R55 on NHE3

CT-treated Caco-2/bbe cells. VPS26 was significantly reduced transport activity and trafficking in human intestinal stem cell-derived Journal of Cell Science

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Fig. 5. CT decreases retromer protein expression. (A) Schematic diagram of an EE illustrating the SNX27–retromer and the WASH complex. Branched tubules (arrow) represents discrete domains into which specific proteins are sorted and targeted to their respective destinations. (B) Expression of retromer proteins VPS35 and VPS26 in untreated and CT-treated (8 h and 4 times over 48 h) 14-day post confluent Caco-2/bbe cells. Western blot analysis of a representative blot is shown above. Quantitative analysis of the blots showing levels of VPS35 and VPS26 normalized to total levels of actin is shown below. Results aremeans ±s.e.m., n=3. *P<0.05 compared to control. (C) Representative maximum intensity projections of HEK293 untreated and CT-treated (8 h) cells immunostained for VPS35 (Alexa Fluor 568) and VPS26 (Alexa Fluor 488). Numbers of VPS35 and VPS26 puncta in untreated (VPS35 60±8 puncta per cell and VPS26 56±12 puncta per cell) and CT-treated (VPS35 35±6 puncta per cell and VPS26 29±18 puncta per cell) cells are shown (n=20 cells studied from each of four separate experiments). Scale bars: 20 μm. Data are presented as means±s.e.m. *P<0.05. enteroid monolayers. For this study, we used differentiated enteroid thereby protecting it from degradation (Ringe and Petsko, 2009). monolayers (5 days post Wnt3A removal) derived from upper small We therefore investigated the effect of R55 on the CT-induced intestine (duodenum) of healthy donors. Enteroid monolayers decrease in the amounts of VPS35 and VPS26 protein. At 14 days (Wnt3A-free; Schuijers et al., 2015; Noel et al., 2017) were treated post plating, confluent Caco-2/bbe cells were treated with R55 and/ with 5 μM R55 or vehicle control (0.2% DMSO) for 48 h and NHE3 or CT for 8 h or 48 h and total changes in the level of core VPS activity was measured. The reported in vitro Kd for R55 was 5 μM complex proteins were measured. Compared to vehicle control, 8 h (Mecozzi et al., 2014). Enteroid monolayers treated with R55 had as well as 48 h of CT treatment, significantly reduced the amount of significantly increased NHE3 basal activity (control, 0.18±0.02 ΔpH/ VPS35 and VPS26, while treatment with R55 alone caused a small min; R55, 0.31±0.02 ΔpH/min; P<0.05) (Fig. 6A). To determine but significant increase in the amounts of VPS35 (25±10%; whether the increase in basal activity was due to increased membrane mean±s.e.m., P<0.05) and VPS26 (29±7%, P<0.05). Moreover, trafficking, we measured NHE3 surface expression in response to treatment with R55 either before CT (CT-treated during last 8 h of R55. R55 treatment significantly increased surface expression of 48 h R55 exposure) or along with CT for 48 h, prevented the NHE3 (131.7±5.4%) (Fig. 6B), without changing total protein amount reduction in the VPS35 and VPS26 amounts, leading to levels that or NHE3 mRNA levels (Fig. S4). A similar increase in NHE3 basal were similar to those in untreated controls (Fig. 7A,B). activity was seen in enteroid monolayers derived from a different Increasing VPS35 levels can increase retromer-mediated donor (data not shown). In addition, a similar increase in surface trafficking (Mecozzi et al., 2014; Small et al., 2005). Therefore, expression of NHE3 (142.4±3.2%) in response to R55 occurred in we tested whether R55 reversed the CT-induced decrease in NHE3 Caco-2/bbe cells. Consistent with this, immunofluorescence analysis surface expression. NHE3 surface expression was measured in R55- also showed a significant increase in apical expression of NHE3 in and/or CT-treated Caco-2/bbe-HA-NHE3 cells. A biotinylation- response to R55 (Fig. 6C). Taken together, these findings demonstrate based surface protein measurement showed that 8 h and 48 h of CT that R55 (a pharmacological chaperone) stimulates basal NHE3 treatment significantly reduced surface NHE3 (−35.0±2.3% and activity by increasing PM expression of NHE3 in human intestinal −19.3±1.2%, respectively versus control; P<0.05). Importantly, CT epithelial cells. did not change total NHE3 expression. This was further confirmed by performing degradation (half-life of total NHE3) assays from R55 prevents the CT effect on the retromer and NHE3, and 0–24 h in control and CT-treated cells in presence of cycloheximide increases NHE3-mediated fluid absorption in CT-treated (100 µM) to block synthesis of new proteins. The half-life of total intestinal epithelial cells NHE3 was not significantly affected by CT treatment (Fig. S5). CT treatment decreased the amount of core VPS proteins (which Furthermore, R55 treatment increased NHE3 (138.0±1.8%, versus destabilizes the trimeric core retromer), while pharmacologic control; P<0.005) surface expression. Moreover, R55 treatment chaperones stabilize the three-dimensional protein structure either before or concurrently with CT treatment prevented the Journal of Cell Science

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Fig. 6. A pharmacological chaperone stimulates NHE3 trafficking to the apical membrane of human enteroids and increases NHE3 activity. (A) Differentiated human enteroid monolayers (5 days post Wnt3A removal) with higher TER than undifferentiated enteroids (1458±110 Ω cm2 vs 451±19 Ω cm2,***P<0.001) were used. Na+/H+ exchange in human enteroid monolayers was measured using the pH-sensitive dye BCECF. A single donor was used for all experiments. Basal NHE3 transport activity was measured as the initial rates of Na+-dependent intracellular alkalinization in R55- (5 μM for 48 h) or vehicle-treated monolayers. A single experiment is shown above and below are shown means±s.e.m., n=4. P values are for the comparison between control and R55 treatment. (B) A differentiated human enteroid monolayer was treated with R55 (5 μM for 48 h) and the amount of NHE3 protein in the PM was determined by surface biotinylation. GAPDH was used as a loading control. A representative experiment is shown above and below are shown results as mean±s.e.m. (n=3 separate studies on monolayers each taken from a separate passage of the same cell line). *P<0.05. (C) NHE3 expression in human enteroid monolayers was detected by immunofluorescence (confocal microscopic) analysis. A differentiated monolayer was treated with R55 as above and stained for NHE3 (Alexa Fluro 488) and phosphorylated ezrin (Alexa Fluro 568). Each panel displays an xz projection; top panels are an xy projection, bottom panels are at the level of the apical PM. The quantification of the NHE3 intensity in control and R55 treated monolayers was performed with Volocity Software. The total NHE3 amount was not increased upon R55 treatment (lower left). The percentage of apical NHE3 in the microvillar region (stacks 1–5, each stack was 1 μminthexz dimension and 20–25 μm total cell height) normalized to total NHE3 (whole image) expression was increased with R55 treatment (lower right). At least four separate images and ten random individual areas (z sections) were analyzed for each group. Scale bar: 10 µm. Results are mean±s.e.m., n=3. P values are comparison with untreated control. decrease in apical membrane expression of NHE3 (Fig. 7C,D). CFTR activity (Dekkers et al., 2013). We modified the assay in Thus, we conclude that the reduction in NHE3 PM expression order to define the contribution of fluid absorption by NHE3 by mediated by CT can be restored by R55 treatment. including conditions in which NHE3 was inhibited with S3226 CT-induced diarrhea involves stimulation of Cl−/fluid secretion (20 μmol/l) (Schwark et al., 1998). Treatment with S3226 caused a via increased activity of the cystic fibrosis transmembrane slight increase in FIS. However, the R55 effect on FIS was totally conductance regulator (CFTR) (Kunzelmann and Mall, 2002) and reversed by the NHE3 inhibitor (Fig. 7F). Unlike the small effect of inhibition of Na+/fluid absorption mediated by NHE3 (Lin et al., R55 on basal enteroid swelling, the R55 effect on forskolin-induced 2011). We investigated the effect of R55 on NHE3-mediated fluid swelling was totally reversed by S3226 (Fig. 7F). Similar results absorption by using the specific NHE3 inhibitor S3226. This was were seen in enteroids tested from another donor (data not shown). investigated in human 3D (closed spheroidal) enteroids using the Of note, the total amount of CFTR and NHE3 was not affect by R55 forskolin-induced swelling (FIS) assay. Forskolin is known to treatment (Fig. 7F). This shows that R55 stimulation of NHE3 stimulate CFTR-mediated fluid secretion into the lumen of small increases intestinal fluid absorption to partially overcome the intestinal enteroids, which can be measured using live-cell forskolin-induced fluid secretion. Cumulatively, these findings microscopy (Dekkers et al., 2013). Human 3D enteroids were show that the retromer stabilizer R55 increases NHE3 surface − preincubated in HCO3 -buffered solution for 1 h and then vehicle expression under basal and CT-inhibited conditions and reduces control and R55-treated (5 μM for 48 h) enteroids were exposed to forskolin-induced intestinal fluid secretion through an effect that forskolin (5 μM) and a time-dependent surface area increase was involves a component of NHE3 stimulation. measured. Forskolin caused a linear increase in the surface area of enteroids for at least 60 min as compared to vehicle control (0.2% DISCUSSION DMSO) (P<0.005), the latter of which did not change significantly In the current study, we provide a more complete description of the over this time. The enteroids pre-incubated with R55 (48 h) showed mechanism by which CT acts in producing diarrhea; we show that a small (but not significant) decrease in basal surface area over time CT inhibits NHE3 exocytosis in addition to its already defined as compared to control. In addition, enteroids treated with both R55 stimulation of endocytosis to reduce its BB expression and activity. (48 h) and forskolin had a partially reduced FIS response compared CT also modifies the PDZ domain of SNX27 by increasing to forskolin alone (∼30%, P<0.05 versus Fsk) (Fig. 7E,F). Previous phosphorylation of Ser49. This is functionally important as the Ser49 reports have demonstrated that the FIS response is dependent on phosphomimetic mutant of SNX27 phenocopied the effect of CT in Journal of Cell Science

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Fig. 7. A pharmacological chaperone rescues CT-dependent retromer destabilization and stimulates fluid absorption by intestinal epithelial cells. (A) Caco-2/bbe cells were treated with either CT (8 h or 48 h) or R55 (5 μM for 48 h) or both, in pre- (CT treatment before R55 treatment) or post- combinations (CT treatment after R55 treatment) for indicated periods of time, and levels of retromer proteins VPS26 and VPS35 were analyzed using western blot analysis. A representative blot is shown. (B) Quantitative analysis of the immunoblots in A. Levels of VPS26 and VPS35 were normalized to total levels of actin (n=4 per group). *P<0.05 versus control, #P<0.05 versus CT for 8 h; $P<0.05 versus CT for 48 h. (C) Polarized Caco-2/bbe-HA-NHE3 cells were treated with CT for indicated periods of time and the effect of R55 (5 μM for 48 h) on NHE3 surface expression was analyzed in a surface biotinylation assay. S-NHE3, surface NHE3; T-NHE3, total NHE3. Results from a single experiment are shown. (D) Quantitative analysis of western blots from four separate experiments, as in C, are shown as a bar graph. The amount of surface NHE3 was normalized to total NHE3 and expressed as percentage of control. Results are means±s.e.m. P values are in comparison with the control conditions related to the individual R55 experiments. (E) FIS assay in Calcein Green-labeled human duodenal enteroids exposed to forskolin and/or R55. Upper panel, a single example of a forskolin-stimulated enteroid. Lower panel, statistical evaluation of results from four separate experiments. Surface areas, expressed as percentage increase relative to that at t=0 (100%) are indicated on the y-axis. Forskolin-stimulated (5 μM) enteroids were pre-incubated with DMSO (0.2%) or R55 (5 μM for 48 h). The surface areas of 30 enteroids were measured in each group. Results are mean±s.e.m. with average from each experiment used as n=1. *P<0.05 versus control, #P<0.05 versus forskolin. (F) Role of NHE3 in the FIS assay response to forskolin and change mediated by R55. Upper panel, a representative blot showing expression of CFTR and NHE3 in 3D enteroids used for swelling assay. Arrows indicate B and C bands of CFTR. Lower panel, normalized forskolin-induced swelling of enteroids pretreatedfor1h with DMSO or the NHE3 inhibitor S3226 (20 μM) with or without R55 (5 μM for 48 h). Contribution of NHE3 was calculated as the change induced by S3226. Results are means±s.e.m. of at least three separate experiments. *P<0.05 versus control; #P<0.05 versus forskolin. terms of having reduced interaction with NHE3, which resulted in Our results show that CT modifies the PDZ domain of SNX27, reduced NHE3 activity as well as reduced NHE3 surface expression. which is atypical in having one additional β-strand compared to SNX27 Ser49 phosphorylation similarly reduced the binding of most other PDZ domains (Gallon et al., 2014), by increasing several other cargoes, with demonstration of effects on Glut-1, phosphorylation at least one site, which in turn inhibits the physical ATP7A and ZO-2. CT also destabilized the retromer by reducing the interaction between NHE3 and SNX27. As with many other protein levels of core retromer complex components. Since the role SNX27-interacting cargoes, SNX27 regulates trafficking of NHE3 of SNX27–retromer in NHE3 trafficking is established, we defined from EE to PM (Singh et al., 2015). Specifically, SNX27 serves as the effects of the pharmacologic chaperone R55 on the retromer an intermediate between the retromer and cargo proteins (Lauffer complex and on NHE3, including the changes induced by CT. R55 et al., 2010; Steinberg et al., 2013) and reduced SNX27–NHE3 stabilized the core retromer complex and increased NHE3 surface binding was associated with a reduction in NHE3 exocytosis, BB expression after CT exposure. R55 stimulated NHE3 activity by NHE3 expression and NHE3 activity. increasing its surface expression under basal conditions and in The CT effect on SNX27–NHE3 binding is not specific for CT-treated intestinal epithelial cells. R55 also stimulated intestinal NHE3 and also occurred for other proteins that, like NHE3, have fluid absorption to partially overcome the forskolin-induced fluid class I PDZ domain recognition domains on their C termini; those secretion in human intestine, suggesting that retromer could be a previously identified as interacting with SNX27 and confirmed here potential drug target for treating diarrhea. include Glut-1, ZO-2 and ATP7A. The demonstrated consequences Regardless of the etiology of the diarrheal disease, inhibition of of the CT-induced phosphorylation of the SNX27-PDZ domain are NHE3 occurs and contributes to the increased stool loss of Na and to reduce SNX27–cargo protein binding and, at least in some cases, water (Hawker et al., 1980). As this study indicates, while we to consequently reduce EE to PM trafficking. These observations understand a great deal mechanistically about how CT contributes to confirm and extend previous observations about the effects of CT. the severe diarrhea by enzymatically activating adenylyl cyclase and CT disrupts tight junction barrier function by inhibiting Rab11/

PKA (Wernick et al., 2010), our understanding remains incomplete. exocyst-mediated trafficking of proteins to cell–cell junctions Journal of Cell Science

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(Guichard et al., 2013). However, it is not known whether this maintenance of PM localization of NHE3 in CT-treated intestinal pathophysiology involves failure of SNX27 binding to critical epithelial cells. This is consistent with previous studies that showed components of the exocyst. SNX27 is known to regulate trafficking that increasing retromer levels enhances retromer-mediated trafficking of misslocalized ZO-2 from the EE back to the tight junctions (MacLeod et al., 2013; Mecozzi et al., 2014; Small et al., 2005). (Zimmerman et al., 2013). Therefore, these data, together, provide Furthermore, R55 treatment did not increase NHE3 binding with the robust evidence for a role of cAMP/PKA in regulating SNX27- S49D mutant of SNX27 (Fig. S6). Therefore, these data together mediated trafficking. suggest that R55 could affect NHE3 without directly involving The observation that the SNX27 PDZ domain phosphorylation is SNX27, since it is known that the retromer can function independently regulated, in this case by CT and forskolin, adds further evidence of of SNX27 in the recycling of membrane proteins back to the cell rapid post-translational regulation at the EE of SNX27 related to PM surface (Steinberg et al., 2013). However, it remains to be determined trafficking, particularly of proteins that contain C-terminal class I PDZ whether R55 can alter trafficking of other SNX27 cargo proteins. domain recognition motifs. While there is some basal phosphorylation The finding of the current study that R55 partially reversed FIS of of SNX27-Ser49, an increase in phosphorylation of this single amino 3D human enteroids by stimulating NHE3-mediated fluid acid reduced binding to multiple substrates and reduced the PM absorption suggests a potential additional use of the FIS assay. expression of each, which supports the idea that stimulation of FIS assays have been successfully used to measure fluid secretion this phosphorylation affects SNX27 function; furthermore, that the rates that involve CFTR as well as restoration of defective CFTR function of increased phosphorylation reduced cargo binding is activity by drugs (Dekkers et al., 2013; Fujii et al., 2016). This assay supported by the effects of the S49D mutation phenocopying the has also been used to test the swelling response of a wide variety of effects of SNX27 KD. Conversely, that there was no significant effect compounds and to efficiently evaluate their potential to induce or with the mutation SNX27-S49A, suggests that basal SNX27 function otherwise inhibit the fluid secretion of intestinal epithelial cells, is not dependent on the level of basal phosphorylation. with all effects assumed to be based on changes in CFTR activity. Phosphorylation of the SNX27 PDZ domain is not the only By using the specific NHE3 inhibitor S3226, we are using this assay circumstance when phosphorylation affects SNX27–cargo association. to evaluate fluid absorption related to stimulated NHE3 activity Changes in phosphorylation of multiple SNX27-binding partners at (Schwark et al., 1998). Of note, further optimization of this their class I C-terminal PDZ domain recognition sequences also affects approach is required to allow the contribution of NHE3 to basal their binding to SNX27 (Clairfeuille et al., 2016). Some SNX27 absorption as only slight and non-significant enteroid swelling ligands constitutively recycle from the EE to the PM while others only occurred with inhibition of basal NHE3 activity. Moreover, all the recycle when they are phosphorylated. NHE3 has both a C-terminal swelling in the FIS assay is CFTR dependent, which likely is a and internal PDZ domain-interacting sequence, with its interaction further demonstration of the influence of CFTR on NHE3 activity with SNX27 involving the C-terminal PDZ class I recognition (Bagorda et al., 2002; Mizumori et al., 2008). sequence (Cha et al., 2017; Singh et al., 2015). NHE3 constitutively The demonstration that a class of retromer stabilizers overcomes recycles from the EE to the PM, but whether this is dependent on basal the CT reduction of retromer proteins VPS35 and VPS26, and at phosphorylation of its C-terminal PDZ domain recognition sequence least partially overcomes the CT inhibition of NHE3 PM expression or correlates with its SNX27 association has not yet been defined. It is and activity, suggests that the retromer might be a drug target for of interest that phosphorylation of the SNX27-PDZ domain-binding treating severe diarrheas. In this regard, oral rehydration solution partners increases SNX27–cargo interactions, while phosphorylation (ORS) treatment of cholera is associated with the ability to rehydrate of the SNX27-PDZ domain itself decreases SNX27–cargo patients without reducing cholera-related diarrhea volume or interactions, at least for the ligands we have characterized. Whether shortening the duration of the diarrhea. Part of the effect of the cargo and SNX27 PDZ domain phosphorylation are coordinately D-glucose-containing ORS-mediated rehydration of patients with regulated and/or involve the same or related kinases acting together is cholera appears to be due to D-glucose/SGLT1-related signaling not known. that stimulates NHE3 activity to partially reverse the CT-inhibited Because SNX27-mediated trafficking of cargo proteins depends NHE3 activity (Lin et al., 2011). Related to potential treatment of upon a stable retromer complex, we tested whether CT affects diarrhea, we speculate that retromer stabilizers might further retromer function. We specifically focused on total protein changes increase NHE3 activity and intestinal Na+ absorption even in the in the core retromer complex. Deficiencies in VPS35 and VPS26 presence of CT. Separately, CFTR has also been shown to bind leading to defective retromer trafficking have been reported in SNX27 (McDermott et al., 2018 preprint); however, the effect of several neurological diseases (Li et al., 2016; Small et al., 2005; retromer stabilizers on CT-mediated stimulation of CFTR has not Tian et al., 2015; Wang et al., 2016). Furthermore, knocking down yet been defined. one protein of the VPS35–VPS26–VPS29 trimeric core complex often leads to secondary reductions in the other retromer proteins MATERIALS AND METHODS (Arighi et al., 2004; Fuse et al., 2015; Vergés et al., 2004). In the Glutathione–Sepharose 4B resin was from GE Healthcare Life Science present study, we observed a CT-induced decrease in the amount of (Pittsburgh, PA). BCECF-AM, nigericin and Hoechst 33342 were from Life core retromer proteins VPS35 and VPS26 suggesting that CT not Technologies (Grand Island, NY). Primary antibodies used in this study only inhibits SNX27–NHE3 binding, but also destabilizes the were: mouse anti-glyceraldehyde-3-phosphate dehydrogenase [Sigma- retromer core complex. In an attempt to increase NHE3 activity, we Aldrich, St. Louis, MO, GAPDH, #G8795; western blotting (WB) used the pharmacological chaperone R55. Consistent with the 1:3000], mouse anti-actin (Sigma-Aldrich, clone AC-74, #A2228, WB previously reported effect of this class of drugs to increase protein 1:3000), rabbit anti-ATP7A (Sigma-Aldrich, #HPA048107, WB 1:250), rabbit anti-GFP [Life Technologies, #A11122, WB 1:1000, stability (Mecozzi et al., 2014), R55 increased the levels of retromer immunofluorescence (IF) 1:200], mouse anti-GST (Cell Signaling proteins in intestinal epithelial cells under basal conditions and Technology, Danvers, MA clone 26H1, #2624, WB 1:2000), mouse anti- prevented the reduction of retromer protein levels caused by CT. GFP (Abcam, Cambridge, MA 9F9.F9, #ab1218, WB 1:3000), mouse Notably, R55 enhanced the function of the retromer, as indicated by anti-SNX27 (Abcam, clone 1C6, #AB77799, IF 1:100; WB 1:1000), goat increase in NHE3 basal activity in human enteroid monolayers and anti-VPS29 (Abcam, #ab10160, WB 1:1000, IF 1:200), mouse anti-VPS35 Journal of Cell Science

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(Abcam, #ab97545, WB 1:2000, IF 1:100), rabbit anti-VPS35 (Abcam, GST–SNX27-FL (full length) and PDZ-PX (GST-tagged N-terminal #ab97545, WB 1:1000, IF 1:100), rabbit anti-VPS26 (Abcam, #ab23892, PDZ-PX, amino acids 1–266) were constructed and purified as previously WB 1:1000, IF 1:100), rabbit anti-Glut-1 (Abcam, #ab15309, WB 1:1000, described (Singh et al., 2015). cDNAs encoding the PDZ-PX fragment of IF 1:100), mouse monoclonal LAMP1 (Developmental Studies Hybridoma SNX27-S49A and -S49D were generated by PCR and inserted into pGEX- Bank, #1DB4, IF 1:500), anti ZO-2 rabbit polyclonal (Invitrogen, #PA5- 4T-1 (GE Healthcare) for expression of GST-fused recombinant proteins. 27847, WB 1:1000), rabbit anti-NHE3 (Novus Biologicals, Littleton, CO, These constructs were transformed into the BL21(DE3) strain (EMD #NBP1-82574, WB 1:1000, IF 1:100). Alexa Fluor 488- and 568-conjugated Millipore). When the bacterial culture reached an optical density (OD) of goat anti-mouse-IgG and anti-rabbit-IgG secondary antibodies, were from ∼0.8, protein expression was induced with 0.3 mM isopropyl-β-D- Life Technologies. IRdye-700- and IRdye-800-conjugated goat anti-mouse- thiogalactoside at 16°C overnight. GST-tagged proteins were purified in a IgG and goat anti-rabbit-IgG secondary antibodies were from Rockland gravity-flow column following the instructions from GE Healthcare. Immunochemicals (Gilbertsville, PA) and were used with the Odyssey system Purified proteins were concentrated with Amicon Ultra-15 Centrifugal (LI-COR, Lincoln, NE) for western blot analysis. R55 TPT-260 (a thiophene filter units (EMD Millipore) supplemented with 10% glycerol and 10 mM thiourea derivative) was from Medkoo Biosciences (Morrisville, NC). DTT, and protein concentrations were measured via a Bradford assay.

Cell culture, shRNA and CT treatment Immunoprecipitation for LC-MS/MS The following cell lines were studied: HEK293T, HeLa, Caco-2/bbe and Immunoprecipitation experiments were performed using lysates from human duodenal enteroids from healthy subjects (see below). All cultures control or CT-treated HEK293 cells stably expressing GFP-tagged rat were routinely tested for mycoplasma. SNX27 as previously described (Singh et al., 2015). Briefly, cell lysates Human embryonic kidney-293 and HeLa cells were cultured in DMEM/ were prepared in lysis buffer (60 mM HEPES pH 7.4, 150 mM NaCl, 3 mM F-12 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine KCl, 5 mM EDTA trisodium, 3 mM lysis buffer ethylene glycol tetraacetic serum (FBS; BD), penicillin (50 mU/ml) and streptomycin (50 μg/ml), at acid, 1 mM Na3VO4, and 1% Triton X-100 with protease inhibitor cocktail; 37°C in a 5% CO2 humidified incubator. HEK293 cells stably expressing Sigma-Aldrich). Aliquots (2 mg of protein) of lysate were incubated with HA–NHE3 were generated previously (Singh et al., 2015). Stable SNX27 20 μl of GFP–nAb agarose resin (Allele Biotechnology) at 4°C for 4 h on a knockdown cells (SNX27-KD; HEK293 and HeLa cells) were generated rotating shaker. Beads were washed five times with the same buffer and using lenti-shRNA constructs, as described previously (Singh et al., 2015). eluted with 1× elution buffer. The input and eluted samples were separated A lentivirus plasmid vector containing shRNA that does not match any by SDS-PAGE. After electrophoresis, the gels were stained with Super Blue known human gene (Sigma-Aldrich) was used as the transduction control. Coomassie stain (Protea Biosciences). Once the gel was adequately stained, Infected cells were maintained under selection pressure of puromycin. a digital image was captured and the immunoprecipitated GFP–SNX27 gel Caco-2/bbe cells were grown in DMEM/F12 50/50 (Gibco/Life band was cut out and sent for LC-MS/MS analysis. Technologies) supplemented with 20% heat-inactivated FBS, Nu-Serum (BD #355500), and a penicillin/streptomycin mix for 14 days at 37°C with 5% LC-MS/MS CO2 and 95% humidity. Caco-2/bbe cell lines stably expressing HA–NHE3 Samples were proteolyzed using the ‘Filter Assisted Sample Preparation’ were generated by lentivirus shRNA transduction and maintained in the (FASP) method (Wisniewskí et al., 2009). Briefly, samples were reduced with presence of puromycin. HA–NHE3wassubclonedintopCDH-MCS-IRES- 5 mM tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 45 min and reduced neo (System Biosciences Inc, Mountain View, CA). Virus was packaged in cysteine residues were blocked using 10 mM iodoacetamide at 25°C for HEK293T cells by co-transfection with pMD2.G and pSPAX2. Media were 15 min. Samples were then buffer exchanged using a 30 kDa Amicon Filter collected, filtered and used to infect Caco-2/bbe cells; 1200 μg/ml G418 were (EMD Millipore) three times with 9 M urea and twice with 50 mM triethyl used to select for the cells in which virus was integrated. ammonium bicarbonate. Samples were then proteolyzed with trypsin/lysC For CT treatment, cells were grown in 12-well Transwell inserts for 14 days. (Promega) for 12 h at 37°C. Peptides were desalted using stage-tip C18 (3 M Purified CT (holo-CT, Sigma, St. Louis, MO) (0.1 μg/ml in 500 μlofmedium) Millipore). For TMT 10-plex comparisons, peptides were resuspended in or 500 μl of control medium was present either for 8 h or added 4 times at 12 h anhydrous acetonitrile and labeled with TMT reagents according to intervals for up to 48 h, and cells were collected after the last treatment. manufacturer’s recommendations (Thermo Fisher Scientific). Protein identification by LC-MS/MS analysis of peptides was performed Tissue collection and enteroid generation on a Q-Exactive mass spectrometer (Thermo Fisher Scientific) interfaced Human enteroid cultures were established from biopsies obtained after with the EasyLC 100 nanoflow LC system. Peptides were separated on a endoscopic or surgical procedures utilizing the methods developed by Sato reversed-phase HPLC on a 75 μm×15 cm PicoFrit column (New Objective) et al. with slight modification (Noel et al., 2017; Sato et al., 2011). Biopsy packed with Magic C18AQ [5 μm, 120 Å (1 Å=0.1 nm), Michrom tissue was obtained from healthy subjects who underwent endoscopies Bioresources]. Peptides were separated using a gradient of 0%–60% with no pathology identified and who provided informed consent. All acetonitrile and 0.1% formic acid over 70 min at a flow rate of 300 nl/min. experimental protocols were approved by the Johns Hopkins University Eluting peptides were sprayed directly into Q-Exactive at 2.0 kV spray Institutional Review Board (IRB# NA_00038329). All clinical investigation voltage. Survey scans were acquired from 350–1800 m/z with up to 15 have been conducted according to the principles expressed in the peptide masses (precursor ions) individually isolated with a 2 Da window Declaration of Helsinki. For this study, enteroids were generated and with 0.5 Da offset and fragmented (MS/MS) using a collision energy of 27 maintained from duodenal biopsies (Noel et al., 2017). Enteroid monolayers and 30 s dynamic exclusion. Precursor and the fragment ions were analyzed were formed onto 0.4 μm pore transparent polyester (PET) membrane at 70,000 and 17,500 resolution, respectively. Peptide sequences were 24-well Transwell cell culture inserts (Transwell; Corning or Millipore) identified from isotopically resolved masses in MS and MS/MS spectra pre-coated with human collagen IV (30 μg/ml; Sigma-Aldrich) and were extracted with and without deconvolution using Thermo Scientific MS2 maintained in Wnt3A-containing non-differentiation medium (NDM) processor and Xtract software. Data were searched against human 2012 at 37°C, 5% CO2 and 95% humidity. Upon confluency (7–14 days), database with oxidation on methionine, deamidation on residues asparagine monolayer differentiation was induced by removal of Wnt3A and Rspo-1 and glutamine, phosphorylation on serine, threonine or tyrosine residues (as for 5 days (Foulke-Abel et al., 2014; Noel et al., 2017). different variable modifications) and carbamidomethyl on cysteine as a fixed modification using Mascot software interfaced with the Proteome Recombinant protein purification Discoverer 1.4 (http://portal.thermo-brims.com/) workflow. For TMT 10- Plasmid pcDNA3.1-HA-NHE3, was constructed previously (Murtazina plex experiments, the TMT label on the peptide N-terminus (fixed et al., 2006). Rat SNX27b (SNX27b) cDNA was kindly provided by Jae modification) and lysine residues (variable modification) was added to the Cheng (Johns Hopkins University, Baltimore, MD). The SNX27b PDZ- database search. Peptide identifications from Mascot searches were S49A and -S49D point mutations were generated using the QuikChange processed within Proteome Discoverer software to identify peptides with site-directed mutagenesis kit (Agilent Technologies). a confidence threshold <1% false discovery rate, based on a concatenated Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218610. doi:10.1242/jcs.218610 decoy database search to calculate the median protein and peptide ratios. Exocytic insertion assays Localization of serine sites was evaluated using PhosphoRS score and To measure exocytic insertion of NHE3, in control and CT-treated Caco-2/ manual inspection. The ratios for the phosphorylated peptides in samples bbe-HA-NHE3 cells, surface protein accessible to NHS-SS-biotin were were calculated from TMT reporter ions after normalization on the median masked by pretreatment with NHS-acetate (1.5 mg/ml) as described ratio from all peptides detected from SNX27. previously (Singh et al., 2015). Then cells were rinsed with ice-cold PBS containing 0.1 mM Ca2+ and 1 mM Mg2+ two times at 4°C and incubated GSH resin pulldown for 0, 15, 30 and 60 min at 37°C to allow exocytosis of NHE3. Cells were For interaction studies, 1 nmol of recombinant GST-tagged protein was used then labeled with sulfo-NHS-SS-biotin (1.5 mg/ml) and treated with lysis as bait. As prey, 1 mg of cell lysate was used as indicated. The volume of the buffer as described previously (Singh et al., 2015). The biotinylated fraction final mixture was adjusted to 500 μl with the lysis buffer (25 mM HEPES, was precipitated with streptavidin–agarose beads. The resultant precipitate pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 0.5% Triton X-100 was subjected to SDS-PAGE, and biotinylated NHE3 was detected by and protease inhibitors). GSH resin (Glutathione–Sepharose 4B resin) was quantitative western blot analysis as described previously (Singh et al., 2015). washed with lysis buffer three times. Each bait–prey mixture was mixed Biotinylated NHE3 was normalized to the total amount of NHE3 present in with 10 μl of resin and incubated at 4°C for 4 h on a rotating shaker. Resin the lysate at each time point and expressed as percentage exocytosed. was washed with the same lysis buffer four times and then eluted with lysis buffer supplemented with 10 mM glutathione. The input and elution Endocytosis internalization samples were analyzed by SDS-PAGE and western blotting. For Endocytosis of NHE3 in response to CT was measured by using a reduced quantification, the densitometric measurement of bands (using ImageJ) glutathione (GSH)–resistant endocytosis assay that was described previously was first normalized to the amount of GST fusion protein pulled down, and (Singh et al., 2015). signal intensities were then normalized to control (GST–PDZ-PX) samples. Total NHE3 degradation assay Measurement of Na+/H+ exchange activity Post confluent Caco-2/bbe cells (14 days; control and CT-treated) were Cellular Na+/H+ exchange activity in HEK293-HA-NHE3 or SNX27-KD treated with 100 µM cycloheximide for the indicated time points. Cells were cells grown to ∼80% confluence on glass coverslips and differentiated human lysed in PBS with 1% (v/v) Triton X-100, and NHE3 levels were determined enteroid monolayers was determined fluorometrically by using the by quantitative western blotting. β-actin fluorescence intensity was used to intracellular pH-sensitive dye 2′,7′-bis(carboxyethyl)5-6-carboxyfluorescein- normalize the detected levels of NHE3. The levels of untreated controls were acetoxymethyl ester (BCECF-AM, 5 μM) as described previously and set to 100%, and the level of detected NHE3 was calculated as the expressed as ΔpHi/minute (Janecki et al., 1998; Levine et al., 1995). percentage of untreated control for each time point.

Immunofluorescence staining and confocal microscopy Quantification of fluid secretion in human enteroids HEK293 or HeLa cells seeded on coverslips or 5 day differentiated human Human enteroids from a 7–10-day-old culture were plated in 50% Matrigel duodenal enteroids grown on Transwell inserts were fixed with 4% (20 μl; Corning Life Sciences, Corning, NY) on glass-bottom 35-mm paraformaldehyde for 1 h. Permeabilization and blocking were carried out dishes. (P35G-1.5-10-C; MatTek, Ashland, MA), containing 50–100 simultaneously in a solution of 15% FBS, 2% BSA and 0.1% saponin (all enteroids and then overlaid with 1.5 ml NDM for 2–3 days. On the day of Sigma-Aldrich) in PBS for 60 min at room temperature. Cells were rinsed imaging, enteroids were incubated with 10 µM Calcein Green- with PBS and incubated overnight at 4°C with primary antibodies diluted acetoxymethyl ester (Invitrogen) in NDM for 60 min. Time-lapse z-stack 1:100 in PBS containing 15% FBS and 2% BSA. Stained cells were then images were collected every 10 min for 1 h with 488-nm excitation using a washed three times for 10 min each with PBS followed by secondary 10× phase-contrast objective on a confocal microscope system (Fluoview antibodies diluted 1:100 in PBS. Secondary antibodies included goat anti- FV10i-LIV; Olympus) at 37°C, 5% CO2, and 95% relative humidity. To rabbit-IgG conjugated to Alexa Fluor 488, goat anti-mouse-IgG conjugated measure the effect of the pharmacological chaperone R55 on fluid to Alexa Fluor 488, goat anti-rabbit-IgG conjugated to Alexa Fluor 568, absorption, enteroids were pre-incubated with 5 μM R55 for 48 h. 0.2% goat anti-Mouse-IgG conjugated to Alexa Fluor 568 (all from Molecular DMSO (control) and R55-treated enteroids were incubated with NHE3 Probes/Invitrogen, USA). Hoechst 33342 (Vector Laboratories) for nuclear specific inhibitor S3226 (20 µM) for 1 h treated with forskolin (5 µM), and labeling was used at a 1:200 dilution in PBS. After incubation, cells were time lapse images were collected and analyzed with MetaMorph version washed three times for 10 min each and mounted in ProLong Gold (Vector 7.7. The confocal section area was defined as the total area (pixels) occupied Laboratories) overnight at 4°C. by the enteroid at a z-plane approximately halfway through the total structure Cells were visualized using a Zeiss LSM-510 META laser scanning height, and each successive measurement over time was made on an image confocal microscope (Zeiss, Germany) running ZEN 2012 (black edition) from the same corresponding z-plane. The percentage change in the imaging software (Zeiss, Germany). All images were captured with a 40× confocal section area is the difference in area at each time point calculated (1.4 NA) oil objective. For quantitative analysis, the same settings were used relative to the area at time=0. to image across samples. Images were acquired and processed using ZEN 2012 (Carl Zeiss) and ImageJ software. Retromer vesicles in cells were Quantitative real-time PCR quantified by counting the number of distinct VPS35 and VPS26 puncta in a Total RNAwas extracted from Caco-2/bbe cell monolayers using a PureLink® maximal projection image. Colocalization analysis was performed with the RNA Mini Kit (Life Technologies, Carlsbad, CA) according to the FIJI plugin, Coloc_2 (http://fiji.sc/Colocalization_Analysis). The degree of manufacturer’s protocol. Complementary DNA (cDNA) was synthesized colocalization between SNX27 (green) and EEA1 (red) was quantified and from 1–2 μg of RNA using SuperScript™ VILO™ MasterMix (Life expressed as a Pearson colocalization coefficient (1, perfect correlation and Technologies, Carlsbad, CA). Quantitative real-time PCR (qRT-PCR) was −1, no correlation). The significance of colocalization was determined by performed using the Power SYBR® Green Master Mix (Life Technologies) Costes’ randomization analysis. on a QuantStudio™ 12K Flex real-time PCR system (Applied Biosystems Inc., Foster City, CA). Each sample was run in triplicate, and 5 ng RNA- Cell surface biotinylation and immunoblotting equivalent cDNA was used for each reaction. The relative fold-change in the NHS-SS-biotin was used to determine the amount of BB NHE3 (Singh mRNA level of proteins were determined using the 2−ΔΔCT method with et al., 2015). All the western blot analyses were performed using Odyssey human 18S rRNA was used as the internal control for normalization. imaging systems, which is known to have a linear dynamic range of at least 104. This wide dynamic range results in marked reduction of detector Statistical analysis saturation for western blotting experiments. All western blot results were All quantified western blot and confocal data are the mean of the indicated normalized to loading controls. number of independent experiments. Statistical analyses were carried out Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218610. doi:10.1242/jcs.218610 using GraphPad Prism 5 software (La Jolla, CA). Either an unpaired and arrestin-like fold interaction reveals mechanistic details of endocytic recycling Student’s t-tests or one-way ANOVA followed by a Dunnett’s post hoc test by SNX27-retromer. Proc. Natl. Acad. Sci. USA 111, E3604-E3613. were performed to determine whether differences between conditions were Guichard, A., Cruz-Moreno, B., Aguilar, B., van Sorge, N. M., Kuang, J., P Kurkciyan, A. A., Wang, Z., Hang, S., Pineton de Chambrun, G. P., McCole, statistically significant. <0.05 is considered statistically significant. D. F. et al. (2013). Cholera toxin disrupts barrier function by inhibiting exocyst- mediated trafficking of host proteins to intestinal cell junctions. Cell Host Microbe Competing interests 14, 294-305. The authors declare no competing or financial interests. Hawker, P. C., McKay, J. S. and Turnberg, L. A. (1980). Electrolyte transport across colonic mucosa from patients with inflammatory bowel disease. Author contributions Gastroenterology 79, 508-511. Conceptualization: V.S., D.E.B., S.A.S., G.P., M.D.; Methodology: V.S., J. Yang, Ikuma, M., Kashgarian, M., Binder, H. J. and Rajendran, V. M. (1999). Differential J. Yin, R.C., M.T., D.E.B., G.P., M.D.; Software: V.S.; Validation: V.S.; Formal regulation of NHE isoforms by sodium depletion in proximal and distal segments of analysis: V.S., J. Yin, M.T., S.A.S.; Investigation: V.S., J. Yang, S.A.S.; Resources: rat colon. Am. J. Physiol. 276, G539-G549. V.S., R.C., S.A.S., G.P., M.D.; Data curation: V.S., J. Yang, J. Yin, R.C., M.T.; Writing Janecki, A. J., Montrose, M. H., Zimniak, P., Zweibaum, A., Tse, C. M., Khurana, - original draft: V.S.; Writing - review & editing: V.S., M.T., D.E.B., M.D.; Visualization: S. and Donowitz, M. (1998). Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon V.S., R.C.; Supervision: V.S., M.D.; Project administration: V.S., M.D.; Funding adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the acquisition: M.D. exchanger. J. Biol. Chem. 273, 8790-8798. Joubert, L., Hanson, B., Barthet, G., Sebben, M., Claeysen, S., Hong, W., Marin, Funding P., Dumuis, A. and Bockaert, J. (2004). New sorting nexin (SNX27) and NHERF This work was supported in part by NIDDK, NIAID and NCATS (National Institutes of specifically interact with the 5-HT4a receptor splice variant: roles in receptor Health) (grants R01-DK26523, R01-DK61765, R24-DK99803, P01-DK072084, targeting. J. Cell Sci. 117, 5367-5379. P01-AI125181, UH3-TR00003, UO1-DK10316 and P30-DK89502), The Hopkins Kunzelmann, K. and Mall, M. (2002). 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