FLCN Is a Novel Rab11a-Interacting Protein That Is Involved in the Rab11a-Mediated Recycling Transport

RESEARCH ARTICLE FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport Lingling Zhao*, Xin Ji*, Xiangxiang Zhang, Lin Li, Yaping Jin‡ and Wei Liu‡

ABSTRACT 2008). This implies that FLCN probably controls certain The Birt–Hogg–Dubé(BHD) syndrome related protein FLCN has fundamental cellular processes that are not unique to higher Drosophila recently been implicated in the vesicular trafficking processes by organisms. Using both (Liu et al., 2013) and the interacting with several Rab family GTPases. In the previous studies, cultured human embryonic kidney HEK293 cells (Wu et al., 2016) we have shown that FLCN could inhibit the binding of overexpressed as the model systems, we discovered that FLCN positively regulates PAT1, which is a membrane-bound amino acid transporter, to the the mTORC1 signaling pathway, in part, by maintaining the lysosome in human embryonic kidney 293 cells. This tends to stabilize stimulatory amino acid signal within the lysosome. In addition, we the lysosomal amino acid pool that is a critical signal to activate the found FLCN performed this function largely by suppressing the mTORC1 signaling pathway. However, the mechanisms of FLCN binding of the amino acid transporter proton-coupled amino acid during this process remain unexplored. Here we report that FLCN can transporter 1 (PAT1; also known as SLC36A1) on the lysosomal bind through its C-terminal DENN-like domain to the recycling transport surface (Wu et al., 2016). These results can reveal a putative regulator, Rab11A. Suppression of either Rab11A or FLCN facilitated function of FLCN during the vesicular trafficking of PAT1. the localization of the overexpressed PAT1 to the lysosome and PAT1 is called the proton-coupled amino acid transporter 1. It inhibited its targeting on the plasma membrane. As a consequence, belongs to the solute carrier protein 36A subfamily that in mammals the mTORC1 was down-regulated. The in vitro GEF activity assay contains four members (PAT1-4, or SLC36A1-4). PAT1 is an eleven- does not support FLCN modifies the Rab11A activity directly. Instead, pass transmembrane protein that can move amino acids, together with we found FLCN promoted the loading of PAT1 on Rab11A. Our data protons, from both the extracellular environment and the intracellular uncover a function of FLCN in the Rab11A-mediated recycling pathway membrane compartments into the cytosol (Thwaites and Anderson, and might provide new clues to understand BHD. 2011). In many examined cell types, PAT1 is enriched on the lysosomal surface, suggesting it has a general function in the luminal This article has an associated First Person interview with the first nutrient homeostasis. Besides, PAT1 was also detected on several author of the paper. other cellular locations, including the plasma membrane (Anderson et al., 2004; Chen et al., 2003; Sagne et al., 2001; Wreden et al., KEY WORDS: PAT1, FLCN, Rab11A, Endocytic recycling, mTORC1 2003), axons (Wreden et al., 2003), podosomes (Cougoule et al., 2005), and even in the nuclei (Jensen et al., 2014), in different cell INTRODUCTION types. This indicates that PAT1 may have diverse physiological Mutations of the tumor suppressor folliculin (FLCN) have been functions, which are related to its specific subcellular localizations. In linked with the Birt–Hogg–Dubé (BHD) syndrome that is clinically addition to its well-characterized role in nutrient homeostasis, PAT1 characterized by benign skin tumors, spontaneous pneumothorax, was also found to regulate mTORC1, which is a multicomponent and kidney cancer (Birt et al., 1977; Schmidt et al., 2005; Toro et al., protein kinase complex that controls cell growth and metabolism by 2008). FLCN was thought to be involved in a number of biological sensing amino acids in both the lysosome and the cytosol. Both processes, including signal transduction, biogenesis of lysosome Sabatini’s group and ours have shown that PAT1 could negatively and mitochondria, cell-cell adhesion, membrane trafficking, energy regulate mTORC1, probably through a mechanism by decreasing the and nutrient homeostasis, and so on (for a recent review, see lysosomal amino acid signal level (Tsun et al., 2013; Wu et al., 2016). Schmidt and Linehan, 2018), but its precise functions related to the More interestingly, we found the localization of PAT1 to the lysosome BHD symptoms are not fully understood. seemed to be dynamic and context-dependent (Wu et al., 2016). The Flcn gene has been highly conserved during evolution. Using the overexpressed PAT1 as a substrate, we found it could FLCN orthologs have been characterized in yeast (Péli-Gulli et al., be transported between the plasma membrane and the lysosome in 2015; Roberg et al., 1997), nematode (Gharbi et al., 2013; Possik HEK293 cells. The distributions of PAT1 on these two sites were et al., 2014), fruit fly (Liu et al., 2013; Singh et al., 2006), zebrafish influenced by two factors, including amino acids and FLCN (Wu (Kenyon et al., 2016) and mouse (Baba et al., 2016; Chen et al., et al., 2016). We have recently shown that amino acids can inhibit the transport of overexpressed PAT1 to the lysosome by inducing the N-terminal protein cleavage, leading to the loss of a tyrosine- Key Laboratory of Animal Biotechnology, the Ministry of Agriculture, College of based lysosomal targeting signal (Ji et al., 2017). This discovery Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China 712100. reveals a putative mechanism that amino acids can stimulate *These authors contributed equally to this work mTORC1 through modulating the localizations of amino acid ‡Authors for correspondence ([email protected]; [email protected]) transporters. So far, the mechanisms of FLCN in the targeting of PAT1 are unknown. Notably, a structural study identified a L.Z., 0000-0002-8907-6495; X.J., 0000-0002-1557-5187; X.Z., 0000-0002-1073- 998X; L.L., 0000-0002-1433-3477; W.L., 0000-0001-6659-1008 differentially expressed in normal and neoplastic cells (DENN)- like domain at the C-terminus of FLCN proteins (Nookala et al.,

Received 9 April 2018; Accepted 2 November 2018 2012). Because some DENN-containing proteins can act as GTP Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218792. doi:10.1242/jcs.218792 exchange factors (GEF) to activate the Rab family of small (Wu et al., 2016). This implies PAT1 may first be sent to the cell GTPases, which are key regulators of vesicular trafficking, FLCN surface, from where it can go into the cell through membrane has been suspected to control the trafficking of membrane proteins invagination and then gradually move to the lysosome. We confirmed (Nookala et al., 2012). However, it is only recently that the evidence this hypothesis by using internalization assay of FLAG-tagged PAT1, of FLCN in protein transport has started to emerge, probably containing 3×FLAG epitope at its C-terminus extending to the because the cargos transported by FLCN were largely unknown. extracellular environment. When the living cells of PAT1-FLAG During the ongoing of this research, two groups reported that FLCN were incubated with a monoclonal anti-FLAG antibody on ice (to promotes the transport of EGFR to the lysosome for degradation by block endocytosis), we detected PAT1-FLAG on the cell surface modulating the activities of Rab7 (Laviolette et al., 2017) and (Fig. 1D, 0 min). After endocytosis was restarted by switching to Rab35 (Zheng et al., 2017), respectively. high temperature (37°C), the pre-labeled PAT1-FLAG on the cell In this study, we looked at overexpressed PAT1 as a substrate and surface could move into the cell and eventually targeted the discovered that FLCN regulates Rab11A-mediated recycling lysosomes (Fig. 1D, 45 min). transport. Our study provides new insights into how FLCN can regulate mTORC1 and might link BHD to the dysregulation of Rab11A regulates the subcellular distribution of Rab11A-regulated recycling pathways. overexpressed PAT1 Of the various localizations of Myc-PAT1, we were particularly RESULTS interested in Rab11A-marked endosomes (Fig. 1A,B). Rab11A is a Overexpressed PAT1 can be sorted into diverse endosomes key regulator of the slow recycling transport pathway (Welz et al., in HEK293 cells 2014) that, in theory, should inhibit the transport of PAT1 to the To understand how FLCN regulates the transport of overexpressed lysosome and promote its targeting on the plasma membrane and/or PAT1 to the lysosome, we generated HEK293 cells constitutively the Golgi network. To study whether Rab11A regulates PAT1 expressing Myc-PAT1, which has been shown to colocalize well localization, we attempted to block Rab11A activity and then with endogenous PAT1 (Ögmundsdottir et al., 2012). Theoretically, quantify PAT1 on both the lysosome and the plasma membrane. targeting of membrane proteins to the lysosomes can be affected First, we used the RNA interference (RNAi) technique to knockdown by two opposing pathways – one that sends them to the lysosome Rab11A. The cells stably expressing PAT1-FLAG (60% confluence) and one that recycles them to the cell surface and/or the Golgi were infected with lentiviruses carrying two different small hairpin network. To study whether Myc-PAT1 travels via these two RNAs (shRNAs) targeting Rab11A (shRab11A). Lysosomes were pathways, we performed fluorescence immunostaining and isolated 48 h later by cell fractionation (see Materials and Methods). analyzed the colocalization of Myc-PAT1 with several important We confirmed that lysosomes could be purified using this method by endosomal markers, including Rab5 (early endosome), Rab7 (late analyzing markers of different cell fractions. In addition, these assays endosome), Rab11 (recycling endosome) and LAMP1 (lysosome). also revealed that starvation could induce accumulation of PAT1- To monitor the different types of endosome, we first used the FLAG on the lysosome (Fig. S1). As shown in Fig. 2A, we found that mCherry-tagged endosomal markers. Myc-PAT1 was stained with a suppression of Rab11A by either of the two shRab11As clearly monoclonal anti-Myc antibody and the mCherry signals were captured promoted localization of PAT1-FLAG to the lysosomes. To measure directly without staining. As anticipated, Myc-PAT1 colocalized well PAT1-FLAG on the plasma membrane, we used biotinylation with LAMP1-mCherry (Fig. 1A). In addition, we also observed many labeling to purify the total cell surface proteins. As a result, we found overlaps between Myc-PAT1 and overexpressed Rab5A, Rab7A or that Rab11A knockdown inhibited targeting of PAT1-FLAG to the Rab11A (Fig. 1A). This indicates that Myc-PAT1 may be transported plasma membrane (Fig. 2B). We also applied a different strategy to along both endocytosis and recycling pathways. Next, we examined suppress the Rab11A activity by overexpressing a dominant negative the endogenous endosomal markers. Consistent with the previous S25N mutant of Rab11A (Rab11A-DN). Consistent with the results reports (Ögmundsdottir et al., 2012; Zoncu et al., 2011), we found of shRab11A, we found that Rab11A-DN promoted localization of Myc-PAT1 was mainly localized to the LAMP1-marked endosomes. PAT1-FLAG to the lysosome and inhibited its localization to the Importantly, we also observed some overlaps between Myc-PAT1 and plasma membrane (Fig. 2C,D). endogenous Rab5, Rab7 or Rab11 (Fig. 1B). To further reveal its We performed double-staining experiments of Myc-PAT1 and trafficking routes, we examined additional endosomal markers, LAMP1. As shown in Fig. 2E, we found that colocalization of Myc- including TGN38 (for the trans-Golgi network; TGN), EEA1 (for PAT1 and LAMP1 was increased by either Rab11A small early endosomes), Chmp5 (for the multivesicular body) and the interfering RNA (siRNA) or Rab11A-DN but decreased by cation-dependent mannose-6-phosphate receptor (M6PR). We found overexpressing a constitutively active Q70L mutant of Rab11A that Myc-PAT1 colocalized more or less with all these endosomal (Rab11A-CA,). Interestingly, after Rab11A-CA overexpression, markers (Fig. 1C). Of note, Myc-PAT1 showed many overlaps with we observed many Myc-PAT1-positive and LAMP1-negative M6PR, which can recognize and deliver many hydrolases from the endosomes that were preferentially located at sub-plasma Golgi to the lysosome, and M6PR itself will be sent back to the Golgi membrane regions (arrowheads in Fig. 2E). We suspect that these for recycling (Braulke and Bonifacino, 2009). Taken together, these endosomes probably recycle to the cell surface. Taken together, our results suggest that Myc-PAT1 can be transported along the classic results demonstrate that Rab11A can modulate the distribution of endocytosis and recycling pathways in HEK293 cells. overexpressed PAT1 on the lysosome and the plasma membrane, probably by promoting its recycling transport pathway. Overexpressed PAT1 can be transported from the cell During these experiments, we noticed that suppression of surface to the lysosome Rab11A often caused a mild decrease of total PAT1 levels. This From the TGN, the membrane proteins can be delivered to the could be reversed by bafilomycin A1 (BafA1), which is a lysosomes through both direct and indirect pathways. Using the cell specific inhibitor of the lysosome-dependent degradation pathway fractionation method, we have shown before that overexpressed (Luo et al., 2017; Yoshimori et al., 1991; Fig. 2F. These data suggest

PAT1 can be localized to the plasma membrane in HEK293 cells that the level of lysosomal PAT1 is probably adjustable. Journal of Cell Science

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Fig. 1. Overexpressed PAT1 can be transported along both endocytosis and recycling pathways. Confocal microscopy images of double-labeling experiments. All scale bars: 10 μm. (A) mCherry-tagged markers were transiently expressed in cells stably expressing Myc-PAT1, followed by staining of the Myc tag (green). mCherry (red) was not stained. (B,C) Myc-PAT1 cells were stained for Myc (green) and endogenous endosomal markers (red). Arrowheads indicate some overlaps of Myc-PAT1 with Rab7 and Rab11. Colocalization was calculated as the Pearson’s correlation coefficient (n>30 cells per assay, see Materials and Methods). (D) PAT1 is transported from the plasma membrane to the lysosome. Living cells coexpressing PAT1-3×FLAG and LAMP1-mCherry were incubated with mouse monoclonal FLAG antibody on ice (endocytosis was stopped) for 1 h. After that, the cells were washed once with ice-cold PBS, and then were either stained (0 min) or first starved with the RPMI 1640 medium (lacking amino acids and serum) at 37°C for 45 min to induce transport of PAT1 to the lysosome. At least 50 cells of each group were analyzed, the representative images are shown.

The inhibitory effect of PAT1 overexpression on mTORC1 can mTORC1 activity, as revealed by the presence of phosphorylated be antagonized by Rab11A ribosomal protein S6 kinase β1 (pS6K1, at T389), was decreased in To reveal the significance of Rab11A-PAT1 relationships, we decided cells stably expressing PAT1-FLAG, compared to wild-type cells to focus on the mTORC1 signaling pathway. It has been shown (Fig. 3A, lanes 1, 2). Interestingly, suppression of Rab11A by previously that overexpression of PAT1 can inhibit mTORC1 when shRNAs caused further reductions of the mTORC1 activity (Fig. 3A, cells were synchronized by starvation, followed by a short time (10 min) lanes 3, 4). A similar result was observed by overexpressing the of nutrient replenishment (Wu et al., 2016; Zoncu et al., 2011). In a Rab11A-DN mutant (Fig. 3B, lane 3). By contrast, overexpressing recent study, we have demonstrated that this result was probably caused Rab11A-CA increased mTORC1 activity in PAT1-overexpressing by incomplete nutrient-induced relocation of lysosomal PAT1, cells (Fig. 3B, lane 4). These results suggest that Rab11A can extending the nutrient stimulation time can reactivate mTORC1 in the antagonize the inhibitory effect of overexpressed PAT1 on mTORC1. PAT1-overexpressing cells (Zhao et al., 2018). We then investigated the influence of Rab11A on mTORC1 in a We starved cells for 50 min and then re-stimulated them with wild-type cell background, and found that mTORC1 activity was nutrient-containing medium for 10 min. As anticipated, the increased by Rab11A-CA but decreased by Rab11A-DN or Journal of Cell Science

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Fig. 2. Rab11A modulates the intracellular distribution of overexpressed PAT1. (A-D) Suppression of Rab11A by either shRNAs (A,B; shRab11A-1 or shRab11A-2) or Rab11A-DN (C,D) increased the lysosomal PAT1 (A,C) but decreased its level on the plasma membrane (B,D). Cells were starved by culturing in RPMI 1640 medium (lacking amino acids and FBS) for 1 h, and stimulated with complete medium for 30 min. Fractions of lysosomes and plasma membranes were isolated, followed by western blotting. N, nonsense shRNA (A,B); pan-cadherin was taken as a control of the plasma membrane proteins (C,D). Note that, due to glycosylation (Zoncu et al., 2011) and hydrophobic interactions (Dorn et al., 2009), there are usually some smear signals above the PAT1 monomers (∼53 kDa). Therefore, total PAT1 signals shown in A (within the enclosed dashed line) were counted. The quantification assays are explained in Materials and Methods, and based on at least 3 biological repeats. **P<0.01. (E) Cells stably expressing Myc-PAT1 were co-stained for Myc (green) and LAMP1 (red). siRNA was used to knock down Rab11A. Notice that Rab11A-CA increased the Myc-PAT1-positive and LAMP1-negative endosomes that are mainly located at sub-plasma membrane regions (arrowheads). Each quantification assay was based on at least 30 cells. Scale bars: 10 μm. (F) Levels of PAT1 were somehow decreased in response to Rab11A suppression and are rescued by addition of BafA1. shRNAs were expressed for 36 h. Then BafA1 was added to the medium at the final concentration of 10 ng/ml. 9 h later, cells were collected and analyzed. **P<0.01. shRab11A (Fig. 3C,D). This raises a question whether Rab11A can the same PAT1 siRNA (i.e. si160) as described before by Heublein regulate mTORC1 through PAT1. Knockdown of PAT1 has been et al., 2010 (Fig. 3E, compare lane 1 with 2). Importantly, the shown to inhibit mTORC1 in HEK293 cells (Heublein et al., 2010). mTORC1 activity did not change upon co-expression of either We suspected that this might be caused by a reduction of the Rab11A-DN and siPAT1, or Rab11A-CA and siPAT1 (Fig. 3E, cytosolic amino acid pool, which is another important signal source lanes 2–6). Considering that Rab11A-CA can stimulate mTORC1 in of mTORC1 (Zhao et al., 2018). We confirmed this result by using wild-type cells (Fig. 3D, lane 2), these results suggest PAT1 can Journal of Cell Science

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Fig. 3. Rab11A antagonizes the inhibitory effect of PAT1 overexpression on mTORC1. (A) Suppression of Rab11A by shRNAs promoted downregulation of mTORC1 in the PAT1-overexpressing cells. pS6K1 is a specific marker of mTORC1 activity. Lanes 1 and 2, mTORC1 activity was decreased in cells stably expressing Myc-PAT1. mTORC1 activity was decreased further by co-suppression of Rab11A (shRab11As, lanes 3, 4). (B) The inhibitory effect of overexpressed PAT1 on mTORC1 (lane 2) was enhanced by Rab11A-DN (lane 3) and suppressed by Rab11A-CA (lane 4). (C,D) Rab11A positively regulates mTORC1 in HEK293 cells. Different forms of FLAG-tagged Rab11A were transiently overexpressed. pS6K1 and p4EBP1 are two different markers of mTORC1 activity. (E) PAT1 is downstream of Rab11A to regulate mTORC1. Lanes 1 and 2, knockdown of PAT1 by RNAi inhibited mTORC1. This result was not changed by either suppression (lanes 3, 4, 6) or activation (lane 5) of Rab11A. (F) Rab5A and Rab7A can antagonize the effect of Rab11A on mTORC1. Lanes 2 and 5, knockdown of Rab11A by two different shRNAs decreased the mTORC1 activity in the PAT1-overexpressing cells. This result could be rescued by blocking endocytosis with Rab7A-DN (lanes 3, 6) or Rab5A-DN (lanes 4, 7). In A and B, cells were starved and stimulated with complete medium for 10 min. In C-F, cells (∼90% confluence) were analyzed directly without starvation and stimulation treatment. In D-F, different forms of FLAG-tagged Rabs were used. For the quantification assays, n=3 biological repeats; *P<0.05, **P<0.01, ***P<0.001. Error bars in graphs indicate the s.e.m. function downstream of Rab11A, possibly by being a Rab11A PAT1-overexpressing cells (compare lane 1 with lanes 2 and 5); substrate. According to this mechanism, we predict that the effect however, this result could be rescued by blocking endocytosis with of Rab11A can be overcome by blocking the transport of PAT1 either the dominant negative T22N mutant of Rab7A (Rab7A-DN) to the lysosome. Indeed, as shown in Fig. 3E, suppression of (Fig. 3E, lanes 3, 6) or the dominant negative S34N mutant of

Rab11A (by using shRab11As) decreased mTORC1 activity in Rab5A (Rab5A-DN) (Fig. 3E, lanes 4, 7). Journal of Cell Science

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FLCN interacts with Rab11A through its DENN domain also recognized an unspecific nuclear signal, as it was still present in Thereare severalpiecesofevidencesuggestingthatFLCNcaninteract the FLCN−/− cells (Fig. S2). A similar result has been reported by with Rab11A. First, FLCN contains a DENN-like domain, and another research group using the same anti-FLCN antibody (Tsun researchers have long suspected that FLCN can bind Rab GTPases and et al., 2013). In the double immunostaining experiments, we modulate their activities (Nookala et al., 2012). Second, both FLCN detected certain overlaps between FLCN and all three Rab proteins (Wu et al., 2016) and Rab11A (Fig. 2A,C) can inhibit the localization in the cytoplasm (Fig. 4A). A quantification assay (the nuclear of overexpressed PAT1 to the lysosome. Third, FLCN was found to signals were excluded) further revealed that FLCN signals overlap bind the cell–cell adhesion protein PKP4 (Medvetz et al., 2012; more with those of GFP-Rab7A and GFP-Rab11A than with those Nahorski et al., 2012), which is known to be a Rab11A-ineracting of GFP-Rab5. factor (Keil and Hatzfeld, 2014). On the basis of these results, we Next, we performed co-immunoprecipitation experiments (co-IP) decided to explore the potential FLCN-Rab11A interaction. to study the physical interaction between FLCN and Rab11A. To First, we wondered whether FLCN can be localized to the this end, we transiently expressed GFP-Rab5A, GFP-Rab7A or Rab11A-marked endosomes. We transiently expressed GFP- GFP-Rab11A in HEK293 cells, and incubated the cell lysates with Rab5A, GFP-Rab7A and GFP-Rab11A in HEK293 cells, and anti-FLCN antibody. Immunoprecipitates were then examined by performed double immunostaining experiments using antibodies western blotting. We found that FLCN interacts with GFP-Rab7A or against GFP and FLCN. We confirmed the specificity of the rabbit GFP-Rab11A but not (or very weakly) with GFP-Rab5A (Fig. 4B). monoclonal anti-FLCN antibody by both western blotting and Similar results were obtained by using the mouse proteins in mouse immunostaining assays (Fig. S2). Notably, this anti-FLCN antibody 3T3 embryonic fibroblast cells (Fig. 4C). Because FLCN has

Fig. 4. FLCN interacts with Rab11A through its DENN domain. (A) HEK293 cells were stained with endogenous FLCN (red) and the GFP-tagged Rabs (green). Note there is an unspecific signal of FLCN in the nuclei (Okimoto et al., 2004; Tsun et al., 2013). In the quantification assays, the nuclear signals were excluded, n>30 cells. Scale bars, 10 μm. (B,C) Indicated cDNAs were transiently expressed in HEK293 (B) or mouse 3T3 cells (C). Cell lysates were immunoprecipitated with the anti-FLCN antibody, followed by western blotting analyses with the indicated antibodies. The empty vector expressing GFP protein (GFP+) was taken as a negative control. (D) GFP-Rab11A and HA-tagged FLCN fragments were transiently expressed, followed by IP using HA antibody. Notice that the FLCN DENN domain is responsible to bind GFP-Rab11A. (E) FLCN interacts with GFP-Rab11B in HEK293 cells. GFP-Rab11B cDNA was transiently expressed in HEK293 cells, followed by IP using anti-FLCN antibody. Journal of Cell Science

6 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218792. doi:10.1242/jcs.218792 recently been found to interact with Rab7A in several human tumor be involved in signal transduction through Rag GTPases (Petit et al., cell lines (Laviolette et al., 2017), we considered the FLCN-Rab7A 2013; Tsun et al., 2013), the signal maintenance function (by FLCN interaction as a positive control in our co-IP experiments. It is and PAT1) must, therefore, lie upstream of the FLCN-mediated probably worth noting that, on the basis of these co-IP data; it seems signal transduction step. that only a small proportion of GFP-tagged Rab proteins co- immunoprecipitated with FLCN. To gain more evidence regarding FLCN promotes the interaction between PAT1 and Rab11A the interaction between FLCN and Rab11A, we tried to map the The next question was how FLCN promotes Rab11A-mediated protein motif(s) that mediate the binding of FLCN to Rab11A. We recycling transport. We think this can be achieved by using one of the found that FLCN preferentially binds to Rab11A through the DENN following two mechanisms: either FLCN can activate Rab11A by domain located at its C-terminus (Fig. 4D). Together, these results functioning as a GEF, or FLCN promotes the cargo loading process. suggested that FLCN physically interacts with Rab11A. By using purified proteins, Tom Blundell’s group previously To examine the specificity of the interaction between FLCN and reported that FLCN does not show clear in vitro GEF activity on Rab11A, we performed co-IP experiments to study whether FLCN Rab11A (Nookala et al., 2012). We performed the similar experiment can interact with Rab11B – another Rab11 family protein that is using His-tagged FLCN and His-tagged Rab11A expressed in mainly expressed in neuronal cells (Lai et al., 1994). Surprisingly, bacteria, and obtained the same result (our unpublished observation). we found that FLCN co-immunoprecipitated with GFP-Rab11B Generally, GEF factors have high affinities with GDP-loaded Rab when transiently expressed in HEK293 cells (Fig. 4E). This proteins. We checked the binding of FLCN to different forms of suggests that FLCN is a common effector of Rab11A and Rab11A Rab11A and found that FLCN preferentially binds Rab11A-CA, i.e. (Horgan and McCaffrey, 2009). GTP-loaded Rab11A (Fig. 6A). Thus, FLCN is probably not a strong GEF of Rab11A. However, FLCN might possess certain GEF FLCN modulates the distribution of PAT1 on the lysosome activity on Rab11A under special conditions. More sensitive and the plasma membrane analyses, such as using FLCN-containing protein complexes The above results led us to speculate that FLCN can regulate the isolated from mammalian cells, in both normal and starvation Rab11A-mediated transport pathway. To test this, we used environments, should be carried out to clarify this issue. overexpressed PAT1 as a substrate and asked whether FLCN can We also carried out experiments to explore whether FLCN can regulate PAT1 localization. interfere with loading of PAT1 on Rab11A, and first checked the Consistent with the previous observations (Wu et al., 2016), we interactions of overexpressed PAT1 with different Rab proteins. As a found that suppression of FLCN by siRNA (siFLCN) increased result, we found that PAT1-FLAG can interact with GFP-Rab5A, localization of PAT1-FLAG on the lysosome (Fig. 5A). GFP-Rab7A and GFP-Rab11A, but not with the control GFP protein Interestingly, this result was accompanied by a decrease of (Fig. 6B). These results support our previous immunostaining data, PAT1-FLAG on the plasma membrane (Fig. 5B). To confirm this which showed that the overexpressed PAT1 localizes to endosomes finding, we generated FLCN-knockout HEK293T cell lines marked with Rab5A, Rab7A or Rab11A (Fig. 1A,B). Next, we (FLCN−/−) by using CRISPR/Cas9-mediated gene-editing checked whether FLCN controls the interaction between PAT1- technique (Fig. S3). Similar to the siFLCN results, we found FLAG and the Rab proteins. Interestingly, we found that interaction PAT1-FLAG was increased on the lysosome but decreased on the between PAT1-FLAG and GFP-Rab11Awas clearly decreased in the plasma membrane in the FLCN−/− cells (Fig. 5C,D). In double FLCN−/− cells. In contrast, FLCN deficiency did not disturb the immunostaining experiments (Fig. 5E), we found the overlaps interaction between PAT1-FLAG and either GFP-Rab5A or GFP- between Myc-PAT1 and LAMP1 were increased by FLCN siRNA, Rab7A (Fig. 6C). Consistent with a role of FLCN in cargo loading, but were decreased by FLCN overexpression (FLCN-HA). Thus, FLCN preferentially bound to Rab11A-CA (Fig. 6A). In an attempt FLCN and Rab11A have the similar influences on the subcellular to check the specificity of this FLCN function, we performed co-IP distribution of overexpressed PAT1. experiments to examine the interaction of Rab11A with transferrin We suspected that FLCN can interfere with overexpressed PAT1 receptor (TfR), which is constitutively recycled by the Rab11A- to regulate mTORC1, as Rab11A does. As shown in Fig. 5F, mediated pathway. Surprisingly, we found that FLCN can interact mTORC1 was downregulated in starved PAT1-overexpressing cells with TfR. Moreover, the interaction between TfR and Rab11A was after 10 min of nutrient stimulation treatment (Fig. 5F, lanes 1, 2). decreased in the absence of FLCN (Fig. 6D). Thus, FLCN promotes Interestingly, mTORC1 activity was further decreased when FLCN binding of Rab11A to its cargos, including PAT1 and TfR. was also suppressed (lane 3) but increased when FLCN was overexpressed (lane 4). These results demonstrate that an increase of DISCUSSION FLCN levels can antagonize overexpressed PAT1 to regulate Previously, a structural study revealed a DENN-like domain at the mTORC1. Given that FLCN overexpression was unable to C-terminus of FLCN. This led to the hypothesis that FLCN binds stimulate mTORC1 the wild-type cells (Fig. 5F, right panel), we Rab GTPases and regulates vesicular trafficking processes (Nookala suspected the influence of PAT1 on mTORC1 to be sensitive to the et al., 2012). In support of this view, three recent reports have shown level of FLCN. Similar observations have been reported by us that FLCN interacts through its DENN domain with Rab34, Rab7A previously, when we found FLCN and overexpressed PAT1 and Rab35 (Starling et al., 2016; Laviolette et al., 2017; Zheng et al., antagonized each other during mTORC1 regulation (fig. 4 in Wu 2017). In one study, Dodding’s group discovered that the FLCN- et al., 2016). On the basis of these results, we propose that FLCN Rab34 interaction regulates the intracellular movement of promotes Rab11A-mediated recycling of PAT1. We also lysosomes in a nutrient-sensitive manner in HeLa cells (Starling investigated how FLCN regulates mTORC1 in the absence of et al., 2016). In another study, Iliopoulos’s group reported that PAT1 (Fig. 5G) and found that compared with suppression of FLCN FLCN is a GAP of Rab7A and accelerates the lysosome-mediated alone, double-suppression of PAT1 and FLCN (siPAT1+siFLCN) degradation of the EGFR protein in several human tumor cell lines did not further decrease mTORC1 activity. However, we do not (Laviolette et al., 2017). Zheng et al. also found that FLCN consider this result to be very surprising because FLCN is known to promotes degradation of EGFR in both HeLa and HEK293T cells, Journal of Cell Science

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Fig. 5. FLCN regulates the intracellular distribution of overexpressed PAT1. (A-D) Loss of FLCN by using siRNA (A,B) or knockout (C,D; FLCN−/−) increased the lysosomal PAT1 (A,C) and decreased the plasma membrane-bound PAT1 (B,D). All cells were transiently transfected with PAT1-3×FLAG and co-transfected with siRNAs (A,B) for 36 h. Then the cells were starved for 1 h and re-stimulated with complete medium for 30 min. The siFLCN has been used in other studies (Petit et al., 2013; Wu et al., 2016). (E) Colocalization of Myc-PAT1 and LAMP1 was increased by FLCN knockdown (siFLCN) and decreased by FLCN overexpression (FLCN-HA). Confocal microscopy images of cells co-stained with Myc (PAT1, green) and LAMP1 (red). Scale bars: 10 μm. (F) The influence of PAT1 on mTORC1 is sensitive to the FLCN level. Cells were transfected with the indicated DNAs for 36 h, followed by starvation and 10 min of nutrient stimulation. Left panel, overexpression of Myc-PAT1 decreased the mTORC1 activity (lanes 1, 2), which was further decreased by siFLCN (lane 3) but was reversed by FLCN overexpression (FLCN-HA, lane 4). Right panel, overexpression of FLCN did not stimulate mTORC1 in the wild-type cell background. (G) HEK293 cells were transfected with indicated siRNAs for 48 h, followed by starvation (50 min) and 10 min of nutrient replenishment. Notice that, compared with siFLCN alone, double suppression of PAT1 and FLCN did not further decrease mTORC1, suggesting the FLCN-mediated signal transduction function is epigenetic to its putative role in signal maintenance. *P<0.05; **P<0.01; ***P<0.001. Error bars in graphs indicate the s.e.m. although, in this case, FLCN was found to be a GEF of Rab35 the late endosome/lysosome (Rab7) or to the fast recycling (Zheng et al., 2017). It is not clear yet how FLCN promotes EGFR endosomes (Rab35). Theoretically, inactivation of Rab7A or degradation in the lysosome through Rab7 and Rab35, because both activation of Rab35 (through FLCN) should inhibit transport of proteins are known to promote the transport of membrane proteins to EGFP to the lysosome. Journal of Cell Science

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Fig. 6. FLCN promotes Rab11A-PAT1 interaction. (A) FLCN preferentially binds Rab11A-CA. The different forms of GFP-Rab11A were transiently expressed in cells stably expressing FLCN-HA for 36 h. The cell lysates were immunoprecipitated with anti-GFP antibody, followed by western blotting. (B) PAT1 binds to Rab5A, 7A and 11A. HEK293 cells were co-transfected with PAT1-3×FLAG and the indicated cDNAs. GFP (lane 1; ∼25 kDa) was taken as a negative control. Notice that PAT1-3×FLAG co-IP with all three Rabs and FLCN, but not GFP (lane 1). (C) Interaction between PAT1 and Rab11A was decreased in FLCN−/− cells. Wild-type 293T (FLCN+) and FLCN−/− cells were transfected with PAT1-3×FLAG and the indicated Rabs. Lysates were precipitated with FLAG (PAT1), followed by western blotting. Notice, in FLCN−/− cells, interactions of PAT1-Rab5A and PAT1-Rab7A stayed the same. (D) Interaction between TfR and Rab11A was decreased in FLCN−/− cells. 293T (FLCN+) or FLCN−/− cells were transfected with GFP-Rab11A. Lysates were immunoprecipitated with IgG (negative control) or anti-TfR antibody. Notice that TfR immunoprecipitated together with FLCN in 293T cells, but this interaction was decreased in the FLCN−/− cells. *P<0.05; ***P<0.001.

In our study here, we have demonstrated that FLCN is a so-far- therefore, suspect that FLCN is likely to require other effectors in unknown Rab11A-interacting protein and that this interaction also order to interact with Rab11A. requires the FLCN DENN domain. To explore the molecular Since FLCN can interact with several Rab proteins, FLCN might mechanism(s) of this interaction, we carried out in vitro GEF inhibit localization of PAT1 to the lysosome through several activity assays by using bacterium-expressed proteins – but no clear mechanisms. For example, FLCN has been found to be a GAP of GEF activity of FLCN on Rab11A was detected. The same result Rab7A (Laviolette et al., 2017). Thus, FLCN might inhibit the has also been reported by Blundell’s group in a previous study transport of PAT1 to the lysosome through inactivating Rab7. In (Nookala et al., 2012). On the basis of these results, it seems that support of this, we found that overexpressed PAT1 has close FLCN does not modify Rab11A activity directly. By using relationships with Rab7A – as revealed by both immunostaining overexpressed PAT1 as a substrate, we found that FLCN promotes (Fig. 1A,B) and co-immunoprecipitation experiments (Fig. 6B). the loading of PAT1 on Rab11A and, consistent with this, FLCN Interestingly, loss of FLCN did not affect the interaction between preferentially binds Rab11A-CA (Fig. 6A). These results suggest PAT1 and Rab7A (Fig. 6C). This result, indeed, supports the that FLCN promotes recycling of PAT1 by mediating the interaction proposed mechanism that FLCN directly modulates Rab7A activity between Rab11A and PAT1. This mechanism would also explain (Laviolette et al., 2017). In another possible mechanism, because the FLCN overexpression results. As shown in Fig. 5F, FLCN is a GEF of Rab35 (Zheng et al., 2017) that, in turn, is a overexpression of FLCN displayed a stimulatory effect on regulator of the fast recycling pathway, FLCN promotes the mTORC1 in PAT1-overexpressing cells (lane 4) but not in wild- transport of PAT1 from early endosomes directly to the recycling type cells (lane 7). We think that, when cargo (i.e. PAT1) is endosomes. As a result, the amount of PAT1 destined to the late increased, more FLCN proteins are needed to promote transport. endosome/lysosome will be reduced. The same mechanism has also been observed for Rab34-regulated How PAT1 regulates mTORC1 is still a mystery. For example, localization of lysosomes (Starling et al., 2016). In that study, overexpression of PAT1 has been shown to either activate or Dodding’s group reported that FLCN did not act as a Rab34 GEF inactivate mTORC1 in HEK293 cells. We have recently discovered but, instead, FLCN promoted the binding of active Rab34 to its a mechanism to explain these controversial observations when effector RILP. We carried out the in vitro pull-down assay by using finding that lysosomal PAT1 is increased in response of starvation purified FLCN protein and overexpressed Rab11A, but no direct and decreased when nutrients are replenished (Wu et al., 2016; Zhao interaction was observed (our unpublished observation). We, et al., 2018). After a short term of nutrient exposure (10 min), Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218792. doi:10.1242/jcs.218792 mTORC1 can be reactivated in wild-type cells but not in PAT1- (Beijing, China); mouse monoclonal anti-β-actin (1:1000; KM9001T) overexpressing cells. However, by extending the nutrient antibody was from Sungene Biotech (Tianjin, China); mouse monoclonal stimulation, mTORC1 can be reactivated in PAT1-overexpressing anti-FLAG (M2) antibody was from Sigma-Aldrich (1:1000; St Louis, cells (Zhao et al., 2018). In this current study, we mainly used MO); mouse monoclonal anti-Rab11 antibody (1:400; #610656) was the PAT1 overexpression system to display the significance of the from BD Biosciences (Franklin Lakes, NJ); rabbit polyclonal anti-Rab11 (1:400; #71-5300) and anti-GFP (1:1000; #A11122) antibodies, and all interaction between Rab11A and FLCN. Consistent with a role of fluorescent secondary antibodies were purchased from Life Technology FLCN during the Rab11A-mediated recycling transport, we found (Carlsbad, CA). that both FLCN and Rab11A can counteract the inhibitory effect of The human FLCN cDNA was kindly provided by Dr L. S. Schmidt (NIH PAT1 overexpression on mTORC1. Although our data support a at Bethesda, MD). The cDNAs of PAT1, Rab5A, Rab7A, Rab11A and negative role of lysosomal PAT1 on mTORC1, we think the LAMP1 were derived from HEK293 or mouse 3T3 cells. For ectopic mTORC1 readout is determined by the relative level of lysosomal expression, the cDNAs were cloned into either pEGFP-C1 (with N-terminal PAT1 (compared with that of total PAT1), because the PAT1 EGFP tag) or pcDNA3.1(+). The resulting constructs were confirmed localizing elsewhere, for example, to the plasma membrane, may by sequencing. have different influences. This view is supported by the finding that overexpression of PAT1 does not necessarily inhibit mTORC1 Cell culture, transfection and generation of stable cell lines Cells were normally cultured in complete medium: Dulbecco’s modified under the normal culture conditions or following extended nutrient Eagle’s medium (DMEM) supplemented with 8% fetal bovine serum replenishment (Zhao et al., 2018). Notably, knockdown of PAT1 by (FBS), 4 mM L-glutamine, 4500 mg/l glucose, and sodium pyruvate. using RNAi can also inhibit mTORC1. To explain this, we suspect For starvation, cells were cultured in complete medium and allowed to that PAT1 knockdown decreases the amino acids input into the grow up to ∼80% confluence. Then, cells were washed with PBS twice, cytosol – from either the environment or the lysosomal lumen, or followed by incubation with RPMI1640 medium lacking both amino acids both. Eventually, this will lead to a reduction of the cytosolic amino and serum (US Biological, R8999-04A) for the indicated period of time. acid pool, which is another important signal of mTORC1 (Zhao Plasmids were introduced into cells using TurboFect transfection reagent et al., 2018). However, we were unable to exclude another (Life Technology). Stable cell lines were selected with G418 (Life possibility, i.e. that PAT1 can positively regulate mTORC1 as s Technology). RNAiMax diluted in OptiMEM (Life Technology) was ′ ′ signal transducer (through Rags) – as suggested by Ögmundsdottir used to deliver the siRNA 5 -GAUAAAGAGACCUCCAUUATT-3 targeting FLCN or the siRNA 5′-AUGCUCCCAUCUUCAUCAATT-3′ et al., 2012. To test this hypothesis, more careful analyses should be targeting PAT1, both of which have been described previously (Petit et al., carried out. Besides, due to the lack of a good anti-PAT1 antibody, it 2013; Wu et al., 2016), into the cells according to the manufacturer’s is still unclear whether endogenous PAT1 exhibits dynamic instructions; shRNAs were cloned into pCD513B-U6 vector and co- lysosomal localizations in response to nutrient fluctuation and transfected into the cells with the helper plasmids (GAG, REV and VSV-G). alterations of Rab11A and FLCN activities. Therefore, it should be Purified and concentrated virus was used to infect cells, which were then noticed that the PAT1 overexpression system does not necessarily selected by using their resistance against puromycin. The following two reflect the in vivo situation. RNAi target sequences of Rab11A were used: #1, 5′-GTAACCTCCTG- FLCN has been found to promote the amino acid signal TCTCGATTTAC-3′ and #2, 5′-GGAGTAGAGTTTGCAACAAGA-3′. transduction through Rags (Petit et al., 2013; Tsun et al., 2013). On the basis of our previous findings (Wu et al., 2016) and the results Generation of FLCN knockout cells shown in this study, we propose that FLCN has another function by We designed two different CRISPR sites on the coding region of the human FLCN genomic locus. The double-strand CRISPR fragments were cloned maintaining the amino acid signal level within the lysosome through into pX330 plasmid. By using one of the targets 5′-GCGTCGAGTCC- amino acid transporters, such as PAT1. Interestingly, this transport AGCAGCCCG-3′, we obtained two independent mutant clones in a function of FLCN seems to be conserved during evolution. For HEK293T background. The mutations were confirmed by both genome example, in yeast, the transport of the general amino acid permease sequencing and western blotting assays (Fig. S3). Gap1p to the vacuole (lysosome) is inhibited by Lst7 (Roberg et al., 1997), an ortholog of FLCN. In addition, Gap1p can be transported Immunofluorescence staining between the cell surface and the lysosome, and this translocation is Cells were fixed with 4% formaldehyde in PBS for 20 min, rinsed once with controlled by several components of the mTORC1 signaling PBS and permeabilized by incubation with PBST for 5 min (0.1% Triton pathway, including LST7 (FLCN), LST4 (FNIP1), LST8 X-100 in PBS). Incubation with either primary or secondary antibody (a component of mTORC1/2), Gtr1/2 (Rag GTPases) and SEC13 was performed at room temperature for 2 h or at 4°C overnight. Nuclei were counterstained with DAPI. Images were captured by using a confocal (a component of the GAP complex targeting the Rag GTPases) microscope (Nikon A1R-si). For colocalization assays, two-channel stacks (Bar-Peled et al., 2013; Roberg et al., 1997). The relationship of each image were analyzed by using the Nikon NIS-Element confocal between trafficking of amino acid transporters and signal microscope program and the Pearson’s correlation coefficient was transduction processes deserves further investigations. calculated. At least 30 cells from three repeated experiments were analyzed in each assay with one way ANOVA followed by Fisher’s least MATERIALS AND METHODS significant difference test (Fisher’s LSD) and using SPSS software (20.0, Antibodies and plasmids SPSS, Inc., Chicago, IL). Antibodies against pS6K1 (1:1000; T389, #9205), S6K1 (1:1000; #9202), FLCN (1:1000; #3697) and Rab5 (1:1000; #3547) were obtained from Cell Lysosome purification and cell surface biotinylation Signaling Technology (Danvers, MA); against Rab7 (1:200; B-3), TGN38 Lysosomes were isolated using LYSISO1 (Sigma-Aldrich). Briefly, the (1:200; B-6), Chmp5 (1:200; F-7), pan-cadherin (1:200; CH-19) and TfR cells were collected by centrifugation at 600 g for 5 min (∼1.5×108 cells in (1:1000; 3B82A1) from Santa Cruz Biotechnology (Santa Cruz, CA); total). The following steps were carried out on ice: cells were suspended in against EEA1 (1:200; 1FB), M6PR (1:200; 22d4) and LAMP1 (1:200; 200 µl of extraction buffer and homogenized with five gentle strokes in a H4A3) from Developmental Studies Hybridoma Bank (Iowa City, IA); and 2 ml dounce glass tissue grinder. After centrifugation at 1000 g for 10 min, against rabbit polyclonal HA (1:500; #9110) and Myc (1:300; #9106) from the supernatant was collected, and the pellet was homogenized in four more Abcam (Cambridge, UK). Mouse monoclonal anti-GFP (1:200; HT801) rounds. The supernatants from all homogenates were then collected and and anti-Myc (1:200, HT101) antibodies were from Transgen Biotech pooled (∼1 ml) and centrifuged at 20,000 g for 20 min. The pellet was Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218792. doi:10.1242/jcs.218792 re-suspended in 50 µl of RIPA lysis buffer, yielding the lysosome fraction. (2013). A tumor suppressor complex with GAP activity for the rag GTPases that Labeling of membrane proteins with biotin was performed as described signal amino acid sufficiency to mTORC1. Science 340, 1100-1106. elsewhere (Okimoto et al., 2004; Tsun et al., 2013); ∼1.5×108 cells Birt, A. R., Hogg, G. R. and Dube, W. J. (1977). Hereditary multiple fibrofolliculomas with trichodiscomas and acrochordons. Arch. Dermatol. 113, (3×90 mm dishes) were used. 1674-1677. To measure the PAT1 signal intensities on the lysosome and cell surface, Braulke, T. and Bonifacino, J. S. (2009). Sorting of lysosomal proteins. Biochim. we used the following two calculation methods (1) [lysosome (PAT1)/ Biophys. Acta 1793, 605-614. lysosome (LAMP1)]/[lysate (PAT1)/lysate (LAMP1)] and (2) [surface Chen, Z., Fei, Y.-J., Anderson, C. M., Wake, K. A., Miyauchi, S., Huang, W., (PAT1)/surface (cadherin)]/[lysate (PAT1)/lysate (cadherin)]. Thwaites, D. T. and Ganapathy, V. (2003). Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2. J. Physiol. 546, 349-361. Western blotting Chen, J., Futami, K., Petillo, D., Peng, J., Wang, P., Knol, J., Li, Y., Khoo, S.-K., Cells were washed once in ice-cold 1×PBS, harvested and lysed with the Huang, D., Qian, C.-N. et al. (2008). Deficiency of FLCN in mouse kidney led to RIPA lysis buffer. The lysates were cleared by centrifugation at 12,000 g for development of polycystic kidneys and renal neoplasia. PLoS ONE 3, e3581. 15 min at 4°C and then were mixed with SDS loading buffer. After that, Cougoule, C., Carreno, S., Castandet, J., Labrousse, A., Astarie-Dequeker, C., samples were either boiled for 5 min or kept at 4°C overnight. The protein Poincloux, R., Le Cabec, V. and Maridonneau-Parini, I. (2005). Activation of the concentration was measured by BCA assay. For western blotting, the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes. Traffic 6, 682-694. samples were separated by SDS-PAGE and transferred to PVDF membrane, Dorn, M., Weiwad, M., Markwardt, F., Laug, L., Rudolph, R., Brandsch, M. and blocked in 5% non-fat milk, and incubated with primary antibodies Bosse-Doenecke, E. (2009). Identification of a disulfide bridge essential for followed by incubation with the HRP-conjugated secondary antibodies. transport function of the human proton-coupled amino acid transporter hPAT1. Immunoreactivity was detected by using ECL and chemiluminescence J. Biol. Chem. 284, 22123-22132. reagents (Bio-Rad, Hercules, CA). Western blot data were quantified by Gharbi, H., Fabretti, F., Bharill, P., Rinschen, M. M., Brinkkötter, S., Frommolt, using the Bio-Rad Quantity One software. For quantification assays, we P., Burst, V., Schermer, B., Benzing, T. and Müller, R.-U. (2013). Loss of the used Student’s t-test with Statistical Package for the Social Sciences (SPSS) Birt-Hogg-Dube gene product folliculin induces longevity in a hypoxia-inducible P factor-dependent manner. Aging Cell 12, 593-603. software (Version 20.0; SPSS, Inc., Chicago, IL). <0.05 was considered Heublein, S., Kazi, S., Ögmundsdóttir, M. H., Attwood, E. V., Kala, S., Boyd, significant. All error bars represent the s.e.m. of at least three repeated C. A. R., Wilson, C. and Goberdhan, D. C. I. (2010). Proton-assisted amino-acid experiments. transporters are conserved regulators of proliferation and amino-acid-dependent mTORC1 activation. Oncogene 29, 4068-4079. Co-immunoprecipitation Horgan, C. P. and McCaffrey, M. W. (2009). The dynamic Rab11-FIPs. Biochem. Soc. Trans. 37, 1032-1036. Cells were rinsed once with ice-cold PBS and lysed with NP40 lysis buffer Jensen, A., Figueiredo-Larsen, M., Holm, R., Broberg, M. L., Brodin, B. and (0.5% NP-40, 25 mM Tris, 200 mM NaCl, 200 mM KCl, 1.5 mM MgCl2, Nielsen, C. U. (2014). PAT1 (SLC36A1) shows nuclear localization and affects 0.5 mM PMSF, 1 mM EDTA, 5% glycerol, pH 7.4), supplemented with growth of smooth muscle cells from rats. Am. J. Physiol. Endocrinol. Metab. 306, protease inhibitor cocktail (Roche, Mannheim, Germany), followed by E65-E74. centrifugation at 12,000 g for 20 min at 4°C. Supernatants were added to Ji, X., Zhao, L., Luo, H., Zhang, X., Jin, Y. and Liu, W. (2017). Amino acids either anti-HA polyclonal, anti-FLCN monoclonal, anti-Tfr monoclonal or suppress the expression of PAT1 on lysosomes via inducing the cleavage of a anti-FLAG monoclonal antibody, and immunoprecipitated with Protein targeting signal. FEBS Lett. 591, 2279-2289. Keil, R. and Hatzfeld, M. (2014). The armadillo protein p0071 is involved in Rab11- A/G Agarose beads (GE Healthcare, Pittsburgh, PA) at 4°C for 2 h. In the dependent recycling. J. Cell Sci. 127, 60-71. quantification assays, the following calculation method was used: Kenyon, E. J., Luijten, M. N. H., Gill, H., Li, N., Rawlings, M., Bull, J. C., [IP(Rabs)/IP(PAT1 or TfR)]/[lysate(Rabs)/ lysate (PAT1 or TfR)], n=3 Hadzhiev, Y., van Steensel, M. A. M., Maher, E. and Mueller, F. (2016). repeated experiments. Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis. BMC Dev. Biol. 16, 23. Acknowledgements Lai, F., Stubbs, L. and Artzt, K. (1994). Molecular analysis of mouse Rab11b: a We thank all the lab members for discussions about the manuscript. new type of mammalian YPT/Rab protein. Genomics 22, 610-616. Laviolette, L. A., Mermoud, J., Calvo, I. A., Olson, N., Boukhali, M., Steinlein, O. K., Roider, E., Sattler, E. C., Huang, D., Teh, B. T. et al. (2017). Negative Competing interests regulation of EGFR signalling by the human folliculin tumour suppressor protein. The authors declare no competing or financial interests. Nat. Commun. 8, 15866. Liu, W., Chen, Z., Ma, Y., Wu, X., Jin, Y. and Hou, S. (2013). Genetic Author contributions characterization of the Drosophila birt-hogg-dubésyndrome gene. PLoS ONE Conceptualization: W.L.; Methodology: L.Z., X.J., X.Z., W.L.; Validation: L.Z., W.L.; 8, e65869. Formal analysis: L.Z., X.J., X.Z., W.L.; Investigation: L.Z., X.J., X.Z., L.L.; Resources: Luo, H., Zhao, L., Ji, X., Zhang, X., Jin, Y. and Liu, W. (2017). Glycosylation affects Y.J.; Data curation: L.Z., X.J.; Writing - original draft: L.Z., W.L.; Writing - review & the stability and subcellular distribution of human PAT1 protein. FEBS Lett. 591, editing: W.L.; Supervision: Y.J., W.L.; Project administration: W.L.; Funding 613-623. acquisition: W.L. Medvetz, D. A., Khabibullin, D., Hariharan, V., Ongusaha, P. P., Goncharova, E. A., Schlechter, T., Darling, T. N., Hofmann, I., Krymskaya, V. P., Liao, J. K. Funding et al. (2012). Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion. This work was supported by a grant from the National Natural Science Foundation of PLoS ONE 7, e47842. China (31372256) to W.L. Nahorski, M. S., Seabra, L., Straatman-Iwanowska, A., Wingenfeld, A., Reiman, A., Lu, X., Klomp, J. A., Teh, B. T., Hatzfeld, M., Gissen, P. et al. (2012). Supplementary information Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with Supplementary information available online at disordered RhoA signalling, epithelial polarization and cytokinesis. Hum. Mol. http://jcs.biologists.org/lookup/doi/10.1242/jcs.218792.supplemental Genet. 21, 5268-5279. Nookala, R. K., Langemeyer, L., Pacitto, A., Ochoa-Montano, B., Donaldson, References J. C., Blaszczyk, B. K., Chirgadze, D. Y., Barr, F. A., Bazan, J. F. and Blundell, Anderson, C. M., Grenade, D. S., Boll, M., Foltz, M., Wake, K. A., Kennedy, D. J., T. L. (2012). Crystal structure of folliculin reveals a hidDENN function in Munck, L. K., Miyauchi, S., Taylor, P. M., Campbell, F. C. et al. (2004). H genetically inherited renal cancer. Open Biol. 2, 120071. +/amino acid transporter 1 (PAT1) is the imino acid carrier: an intestinal nutrient/ Ögmundsdottir, M. H., Heublein, S., Kazi, S., Reynolds, B., Visvalingam, S. M., drug transporter in human and rat. Gastroenterology 127, 1410-1422. Shaw, M. K. and Goberdhan, D. C. I. (2012). Proton-assisted amino acid Baba, M., Toyama, H., Sun, L., Takubo, K., Suh, H.-C., Hasumi, H., Nakamura- transporter PAT1 complexes with Rag GTPases and activates TORC1 on late Ishizu, A., Hasumi, Y., Klarmann, K. D., Nakagata, N. et al. (2016). Loss of endosomal and lysosomal membranes. PLoS ONE 7, e36616. folliculin disrupts hematopoietic stem cell quiescence and homeostasis resulting Okimoto, K., Sakurai, J., Kobayashi, T., Mitani, H., Hirayama, Y., Nickerson, in bone marrow failure. Stem Cells 34, 1068-1082. M. L., Warren, M. B., Zbar, B., Schmidt, L. S. and Hino, O. (2004). A germ-line Bar-Peled, L., Chantranupong, L., Cherniack, A. D., Chen, W. W., Ottina, K. A., insertion in the Birt-Hogg-Dube (BHD) gene gives rise to the Nihon rat model of

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