Research Article 459 LAPTMs regulate lysosomal function and interact with mucolipin 1: new clues for understanding mucolipidosis type IV

Silvia Vergarajauregui, Jose A. Martina and Rosa Puertollano* Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA *Author for correspondence ([email protected]) Accepted 8 October 2010 Journal of Cell Science 124, 459-468 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.076240

Summary Loss-of-function mutations in mucolipin 1 (MCOLN1) result in mucolipidosis type IV (MLIV), a lysosomal storage disorder characterized by severe mental and psychomotor retardation. MCOLN1 is a lysosomal that belongs to the transient receptor potential (TRP) superfamily. To better understand the cellular function of MCOLN1, a split-ubiquitin yeast two-hybrid screen was performed with the purpose of revealing new MCOLN1 interaction partners. The screen identified two members of the lysosome- associated transmembrane (LAPTM) family as novel interaction partners of MCOLN1. The binding between MCOLN1 and LAPTM members (LAPTMs) was confirmed by co-immunoprecipitation and yeast two-hybrid assays. In addition, MCOLN1 and LAPTMs extensively colocalize at late endosomes and lysosomes. Overexpression of LAPTM4b caused enlargement of lysosomes and defective lysosomal degradation, indicating that LAPTMs are important for proper lysosomal function. Interestingly, lysosomal swelling induced by LAPTM4b was rescued by expression of MCOLN1, suggesting a functional connection between the two . Finally, depletion of endogenous LAPTMs by siRNA induced accumulation of concentric multi-lamellar structures and electron-dense inclusions that closely resemble the structures found in MLIV cells. Overall, our data provide new insight into the molecular mechanisms of MCOLN1 function and suggest a potential role for LAPTMs in MLIV pathogenesis.

Key words: Mucolipin, LAPTM, TRPML, MLIV, Lysosomes

Introduction through interactions with clathrin adaptors (Miedel et al., 2006; Mucolipidosis type IV (MLIV) is an autosomal recessive disorder Pryor et al., 2006; Vergarajauregui and Puertollano, 2006).

Journal of Cell Science characterized by severe neurologic and ophthalmologic MCOLN1 undergoes several post-translational modifications abnormalities (Altarescu et al., 2002; Amir et al., 1987; Bach, that regulate its function. For example, palmitoylation and 2001). Symptoms appear during the first year of life and include phosphorylation at the C-terminal tail modulate trafficking and intellectual disabilities, psychomotor delay, weak muscle tone channel activity, respectively, whereas cleavage at the first luminal (hypotonia), corneal clouding and progressive retinal degeneration loop inactivates the protein (Kiselyov et al., 2005; Vergarajauregui (Berman et al., 1974; Riedel et al., 1985). In addition, MLIV et al., 2008b; Vergarajauregui and Puertollano, 2006). patients show impaired secretion of gastric acid (achlorhydria) According to previous reports, analysis of fibroblasts from MLIV leading to defective iron absorption and anemia (Schiffmann et al., patients by electron microscopy revealed the accumulation of 1998). By their early teens, most affected individuals are unable to enlarged vacuolar structures that contain mucopolysaccharides and walk, present limited or no ability to speak and have severe vision lipids forming characteristic multiconcentric lamellae (Bach et al., loss or blindness. 1975; Bach et al., 1977; Crandall et al., 1982; Tellez-Nagel et al., MLIV is caused by loss-of-function mutations in MCOLN1 1976). These enlarged vacuoles are present not only in fibroblasts (also referred to as TRPML1), an ion channel that belongs to the but in every tissue and organ of MLIV patients, suggesting a TRP superfamily (Bargal et al., 2000; Bassi et al., 2000; general impairment of lysosomal function. Accumulation of Slaugenhaupt et al., 1999; Sun et al., 2000). MCOLN1 is a protein defective lysosomes appears to affect other cellular processes such with six transmembrane-spanning domains with both amino- and as . Increased levels of insoluble p62, ubiquitinated C-terminal tails oriented toward the cytosol and the pore located aggregates and autophagosomes have been observed in MLIV between transmembrane segments 5 and 6. Electrophysiological fibroblasts, indicating that autophagy-mediated clearance of harmful studies indicate that MCOLN1 is an inwardly (from lumen to cellular products is impaired in MLIV patients (Vergarajauregui et cytoplasm) rectifying channel permeable to Ca2+, Na+, K+ and Fe2+ al., 2008a). Generation of two different MLIV animal models in or Mn2+, and whose activity is modulated by pH and Ca2+ (Cheng Drosophila and mouse confirmed that absence of MCOLN1 results et al., 2010; Puertollano and Kiselyov, 2009). However, the in defective autophagy (Micsenyi et al., 2009; Venkatachalam et selectivity and mechanisms of activation of MCOLN1 under al., 2008). However, these observations are based on studies physiological conditions remain unknown. characterizing cellular effects resulting from the loss of MCOLN1. MCOLN1 is localized at late endosomes and lysosomes. Two Thus, it is unclear whether the observed phenomena directly result acidic di-leucine consensus motifs located at the N- and C-terminal from the absence of MCOLN1 or whether they are a secondary tails regulate trafficking of MCOLN1 to the late endosomal pathway consequence of lipid accumulation in lysosomes. 460 Journal of Cell Science 124 (3)

In order to gain insights into MCOLN1 function, a yeast two- hybrid screen was performed to identify proteins that interact with MCOLN1. Here, we report a novel interaction between MCOLN1 and the members of the LAPTM family. Although the cellular function of LAPTMs is not well understood, it has been suggested that LAPTMs might participate in the transport of small molecules across intracellular membranes (Hogue et al., 1996; Hogue et al., 1999). We found that MCOLN1 and LAPTMs colocalize to late endosomes and lysosomes and confirmed the interaction by co- immunoprecipitation in human cells. Overexpression of LAPTMs caused enlargement of lysosomes and defective lysosomal degradation, whereas depletion of endogenous LAPTMs induced accumulation of concentric multi-lamellar structures and electron- dense inclusions that closely resemble the structures found in Fig. 1. MCOLN1 interacts with the three members of the LAPTM family MLIV cells. Overall, our data provide new insight for understanding in yeast two-hybrid assays. A split-ubiquitin yeast two-hybrid assay was MCOLN1 function and reveal a novel role for LAPTMs in the performed to corroborate the interaction of LAPTM proteins with MCOLN1. regulation of lysosomal function. Interactions were tested by monitoring the growth of yeast cells expressing MCOLN1-Cub or MCOLN3-Cub bait with various Nub-LAPTM fusion Results proteins on agar plates lacking tryptophan and leucine (top), or tryptophan, Identification of LAPTMs as novel MCOLN1 binding leucine and histidine and containing 10 mM 3-aminotriazole (3AT; bottom). partners The vectors pAl-Alg5 and pDL2-Alg5 were used as positive and negative In order to further understand the cellular function of MCOLN1, controls, respectively. we searched for novel binding partners of MCOLN1. Given that MCOLN1 is a highly hydrophobic transmembrane protein that oligomerizes and undergoes post-translational modifications, we FLAG antibody directly coupled to protein-G–agarose beads. Co- used the split-ubiquitin membrane-based yeast two-hybrid system. immunoprecipitation of LAPTMs was detected by immunoblot This system uses the split-ubiquitin approach, in which using polyclonal anti-GFP antibodies. As shown in Fig. 2A, GFP– reconstitution of two ubiquitin halves (Nub and Cub) is mediated LAPTM4b was present in immunoprecipitates of cell lysates by a protein–protein interaction, resulting in the release of a transfected with MCOLN1–FLAG cDNA but not in those collected transcription factor and expression of reporter (Johnsson and after transfection with the FLAG vector alone, suggesting that both Varshavsky, 1994). The advantage of this approach is that it allows proteins interact in vivo. Co-immunoprecipitation experiments also us to use full-length MCOLN1 as bait and reveals interactions that confirmed the interaction of MCOLN1 with GFP–LAPTM4a and take place at the organelle where the protein typically localizes (in GFP–LAPTM5 (Fig. 2A,B). In support of our two-hybrid data, this case the vacuole). MCOLN3 did not interact with LAPTMs (Fig. 2B). Western blot

Journal of Cell Science To generate the MCOLN1 bait, we cloned the full-length human profiles of LAPTM4a, LAPTM4b and LAPTM5 showed the MCOLN1 protein into the pBTE-STE vector, thus generating presence of low molecular mass bands that probably correspond to MCOLN1–Cub. The bait was screened against a human adult proteolytic fragments (Fig. 2, arrows). In addition, MCOLN1 and brain library of cDNAs fused to the mutated form of N-ubiquitin LAPTMs did not interact with LAMP1, another lysosomal in the pPR3-N vector (NubG-x) and was carried out by Dualsystems transmembrane protein used as a negative control (supplementary Biotech AG (Schlieren, Switzerland). Among 277 positive clones material Fig. S1). Overall, our data reveal that MCOLN1 interacts isolated, two independent clones encoded members of a family of with the three members of the LAPTM family both in yeast and endosomal and lysosomal transmembrane proteins named human cells. LAPTMs. The clones included the first 217 amino acids (aa) of LAPTM4a and the N-terminal sequence (aa 27–47) of LAPTM4b, MCOLN1 and LAPTMs colocalize to late endosomes and respectively. Both clones were in-frame with the N-terminal half lysosomes of ubiquitin. The function of LAPTMs is not completely understood Next, we asked whether MCOLN1 and LAPTMs colocalize, which but it has been suggested that they are transporters involved in the would indicate that the proteins have the ability to interact at the subcellular compartmentalization of different compounds (Hogue physiological level. To analyze the colocalization between LAPTMs et al., 1996; Hogue et al., 1999). MCOLN1 protein binding to and MCOLN1, ARPE-19 cells were transiently co-transfected with LAPTMs was confirmed by performing additional yeast two- GFP–LAPTMs and Cherry–MCOLN1 constructs and observed by hybrid experiments. As seen in Fig. 1, MCOLN1 interacted with confocal microscopy. As seen in Fig. 3, all the members of the the three members of the LAPTM family (LAPTM4a, LAPTM4b LAPTM family (LAPTM4a, LAPTM4b and LAPTM5) showed a and LAPTM5). By contrast, MCOLN3, another member of the very high degree of colocalization with MCOLN1. In fact, nearly mucolipin family responsible for the varitint-waddler phenotype in all LAPTM-containing vesicles were also labeled for MCOLN1. mice, did not show any significant binding to LAPTMs (Fig. 1). We have previously described that MCOLN1 localizes to late endosomes and lysosomes and identified two di-leucine-sorting Confirmation of the LAPTM–MCOLN1 interaction by in consensus motifs that mediate the transport of MCOLN1 to late- vivo binding assays endocytic compartments (Vergarajauregui and Puertollano, 2006). To confirm the association between LAPTMs and MCOLN1, we Therefore, these results suggest that the interaction between coexpressed full-length MCOLN1-FLAG and GFP–LAPTMs in MCOLN1 and LAPTMs probably occurs at late endosomes and HeLa cells and performed immunoprecipitation using an anti- lysosomes. These data are also consistent with previous reports Role of LAPTMs in lysosomal function 461

Fig. 2. Physical interaction of MCOLN1 with the three members of the LAPTM family. (A,B)The interaction between MCOLN1 and LAPTM family members was tested by co-immunoprecipitation. HeLa cells were transfected with various combinations of the indicated vectors. FLAG-tagged MCOLN1 and MCOLN3 were immunoprecipitated using the anti-FLAG mAb and samples were assessed by western blotting using anti- FLAG antibody or anti-GFP antibody. The results shown are representative of three independent experiments. The predicted molecular weight is approximately 53.7 kDa for GFP–LAPTM4a, 62 kDa for GFP–LAPTM4b, 56.8 kDa for LAPTM5 and 66 kDa for MCOLN1-FLAG. Arrowheads indicate full-length proteins, arrows correspond to proteolytic fragments and asterisks represent oligomers.

describing the distribution of LAPTM4a and LAPTM5 in late ARPE-19 cells and performed co-staining with different endogenous endosomes and/or lysosomes (Cabrita et al., 1999; Pak et al., markers. As shown in Fig. 4, most of the LAPTM4b-positive vesicles 2006). also contain the late-endosomal and -lysosomal marker CD63. By To examine the subcellular localization of LAPTM4b in more contrast, colocalization of LAPTM4b with early endosomal markers detail and to ensure that its distribution was not altered by such as Hrs was very limited, thus confirming the distribution of coexpression of MCOLN1, we expressed GFP–LAPTM4b alone in LAPTM4b at the late-endocytic pathway (Fig. 4). Journal of Cell Science

Fig. 3. MCOLN1 and LAPTMs colocalize to late endosomes and lysosomes. ARPE-19 cells were transiently transfected with Cherry-MCOLN1 (red) and either GFP–LAPTM4a, GFP–LAPTM4b or GFP–LAPTM5 (green). Twenty hours after transfection, cells were fixed and analyzed by confocal fluorescence microscopy. Insets show a 3- fold magnification of the indicated region. Scale bar: 10mm. 462 Journal of Cell Science 124 (3)

Fig. 4. Distribution of LAPTM4b into late endosomes and lysosomes. ARPE-19 cells transiently transfected with GFP–LAPTM4b were fixed, permeabilized, immunostained with the indicated antibodies and analyzed by confocal fluorescence microscopy. Yellow indicates colocalization between LAPTM4b (green) and CD63 (red), whereas white indicates colocalization of LAPTM4b, Hrs (blue) and CD63. Insets show a 4-fold magnification of the indicated region. Scale bar: 10mm.

LAPTM4b undergoes proteolytic cleavage in late endosomes and lysosomes As mentioned previously, expression of LAPTMs in ARPE-19 cells resulted in two major protein products: the predicted full- Fig. 5. LAPTM4b undergoes proteolytic cleavage following delivery to the lysosomes. ARPE-19 cells were grown on coverslips (A) or plates (B) and Journal of Cell Science length protein and a shorter form that we propose might correspond transfected with a plasmid encoding GFP–LAPTM4b. Six hours after to a proteolytic fragment. To test this hypothesis, we followed the transfection, cells were incubated for an additional 12 hours in the presence of fate of newly synthesized LAPTM4b by performing ‘pulse-chase’ 2mg/ml BFA to accumulate the newly synthesized protein in the ER. BFA was experiments upon treatment with brefeldin A (BFA). This approach then removed and the cells were incubated for the indicated periods of time at has been previously used to monitor the transport of MCOLN1 to 37°C in the absence or presence of 20mM leupeptin. Cells were then fixed, lysosomes (Vergarajauregui and Puertollano, 2006). ARPE-19 cells stained with the late-endosomal and -lysosomal marker CD63 and analyzed by were transfected with GFP–LAPTM4b and, 6 hours after confocal microscopy (A) or lysated and subjected to western blotting (B) to transfection, cells were incubated with BFA for 12 hours to detect GFP–LAPTM4b and actin (loading control). Scale bar: 10mm. accumulate the newly synthesized proteins in the endoplasmic reticulum (ER; pulse) (Lippincott-Schwartz et al., 1989). Proteins LAPTM4b might form homo-oligomers upon reaching the were then released from the ER by incubation in medium without lysosomes. BFA (chase) and cells were analyzed at different time-points by either immunofluorescence or western blot. As presented in Fig. LAPTMs and MCOLN1 are transported to lysosomes 5A, LAPTM4b started showing colocalization with CD63 3 hours independently of each other after BFA removal, whereas colocalization between LAPTM4b Next, we addressed whether the binding of LAPTMs to MCOLN1 and CD63 was nearly 100% complete after 6 hours. As expected, is required for the proper transport of LAPTMs to late endosomes delivery of LAPTM4b to late endosomes and lysosomes correlated and lysosomes. For that, we generated a stable cell line in which with the appearance of the low molecular mass band, consistent the levels of endogenous MCOLN1 were reduced by lentivirus- with the idea that it is the result of proteolytic cleavage (Fig. 5B). mediated shRNA interference. Quantitative RT-PCR showed that The fragment was not observed when LAPTM4b was retained at the lentivirus-mediated shRNA against MCOLN1 caused a greater the ER or shortly after release of the protein from the ER, but it than 80% reduction in MCOLN1 mRNA levels compared with a was most abundant at 6 hours chase when most of the protein had lentivirus-expressing control shRNA (data not shown). Delivery of reached the late endosomes and lysosomes. Appearance of the LAPTMs to late endosomes and lysosomes was not affected by proteolytic fragment was inhibited by incubation with leupeptin, depletion of MCOLN1, as assessed by the appearance of LAPTM an inhibitor of lysosomal proteases, thus confirming that cleavage proteolytic fragments (Fig. 6A) and by confocal microscopy (data of LAPTM4b occurs at lysosomes. In addition, the amount of high not shown). These results indicate that the binding of LAPTMs to molecular mass bands increased over time, suggesting that MCOLN1 does not regulate sorting of LAPTMs along the Role of LAPTMs in lysosomal function 463

Fig. 6. Sorting of LAPTMs to late endosomes and lysosomes is independent of MCOLN1. (A)Stable HeLa cell lines expressing control non- target shRNA (shControl) or shRNA against MCOLN1 (shMCOLN1) were transfected with GFP–LAPTM4a, GFP–LAPTM4b or GFP–LAPTM5. Fig. 7. MCOLN1 rescues the enlargement of late endosomes and Twenty-four hours after transfection, cells were analyzed by western blot using lysosomes induced by LAPTM4b overexpression. (A)ARPE-19 cells were anti-GFP antibody. The presence of proteolytic fragments (arrows) indicates infected with adenovirus encoding GFP–LAPTM4b (top panel) or GFP alone delivery of LAPTMs to the lysosomes. (B)HeLa cells treated with siRNAs (bottom panel). Forty hours after infection, cells were fixed and against both LAPTM4a and LAPTM4b were transfected with a plasmid immunostained with antibodies against CD63. Scale bar: 10mm. (B)ARPE-19

Journal of Cell Science encoding GFP–MCOLN1. Sorting of MCOLN1 to late endosomes and cells were infected with adenovirus expressing GFP–LAPTM4b (top panel) or lysosomes was assessed by colocalization with CD63. Scale bar: 10mm. co-infected with adenovirus expressing GFP–LAPTM4b and MCOLN1– FLAG (bottom panel). Forty hours after infection, cells were fixed, immunostained with antibodies against FLAG and analyzed by confocal endocytic pathway. Similarly, MCOLN1 efficiently reached late microscopy. Insets represent a 2-fold magnification. Scale bar: 10mm. endosomes and lysosomes in cells depleted of LAPTM4a and LAPTM4b (LAPTM5 is not expressed in HeLa cells; Fig. 6B). The levels of LAPTM4a and LAPTM4b mRNAs were reduced by mock-infected cells and 0.46 mm (±0.15 s.d., n98) in Ad-GFP- more than 90% in cells treated with specific siRNAs when infected cells. By contrast, the average diameter of late endosomes compared with control cells (data not shown). Therefore, our results and lysosomes increased to 1.33 mm (±0.28 s.d., n89) in indicate that LAPTMs and MCOLN1 are sorted independently of LAPTM4b-expressing cells (supplementary material Fig. S2). each other. Interestingly, the enlargement of late endosomes and lysosomes caused by the overexpression of LAPTM4b was rescued by Overexpression of LAPTM4b causes enlargement of late coexpression of MCOLN1. As seen in Fig. 7B, co-infection of endosomes and lysosomes ARPE-19 cells with Ad-LAPTM4b and Ad-MCOLN1–FLAG Next, we sought to determine the effect of LAPTM4b prevented the accumulation of swelled late endosomes and overexpression on the late-endosomal pathway. In order to achieve lysosomes. The presence of MCOLN1 did not affect the localization high levels of protein expression, we generated recombinant of LAPTM4b with CD63 but induced a 60% reduction in the size adenoviruses expressing GFP–LAPTM4b (Ad-LAPTM4b) or an of late endosomes and lysosomes, changing from 1.33 mm in adenovirus expressing GFP alone (Ad-GFP) as a control. As shown LAPTM4b-expressing cells to 0.53 mm (±0.12 s.d., n76) in cells in Fig. 7A, overexpression of GFP–LAPTM4b caused a significant co-expressing LAPTM4b and MCOLN1 (supplementary material change in the size and distribution of CD63-positive vesicles. Late Fig. S2). These results indicate that the interaction between endosomes and lysosomes appeared enlarged and, in some cases, MCOLN1 and LAPTM4b is functionally relevant in cells. clustered to the perinuclear region when compared with non- infected cells. Expression of Ad-GFP did not affect the extent of Autophagy is altered in LAPTM4b-expressing cells distribution of late endosomes or lysosomes. The average diameter To analyze whether the enlargement of late endosomes and of CD63-positive structures was 0.45 mm (±0.12 s.d., n56) in lysosomes upon LAPTM4b overexpression correlates with defects 464 Journal of Cell Science 124 (3)

infected cells show an average of 20 LC3-positive vesicles per cell (18.82±4.93, n40 in mock-infected cells and 19.46±5.14, n46 in cells expressing Ad-GFP, where n represents the number of cells). By contrast, there was a greater than 6-fold increase in the average number of autophagomes in cells infected with Ad-LAPTM4b (123±28.39, n36; Fig. 8B). Autophagy can also be monitored by immunoblot. LC3 is initially synthesized in an unprocessed form that is rapidly cleaved in its C-terminal region to generate soluble LC3I. During autophagy induction, the LC3I isoform is converted into LC3II by conjugation of a phosphatidylethanolamine group. This post-translational modification allows LC3 to translocate to autophagosomal membranes. It is well established that the LC3II:LC3I ratio correlates with the extent of autophagosome formation (Klionsky et al., 2007). We prepared lysates from mock-infected cells and from cells infected with adenovirus expressing GFP or GFP– LAPTM4b. As seen in Fig. 8C, a robust increase in the level of LC3II was observed in cells expressing GFP–LAPTM4b, confirming disruption of the autophagic process. Quantification of three independent experiments revealed an approximately threefold increase (2.85±0.2) in the LC3II:LC3I ratio in LAPTM4b-expressing cells (Fig. 8D). To further confirm that basal autophagic flux is interrupted upon overexpression of LAPTM4b, we examined the levels of ubiquitinated proteins. As expected, a remarkable accumulation of ubiquitinated proteins was observed by western blot (Fig. 8E) and immunofluorescence (supplementary material Fig. S3). Therefore, our results indicate that appropriate levels of LAPTM4b are crucial for optimal lysosomal function because overexpression of the protein results in enlargement of late endosomes and lysosomes and defective autophagosome maturation. Fig. 8. Overexpression of LAPTM4b causes accumulation of autophagosomes. (A)ARPE-19 cells were infected with adenovirus encoding Depletion of LAPTMs induces accumulation of electron- GFP–LAPTM4b (top panel) or GFP alone (bottom panel). Forty hours after dense and multi-laminar structures

Journal of Cell Science infection, cells were fixed and immunostained with antibodies against LC3. In MLIV, absence of MCOLN1 leads to the buildup of membranous Scale bar: 10mm. (B)The number of LC3-positive vesicles per cell was and electron-dense organelles containing undigested lipid products. counted in control (mock-infected) cells or in cells expressing either Ad-GFP This heterogeneous storage is readily identified by electron or Ad-LAPTM4b. Bars represent mean values + s.d. (C)ARPE-19 cells mock- microscopy and suggests that proper MCOLN1 function is essential infected or infected with Ad-GFP or Ad-LAPTM4b were harvested and for the maintenance of lysosomal integrity. To further confirm the subjected to western blotting to detect LC3. The blot shown is representative role of LAPTMs in lysosomal function, we depleted endogenous of three independent experiments. (D)Quantification of the LC3II:LC3I ratio. LAPTMs by treatment with specific siRNAs. Quantitative RT- Results are the mean + s.d. from three independent experiments. (E)Lysates from mock-infected ARPE-19 cells or cells expressing either Ad-GFP or Ad- PCR revealed that only LAPTM4a and LAPTM4b are expressed LAPTM4b were subjected to western blotting using a monoclonal antibody to in HeLa cells. These results are in agreement with previous reports ubiquitin. suggesting that LAPTM5 is preferentially expressed in hematopoietic cells (Adra et al., 1996; Scott et al., 1996). After two 72-hour consecutive transfections with a combination of LAPTM4a and LAPTM4b siRNAs, we were able to achieve a greater than in lysosomal function, we monitored autophagic flux. Autophagy 90% reduction in the levels of LAPTM4 (a+b) mRNA, whereas no is a crucial clearance mechanism that prevents accumulation of variations were observed in cells transfected with control non- protein aggregates and abnormal organelles (Cuervo, 2004; target siRNA. Ultrastructure analysis revealed a significant Martinez-Vicente and Cuervo, 2007). It has been described that accumulation of lysosomal inclusions in LAPTM4 knockdown alterations in lysosomal function result in defective autophagosome cells when compared with control cells (Fig. 9A,B). These storage degradation. Interestingly, we found a remarkable accumulation of bodies, composed of electron-dense materials and concentric multi- LC3-positive structures in cells expressing LAPTM4b, whereas laminar (fingerprint) organelles (Fig. 9E; supplementary material accumulation of autophagosomes was not observed in mock- Fig. S4), closely resemble those found in MLIV patients (Fig. infected cells or in cells expressing Ad-GFP (Fig. 8A). LC3 is 9D,G; supplementary material Fig. S4). Quantification experiments considered to be one of the most specific markers for confirmed that accumulation of lysosomal inclusions in LAPTM4 autophagosomes (Kabeya et al., 2000); therefore, our data indicate knockdown cells is highly significant when compared with control that autophagosome maturation is severely diminished by cells (Fig. 10). For example, only 19% (n54) of the cells treated overexpression of LAPTM4b. Quantification of the number of with control siRNA showed more than 10 electron-dense structures, autophagosomes per cell revealed that mock-infected and Ad-GFP- whereas 78% (n50) of the LAPTM4-depleted cells have over 10 Role of LAPTMs in lysosomal function 465

Fig. 10. Simultaneous depletion of LAPTMs and MCOLN1 increases lysosomal inclusion size. (A,B) Quantification of the number and size of the electron-dense and multiconcentric structures accumulated under the following conditions (n represents the number of cells analyzed per condition): HeLa cells treated with control non-target siRNA (siControl, n54), HeLa cells treated with siRNAs against both LAPTM4a and LAPTM4b (siLAPTM, n51), stable HeLa cell line infected with lentivirus-mediated shRNA against MCOLN1 (shMCOLN1, n16), stable HeLa MCOLN1-deficient cell line transfected with siRNAs against LAPTM4a and LAPTM4b (shMCOLN1+ Fig. 9. Depletion of LAPTMs results in accumulation of storage bodies. siLAPTM, n14). (A–D) Electron micrographs showing HeLa cells treated with non-target siRNA (siControl; A), HeLa cells depleted of endogenous LAPTM4a and LAPTM4b (B), HeLa cells depleted of both LAPTMs and MCOLN1 (C) and MLIV human fibroblasts (D). Note the accumulation of electron-dense and and intracellular organelles (Hogue et al., 1996). Further work

Journal of Cell Science concentric multi-laminar membrane structures in B–D. Scale bar: 2mm. suggested that LAPTMs might have a more general role in the (E–G) High-magnification images of the fingerprint structures shown in B–D. transport of structurally unrelated amphiphilic molecules (Hogue Scale bar: 200 nm. et al., 1999). Thus, drug-sensitive yeast expressing mouse LAPTM4a were found to have increased resistance to several compounds such as , , erythromycin, and rhodamine 123, whereas they showed increased inclusions (Fig. 10A). Interestingly, simultaneous depletion of sensitivity to 5-fluorouracil, 5-fluorouridine and trifluoperazine. LAPTM4 (a+b) and MCOLN1 caused a greater than twofold Moreover, the finding that LAPTM4a and LAPTM5 located to increase in the size of the storage bodies when compared with cells lysosomes (Cabrita et al., 1999; Pak et al., 2006) suggested that lacking just LAPTM4 (Fig. 9C,F; Fig. 10B). Overall, our results LAPTMs mediate transport of one or more substrates across confirm that endogenous LAPTMs are important for the lysosomal membranes. maintenance of lysosomal integrity and indicate that defects in In this study, we described for the first time that LAPTM4b also LAPTM function lead to accumulation of storage material in localizes to late endosomes and lysosomes. LAPTM4b is a lysosomes that mimics the phenotype found in MLIV patients. candidate oncogene whose expression is increased in hepatocellular carcinoma, ovarian cancer and gastric cancer (Kasper et al., 2005; Discussion Liu et al., 2007; Yang et al., 2010). Furthermore, expression of Mutations in MCOLN1 are the cause of MLIV, a devastating LAPTM4b contributes to resistance in breast cancer lysosomal storage disorder that affects children in their first year by preventing transport of the doxorubicin to the of life. To better understand the cellular function of MCOLN1, we nucleus (Li et al., 2010). Therefore, this could suggest that performed a split-ubiquitin yeast two-hybrid screen with the goal sequestration of doxorubicin in the lysosomal lumen mediated by of revealing novel interaction partners of MCOLN1. As a result of LAPTM4b is the cause of anthracycline chemoresistance. the screen, we found that MCOLN1 physically interacts with the We are also the first to describe that LAPTMs undergo three members of the LAPTM family, and we observed extensive proteolytic cleavage upon reaching lysosomes. The approximate colocalization between MCOLN1 and LAPTMs in late-endosomes molecular masses of the proteolytic products were 8.5 kDa for and lysosomes. LAPTM4a, 17 kDa for LAPTM4b and 7 kDa for LAPTM5, Initial studies identified LAPTM4a as a transporter that transfers indicating that the cleavage probably occurs at the first intraluminal nucleosides (and/or nucleoside metabolites) between the cytosol loop. Interestingly, injection of a truncated version of mouse 466 Journal of Cell Science 124 (3)

LAPTM4a (lacking the first 83 residues) into Xenopus oocytes storage diseases are caused by deficiencies of soluble lysosomal stimulated uptake of thymidine, whereas full-length mouse hydrolases or lysosomal transporters that cause accumulation of LAPTM4a did not show transport activity (Hogue et al., 1996). specific molecules in the lumen of lysosomes. In support of this, These data suggest that LAPTMs might be activated by proteolytic expression of LAPTM4a and LAPTM5 in MLIV cells is increased cleavage only after being delivered to lysosomes. The first luminal 3-fold and 7-fold, respectively (Bozzato et al., 2008). This increase loop of MCOLN1 is also proteolytically cleaved in lysosomes. might represent an attempt by MLIV cells to compensate for the However, in the case of MCOLN1, it has been suggested that lack of LAPTM activity. proteolytic cleavage might induce inactivation of the protein Further studies will try to identify the physiological ligands (Kiselyov et al., 2005). In addition, we did not observe significant transported by LAPTMs as well as discover compounds that homology between the loops of MCOLN1 and LAPTMs, thus modulate LAPTM activity. This information will further improve suggesting that the cleavage might be mediated by different our understanding of MLIV and might open new and exciting lysosomal hydrolases. avenues for the development of a treatment for the disease. Overexpression of LAPTM4b has a profound effect on the morphology and functionality of lysosomes. Infection of ARPE-19 Materials and Methods cells with Ad-LAPTM4b caused enlargement of CD63-positive Antibodies and reagents The primary antibodies used were: mouse monoclonal anti-CD63 (H5C6; BD vesicles, accumulation of ubiquitinated proteins and defective Pharmingen, San Jose, CA), mouse monoclonal anti-actin (clone Ab-5; BD autophagosome maturation. These data are consistent with the Biosciences, San Jose, CA), mouse monoclonal anti-GFP (Covance, Princeton, NJ), proposed role for LAPTMs in the transport of molecules across the mouse monoclonal anti-Lamp1 (clone H4A3, Developmental Studies Hybridoma lysosomal membrane, as the accumulation of certain substrates is Bank, Iowa City, IA), rabbit polyclonal anti-LC3 (Sigma, St Louis, MO), rabbit anti- GFP and mouse monoclonal anti-ubiquitin (clone FK2) (MBL International, Woburn, likely to impair lysosomal function. The data are also in agreement MA), rabbit polyclonal anti-Hrs (Novus Biologicals, Littleton, CO) and mouse with recent evidence showing that overexpression of LAPTM5 monoclonal anti-FLAG M2 (Sigma). The secondary antibodies used were: goat anti- associates with spontaneous regression of neuroblastomas by mouse or goat anti-rabbit conjugated to Alexa Fluor 488 or 555, goat anti-mouse conjugated to Alexa Fluor 633 (Molecular Probes, Eugene, OR) and HRP-conjugated inducing lysosomal cell death with impaired autophagy (Inoue et anti-mouse or anti-rabbit IgG (Amersham Bioscience, Piscataway, NJ). FuGENE-6 al., 2009). We and others have previously described that reagent and protease inhibitor cocktail tablets were obtained from Roche Applied overexpression of MCOLN1 also causes accumulation of enlarged Science (Indianapolis, IN). BFA and leupeptin were from Sigma and were used aberrant lysosomes and impaired autophagy, thus indicating that according to the manufacturer’s instructions. LAPTMs and MCOLN1 are required to maintain adequate Split-ubiquitin yeast two-hybrid assays lysosomal function (Vergarajauregi et al., 2008; Manzoni et al., Screening of an adult human brain cDNA library with full-length MCOLN1 was 2004). performed by Dualsystems Biotech AG (Schlieren, Switzerland). Full-length MCOLN1 was inserted into the bait expression vector pBT3-STE (Dualsystems The importance of LAPTMs in lysosomal function was further Biotech, Switzerland) with Cub-LexA-VP16 downstream of MCOLN1. The bait corroborated by the accumulation of storage bodies in cells depleted vector carries the LEU2 for auxotrophic selection. A human adult brain cDNA of endogenous LAPTMs. Storage material included fine lamellated library of 50.3ϫ106 clones fused to a mutated version of the amino-terminal portion of ubiquitin (NubG-x) in their amino-terminal sequence in the pPR3-N (prey vector) membrane structures (fingerprints), as well as electron-dense was used to screen for potential positive interactions. The prey vector carries the bodies. Interestingly, the same types of lysosomal inclusions are TRP1 (TRNAP1) gene for auxotrophic selection. The screening was carried out by the mating of NMY32 yeast transformed with the bait and prey constructs. A total Journal of Cell Science found in MLIV cells. It has been suggested that the ultrastructural of 277 positive clones representing 23 distinct human proteins were isolated. One appearance of storage material might be specific to the type of clone was found to encode the first 217 amino acids of LAPTM4a and a second one lipid accumulated in each lysosomal storage disorder (Alroy and encodes amino acids 27–47 of LAPTM4b. Selection of positive interactions was Ucci, 2006). For example, fingerprint structures often result from performed by the ability of yeast to grow on selective synthetic dropout (SD) agar the accumulation of gangliosides and other glycolipids, and have plates [SD –tryptophan –leucine –histidine (Clontech, Palo Alto, CA)] containing 10 mM 3-amino-1,2,4-triazole (3AT). The selected positive clones were sequenced and been described in MLIV, Fabry disease, fucosidosis, GM1 and the resulting nucleotide sequences were analyzed using the NCBI BLAST algorithm. GM2 gangliosidosis, galactosialidosis and sialidosis. Therefore, the appearance of concentric membranes in the absence of either Plasmids and adenovirus preparation The complete open reading frames (ORFs) of human LAPTM4a and LAPTM5 were MCOLN1 or LAPTMs could indicate accumulation of the same PCR-amplified from a placenta cDNA library using specific primers and cloned into type of storage material. the EcoRI and SalI sites of the pEGFP-C2 vector (Clontech). The ORF of human The presented evidence suggests that MCOLN1 and LAPTMs LAPTM4b was PCR-amplified from a cDNA clone from OriGene (NM_018407) using specific primers to introduce an EcoRI site at the 5Ј end and an XhoI site at are functionally connected. The fact that MCOLN1 and LAPTMs the 3Ј end and cloned into the EcoRI and SalI sites of pEGFP-C2. To generate interact and colocalize to late endosomes and lysosomes, the ability Cherry-MCOLN1, full-length MCOLN1 was cloned into the EcoRI and SalI sites of of MCOLN1 to rescue the enlargement of lysosomes caused by the pCherry vector (Clontech). MCOLN3–FLAG was cloned into the EcoRI–SalI LAPTM4b overexpression, the accumulation of the same type of sites of the pCMV-TAG-2B vector. Cloning of MCOLN1–FLAG has been described previously (Vergarajauregui et al., 2008b). MCOLN1, MCOLN3, LAPTM4a, storage material upon depletion of either LAPTMs or MCOLN1 LAPTM4b and LAPTM5 were cloned into the SfiI sites of the pBT3-STE, pPR3-N and the increase in the size of lysosomal inclusions by simultaneous and pPR3-STE vectors (Dualsystems Biotech AG, Switzerland) according to the depletion of both MCOLN1 and LAPTMs suggest that MCOLN1 manufacturer’s directions. We used pDL2-Alg5 as a negative control for the split- ubiquitin two-hybrid system. To check if the bait was properly expressed and might regulate LAPTM function. We speculate that the release of functional, we used the control prey pAl-Alg5. All constructs were confirmed by ions by MCOLN1 might provide energy for the LAPTM-dependent DNA sequencing. Adenoviruses expressing GFP–LAPTM4b and MCOLN1–FLAG transport of specific molecules across lysosomal membranes. were prepared, amplified and purified by Welgen (Worcester, MA).

Alternatively, binding of LAPTMs to MCOLN1 might induce shMCOLN1 cell line changes in the conformation and/or activity of the transporters. Control and MCOLN1 lentiviral shRNAs were obtained from Sigma (MISSION Our model has important implications for the understanding of shRNA Lentiviral Transduction Particles). HeLa cells were infected with either the MLIV. Alterations in the transport activity of LAPTMs caused by MCOLN1-lentivirus (MISSION shRNA TRCN0000083297) or the control lentivirus (MISSION Non-Target shRNA Control SHC002) together with 8 mg/ml of Polybrene. the absence of MCOLN1 might lead to the storage disorder Clones were selected using 1 mg/ml puromycin. Q-RT-PCR was used to assay for observed in MLIV cells. Studies have shown that most lysosomal knockdown of MCOLN1. Role of LAPTMs in lysosomal function 467

Cell culture, transfection, siRNA knockdown and adenoviral infection We thank Mathew P. Daniels and Patricia S. Connelly from the HeLa and ARPE19 cells (American Type Culture Collection, Manassas, VA) were NHLBI Electron Microscopy Core Facility for assistance. We also grown at 37°C in DMEM medium or in a 1:1 mixture of DMEM and Ham’s F12 appreciate the editorial advice of the NIH Fellows Editorial Board. media (Invitrogen, Carlsbad, CA), respectively, supplemented with 10% fetal bovine This project was supported by the Intramural Research Program of the serum, 2 mM Glutamax, 100 U/ml penicillin and 100 mg/ml streptomycin in a NIH, National Heart, Lung and Blood Institute (NHLBI). The authors humidified 5% CO2 atmosphere. Cells were transiently transfected using FuGENE- 6 reagent according to the manufacturer’s instructions. Transfected cells were have no conflicts of interest to declare. Deposited in PMC for release analyzed 24–36 hours post-transfection. after 12 months. 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