Lysosomal Localization of TRPML3 Depends on TRPML2 and the Mucolipidosis-associated Protein TRPML1

Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa. Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa. Mucolipidosis type IV (MLIV) 2The abbreviations used are: MLIV, mucolipidosis type IV; AP180, Adaptor protein 180; AMO, Almost there; CFP, cyan fluorescent protein; Dyn1a, Dynamin 1a; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; MVB, multivesicular bodies; Sun, Sunglasses; TRP, transient receptor potential channels; YFP, yellow fluorescent protein; HA, hemagglutinin; DAPI, 4′,6-diamidino-2-phenylindole. is a developmental disorder with a variety of clinical manifestations ranging from achlorhydria to neurodegeneration, psychomotor retardation, and visual impairments (1Bach G. Mol. Genet. Metab. 2001; 73: 197-203Crossref PubMed Scopus (154) Google Scholar, 2Bach G. Pfluegers. Arch. 2005; 451: 313-317Crossref PubMed Scopus (79) Google Scholar, 3Altarescu G. Sun M. Moore D.F. Smith J.A. Wiggs E.A. Solomon B.I. Patronas N.J. Frei K.P. Gupta S. Kaneski C.R. Quarrell O.W. Slaugenhaupt S.A. Goldin E. Schiffmann R. Neurology. 2002; 59: 306-313Crossref PubMed Scopus (140) Google Scholar). The disease is a lysosomal storage disorder associated with lysosomal accumulation of macromolecules such as sphingolipids, phospholipids, and mucopolysaccharides (1Bach G. Mol. Genet. Metab. 2001; 73: 197-203Crossref PubMed Scopus (154) Google Scholar). It was reported previously that MLIV does not appear to be due to defects in the activities of lysosomal enzymes but rather from perturbations in membrane sorting and trafficking during late steps in endocytosis and lysosomal biogenesis (4Chen C.S. Bach G. Pagano R.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6373-6378Crossref PubMed Scopus (167) Google Scholar). However, a recent study indicates that cells obtained from patients with MLIV are characterized by over-acidified lysosomes and reduced acidic lipase activity (5Soyombo A.A. Tjon-Kon-Sang S. Rbaibi Y. Bashllari E. Bisceglia J. Muallem S. Kiselyov K. J. Biol. Chem. 2006; 281: 7294-7301Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). MLIV is a consequence of mutations in a member of the group 2 subset of TRP cation channels (6Montell C. Sci. STKE. 2005; 2005: re3PubMed Google Scholar) referred to as TRPML1 (Mucolipin1) (7Bargal R. Avidan N. Ben-Asher E. Olender Z. Zeigler M. Frumkin A. Raas-Rothschild A. Glusman G. Lancet D. Bach G. Nat. Genet. 2000; 26: 118-123Crossref PubMed Scopus (306) Google Scholar, 8Bassi M.T. Manzoni M. Monti E. Pizzo M.T. Ballabio A. Borsani G. Am. J. Hum. Genet. 2000; 67: 1110-1120Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 9Sun M. Goldin E. Stahl S. Falardeau J.L. Kennedy J.C. Acierno Jr., J.S. Bove C. Kaneski Nagle C.R. Bromley J. Colman M.C. Schiffmann M. Slaugenhaupt R.S.A. Hum. Mol. Genet. 2000; 9: 2471-2478Crossref PubMed Scopus (345) Google Scholar). The group 2 TRPs also include TRPP proteins, two of which are disrupted in autosomal dominant polycystic kidney disease (6Montell C. Sci. STKE. 2005; 2005: re3PubMed Google Scholar). Members of the five group 1 TRP subfamilies (TRPC, TRPV, TRPM, TRPA, and TRPN) are only distantly related to the TRPML and TRPP proteins, although all TRP channels contain six transmembrane segments. TRPML1 has been localized to late endosomes and lysosomes (10Kiselyov K. Chen J. Rbaibi Y. Oberdick D. Tjon-Kon-Sang S. Shcheynikov N. Muallem S. Soyombo A. J. Biol. Chem. 2005; 280: 43218-43223Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 11Manzoni M. Monti E. Bresciani R. Bozzato A. Barlati S. Bassi M.T. Borsani G. FEBS Lett. 2004; 567: 219-224Crossref PubMed Scopus (70) Google Scholar), and this spatial distribution, combined with phenotypic analyses of cells isolated from MLIV patients, led to the proposal that TRPML is required for lysosomal reformation/biogenesis (11Manzoni M. Monti E. Bresciani R. Bozzato A. Barlati S. Bassi M.T. Borsani G. FEBS Lett. 2004; 567: 219-224Crossref PubMed Scopus (70) Google Scholar). This conclusion is further supported by functional analyses of the Caenorhabditis elegans TRPML homolog, CUP-5 (12Fares H. Greenwald I. Nat. Genet. 2001; 28: 64-68Crossref PubMed Google Scholar, 13Hersh B.M. Hartwieg E. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4355-4360Crossref PubMed Scopus (97) Google Scholar, 14Treusch S. Knuth S. Slaugenhaupt S.A. Goldin E. Grant B.D. Fares H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4483-4488Crossref PubMed Scopus (192) Google Scholar, 15Schaheen L. Dang H. Fares H. Dev. Biol. 2006; PubMed Google Scholar). Recently, TRPML1 has been reported to be a proton-leak channel in the lysosomes, thereby preventing excessive acidification of the lysosomal lumen (5Soyombo A.A. Tjon-Kon-Sang S. Rbaibi Y. Bashllari E. Bisceglia J. Muallem S. Kiselyov K. J. Biol. Chem. 2006; 281: 7294-7301Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). In addition to TRPML1, mammals encode two other highly related proteins, TRPML2 and TRPML3, although the subcellular distributions of these latter proteins have not been defined. Nevertheless, mutations in mouse TRPML3 (encoded by mcoln3) have been shown to be responsible for the hearing, vestibular, and pigmentation defects associated with varitint-waddler mice (16Di Palma F. Belyantseva I.A. Kim H.J. Vogt T.F. Kachar B. Noben-Trauth K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14994-14999Crossref PubMed Scopus (185) Google Scholar). A feature of many TRPs is their ability to heteromultimerize with closely related members within the same subfamily (17Hellwig N. Albrecht N. Harteneck C. Schultz G. Schaefer M. J. Cell Sci. 2005; 118: 917-928Crossref PubMed Scopus (246) Google Scholar, 18Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (634) Google Scholar, 19Chubanov V. Waldegger S. Mederos y Schnitzler M. Vitzthum H. Sassen M.C. Seyberth H.W. Konrad M. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2894-2899Crossref PubMed Scopus (321) Google Scholar, 20Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 21Xu X.Z. Chien F. Butler A. Salkoff L. Montell C. Neuron. 2000; 26: 647-657Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 22Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 2003; 278: 39014-39019Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 23Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). However, it is not known whether group 2 TRP channels, such as the TRPMLs, share this characteristic. In this report we used a fluorescence resonance energy transfer (FRET)-based approach to demonstrate that each TRPML protein was capable of forming homo- and heteromultimers with other members of the TRPML subfamily. Furthermore, we show that the subcellular distribution of TRPML3 is dictated by TRPML1 or TRPML2. When expressed individually, TRPML1 and TRPML2 were lysosomal membrane proteins whereas TRPML3 was retained in the ER. In contrast, when TRPML3 was coexpressed with either TRPML1 or TRPML2, it translocated to the lysosomes. Mislocalization of TRPML1 or TRPML2 to the plasma membrane, due to mutations in lysosomal targeting sequences or as result of interfering with clathrin-mediated endocytosis, caused a similar plasma membrane distribution of TRPML3. Since TRPML3 did not influence the localization of TRPML1 or TRPML2, these latter TRPML family members were dominant over TRPML3 with respect to trafficking. Plasmids and cDNAs—The TRPML1 (GenBank™ accession number NM020533) and TRPML3 (GenBank™ accession number NM134160) expressed sequence tags (Invitrogen) cDNAs were subcloned into the pcDNA3.1 vector (Invitrogen). The TRPML2 clone was a gift from Sharon Matthews and Dr. Andrew Scharenberg (University of Washington). The truncated mutants described in supplemental Fig. S5 are TRPML1Δ1 (removal of C-terminal LLVN; supplemental Fig. S5B), TRPML1Δ2 (removal of the first 15 amino acids following the initiation methionine; supplemental Fig. S5C), TRPML1Δ3 (combination of the C- and N-terminal deletions; supplemental Fig. S5D) and TRPML2Δ1 (removal of the C-terminal DRLILID; supplemental Fig. S5F) were generated by PCR-mediated mutagenesis and subcloned into pcDNA3.1 (Invitrogen). The amo cDNA was previously described (24Watnick T.J. Jin Y. Matunis E. Kernan M.J. Montell C. Curr. Biol. 2003; 13: 2179-2184Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). To fuse TRPML1, -2, and -3 or AMO with enhanced YFP or enhanced CFP their translation STOP codons were replaced by in-frame XhoI or EcoRI restriction sites and then subcloned into the appropriate pcDNA3-CFP or -YFP fusion vectors (18Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (634) Google Scholar). To fuse TRPML1-3, TRPML1Δ1-3, and TRPML2Δ1 with the HA tag, their STOP codons were replaced by the sequence encoding the HA tag followed by an in-frame XhoI restriction site and subcloned into pcDNA3.1. and have been described previously H. E. A. Li H.S. Montell C. J. 2004; PubMed Scopus (97) Google Scholar). The cDNA was by Dr. S. of was generated by the sequence of with a sequence and into was by Dr. A. of was described previously T. Schaefer M. Harteneck C. Gudermann T. Schultz G. PubMed Scopus Google Scholar). was by Dr. C. of cDNA was obtained from the and was to the HA Plasmids for of Dynamin and Dynamin were obtained from Dr. A. (University of of cDNA were obtained from H. and E. and mouse were from and were from and all and were from Cell and cells were in (Invitrogen) with (Invitrogen) and to of was used of used for were used the 1 of each was used in an to of the TRPML proteins, which were to either CFP or YFP, were coexpressed in The vectors encoding the were a to the of The cells were an with a and a and were by in the and In a subset of an with a IV an a and a were as previously described (18Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (634) Google Scholar, 19Chubanov V. Waldegger S. Mederos y Schnitzler M. Vitzthum H. Sassen M.C. Seyberth H.W. Konrad M. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2894-2899Crossref PubMed Scopus (321) Google Scholar). the cells were for for CFP and for for YFP an additional was to each This in of of the YFP within the A of were were and data were from of A of in the of the was as were as the in CFP fluorescence a of the YFP In the of the the of CFP and YFP was by the CFP the YFP and were in for the cells were with for in with for 1 with and for 1 The cells were with all steps and with To with was To YFP and we to the and the cells for 2 the cells as were obtained with the with an a and a was to of as as The was used to and and to analyses of in the due to in the and of the was the same of each to of the the for the analyses to and of which to to and the cells the to of a these of two or did not their fluorescence with when are expressed supplemental Fig. and analyses for the of were with in which their were to of the of the with did not the fluorescent of YFP and in these not and did not cells were and and and was to of TRPML1 or was a protein and as the The and were and to and were was used as the and was used as the The were to for 1 the of the were by The of the were to of the was from the was an were a and a The and were in and The TRPML1, -2, and -3 cDNA and of the activities of and and were used of and The were in for and with a the same whether the TRPML proteins interact to form homo- and we a either yellow fluorescent protein or cyan fluorescent protein to the of each of the TRPMLs and coexpressed these proteins in cells in The presence of two proteins is characterized by an in the CFP of YFP and indicates that the proteins are in fluorescence resonance energy transfer only when the are by J. Biol. PubMed Scopus (306) Google Scholar). that there were each of the TRPML proteins in all that the TRPMLs form homo- and and a the CFP and YFP as has been for other TRPs (18Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (634) Google Scholar, 19Chubanov V. Waldegger S. Mederos y Schnitzler M. Vitzthum H. Sassen M.C. Seyberth H.W. Konrad M. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2894-2899Crossref PubMed Scopus (321) Google Scholar, H. Schultz G. Schaefer M. Cell 2003; PubMed Scopus Google Scholar). In to these of TRPMLs with two other proteins with transmembrane did not result in These include the TRPP protein AMO (24Watnick T.J. Jin Y. Matunis E. Kernan M.J. Montell C. Curr. Biol. 2003; 13: 2179-2184Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, Z. Curr. Biol. 2003; 13: Full Text Full Text PDF PubMed Scopus Google Scholar) and which is also a group 2 TRP, and the lysosomal associated referred to as Sun H. E. A. Li H.S. Montell C. J. 2004; PubMed Scopus (97) Google Scholar) and The of the TRPML and either AMO or Sun was not due a to these latter proteins, as there were and Since TRPML1 and Sun are lysosomal membrane proteins, the the TRPMLs did not appear to be a consequence of in the same the TRPMLs form then be in with this we that the encoding all TRPMLs were coexpressed in an Fig. TRPML1 and TRPML2 whereas TRPML3 in the the TRPML proteins that the same or highly spatial TRPML1 has been reported to be a lysosomal protein (10Kiselyov K. Chen J. Rbaibi Y. Oberdick D. Tjon-Kon-Sang S. Shcheynikov N. Muallem S. Soyombo A. J. Biol. Chem. 2005; 280: 43218-43223Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 11Manzoni M. Monti E. Bresciani R. Bozzato A. Barlati S. Bassi M.T. Borsani G. FEBS Lett. 2004; 567: 219-224Crossref PubMed Scopus (70) Google the of TRPML2 and TRPML3 have not been To the subcellular distribution of each of the TRPML proteins, we the spatial distributions of the TRPMLs with that and the of by the lysosomal and in the lumen of acidic such as lysosomes and multivesicular L. J. Cell Biol. PubMed Scopus Google Scholar). TRPML1 and TRPML2 are in the of these rather in the lumen and proteins that the are PubMed Scopus Google Scholar) The in the of by and is for cells and does not appear to be a consequence of TRPML as the of similar in cells Fig. TRPML1 TRPML2 with other such as the Fig. M.J. J.C. J. PubMed Scopus Google Fig. and endosomes M. H. Nat. Mol. Cell. Biol. 2001; PubMed Scopus Google Fig. and late endosomes M. H. Nat. Mol. Cell. Biol. 2001; PubMed Scopus Google Fig. and and M. H. Nat. Mol. Cell. Biol. 2001; PubMed Scopus Google Fig. and that TRPML1 and TRPML2 were lysosomal membrane proteins was that HA of these proteins with other and membrane proteins associated with lysosomal These include the G. J.A. G. Mol. Biol. Cell. 2002; 13: PubMed Scopus Google Scholar), which of transmembrane Fig. and and the and which is associated with the of lysosomes and in addition to A. M.T. 2005; PubMed Scopus Google Scholar). we that TRPML1 and TRPML2 are lysosomal associated membrane In to TRPML1 and TRPML2, we that the with an membrane Fig. TRPML3 with lysosomes Fig. and Fig. and supplemental Fig. endosomes supplemental Fig. late endosomes supplemental Fig. and supplemental Fig. These were the TRPML3 and the other TRPML The of of the TRPMLs with the was by the of the TRPMLs with each TRPML1 and TRPML2 the with the and and whereas TRPML3 a comparably only with the The of the TRPMLs with these did not appear to be by in due to with the proteins, the of TRPML3 was similar expressed or in with other proteins Fig. with TRPML1 or TRPML2 TRPML3 to in the is that TRPML3 interact with either TRPML1 or TRPML2, it a subcellular localization from TRPML1 and TRPML2. To this we whether the spatial distribution of TRPML3 was with either TRPML1 or TRPML2. that TRPML3 with either TRPML1 or TRPML2 and Furthermore, such in a in the localization of TRPML3 and a in retention These data that TRPML3 is with either TRPML1 or TRPML2 to the as a result of Moreover, the spatial distributions of TRPML1 or TRPML2 were when coexpressed with TRPML3 not either TRPML1 or TRPML2 the localization of TRPML3 but not vice versa. The localization of TRPML3 in lysosomes, when coexpressed with other TRPMLs, did not appear to be by as the of the TRPML3 were similar in the presence or of TRPML1 Fig. A and To whether the were we the in a In with the in we that TRPML1 and TRPML2 were lysosomal membrane proteins with Fig. and whereas TRPML3 localized to the when expressed in the of either TRPML1 or TRPML2 with Fig. with either TRPML1 or TRPML2, we that TRPML3 was in the lysosomes, as by with and Mislocalization of TRPML3 by of additional that TRPML3 with TRPML1 and TRPML2 we to whether mutations that the lysosomal localization of TRPML1 and TRPML2 in cause a similar of TRPML3. we the TRPML1 and TRPML2 sequences for similar to known lysosomal targeting J.S. 2003; PubMed Scopus Google Scholar). TRPML1 and TRPML2 type of the and of TRPML1 Fig. and and the of TRPML2 Fig. of the C-terminal sequence of TRPML1 Fig. did not lysosomal localization However, of either the N-terminal or the and C-terminal sequences Fig. and TRPML1Δ2 and disrupted lysosomal localization and enhanced distribution as by with the plasma membrane cation channel T. Schaefer M. Harteneck C. Gudermann T. Schultz G. PubMed Scopus Google Scholar) and of the C-terminal of TRPML2 Fig. also disrupted lysosomal localization and in a distribution similar to that of the subcellular distribution of TRPML3 is by TRPML1 or TRPML2, then TRPML3 an distribution when coexpressed with either TRPML1Δ2 or with this we that the of when it was coexpressed with TRPML1Δ2 or TRPML2Δ1 and Mislocalization of TRPML3 by with data that the distribution of TRPML3 that of either TRPML1 or TRPML2. To further to this we to the of TRPML1 or TRPML2 by the proteins in endocytosis and lysosomal rather by the TRPML To with clathrin-mediated endocytosis, we used a dominant form of Dynamin a that the energy of to cause of during clathrin-mediated endocytosis the plasma membrane or J.S. J. Cell Biol. PubMed Scopus Google Scholar, Nat. Mol. Cell Biol. 2004; PubMed Scopus Google Scholar). The dominant of is of and clathrin-mediated endocytosis J.S. J. Cell Biol. PubMed Scopus Google Scholar, E. B. Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar). of with did not lysosomal localization A and However, of with lysosomal localization and in distribution of the of TRPML1Δ2 and TRPML1Δ3 were obtained with TRPML2 is protein that clathrin-mediated endocytosis B.M. Y. A. C.R. 2001; PubMed Scopus Google Scholar). and it to be into a thereby preventing and clathrin-mediated endocytosis B.M. Y. A. C.R. 2001; PubMed Scopus Google Scholar). When expressed with either or lysosomal targeting and localization and In to the TRPML1 and TRPML2, Dyn1a, and the localization of TRPML3 when expressed in the of other TRPMLs However, caused localization of TRPML3 when it was coexpressed with either TRPML1 or TRPML2 and were obtained when TRPML3 was coexpressed with TRPML1 or TRPML2 and and when expressed the spatial distribution of TRPML3 that of either TRPML1 or TRPML2. TRPML1 into the to the mislocalized TRPML1 is into the plasma membrane or whether it in to the plasma The of lysosomal TRPML1 has been reported previously (10Kiselyov K. Chen J. Rbaibi Y. Oberdick D. Tjon-Kon-Sang S. Shcheynikov N. Muallem S. Soyombo A. J. Biol. Chem. 2005; 280: 43218-43223Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Here we show that of clathrin-mediated endocytosis with or the of TRPML1 and Fig. and The in of TRPML1Δ3 due to of the lysosomal targeting is Fig. and These that TRPML1 into the plasma membrane of clathrin-mediated endocytosis or as a result of of lysosomal targeting data of the that the TRPML proteins form heteromultimers and such dictate the subcellular localization of TRPML3. a approach we that the TRPML proteins in all the localization of TRPML3 was in the presence of TRPML1 or TRPML2. When expressed TRPML3 was in the ER. However, in the presence of the TRPML1 or TRPML2 proteins, we TRPML3 in the lysosomes. These latter were with the presence of lysosomal targeting in TRPML1 and TRPML2 but not TRPML3. mutations in the which localization of TRPML1 or TRPML2, cause a similar in the spatial distribution of coexpressed TRPML3. TRPML3 an when coexpressed with either TRPML1 or TRPML2 with proteins that with clathrin-mediated a of protein lysosomal protein for appropriate localization is of the of the of the to the lysosomes. of the and the of the disease A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). the localization of TRPML3 was in the presence of either of the other TRPMLs, TRPML3 did not the localization TRPML1 or TRPML2. there was a hierarchy of the spatial such that TRPML1 and TRPML2 were dominant over TRPML3. This hierarchy be TRPML1 and TRPML2 have to the lysosomes, which are in TRPML3. with this we that the of TRPML1 or TRPML2 and which in the when expressed are to the lysosomes with TRPML1 or TRPML2 not However, it is that with either TRPML1 or TRPML2 also of in TRPML3. with the proposal that TRPMLs form heteromultimers in we that all TRPML were expressed in a as by However, due to the of it to be shown in cells whether the TRPML proteins are coexpressed and whether the localization of TRPML3 TRPML1 and TRPML2. The the that trafficking of TRPML3 to the lysosomes is required for lysosomal and that of the of MLIV be to defects in lysosomal localization of TRPML3. the localization of TRPML3 is disrupted in patients with mutations that or the of TRPML1 (1Bach G. Mol. Genet. Metab. 2001; 73: 197-203Crossref PubMed Scopus (154) Google Scholar, R. Avidan N. Olender T. E. Zeigler M. Raas-Rothschild A. Frumkin A. Y. Lancet D. Bach G. Hum. 2001; PubMed Scopus Google Scholar, R. E. Bach G. 2002; PubMed Scopus Google Scholar) the of that the TRPML3 H. M. and R. for and Dr. A. Scharenberg and S. Matthews for the TRPML2 with