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• W2022166639 abstract "MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu66-Leu67) and a tyrosine residue (Tyr89) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s). MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu66-Leu67) and a tyrosine residue (Tyr89) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s). low density lipoprotein steroidogenic acute regulatory protein StAR homology domain StAR-related lipid transfer Chinese hamster ovary phosphate-buffered saline Niemann-Pick C Cholesterol, the essential sterol found in vertebrates, has several functions, which include modulating the fluidity and permeability of membranes, serving as precursor for steroid hormones and bile acid synthesis, and covalently modifying proteins. Animal cells obtain cholesterol by de novo synthesis in the endoplasmic reticulum or receptor-mediated uptake of plasma lipoproteins (1Liscum L. Munn N.J. Biochim. Biophys. Acta. 1999; 1438: 19-37Crossref PubMed Scopus (294) Google Scholar). Most of cells acquire cholesterol from low density lipoprotein (LDL).1 LDL is endocytosed and transported to early endosomes and then to late endosomes/lysosomes for degradation. Free cholesterol generated from LDL in late endosomes/lysosomes is then redistributed in the cell. Although the regulation of cholesterol content in cells has been extensively studied, little is known about the mechanisms of its intracellular transport. Only a few molecules involved in this pathway have been identified, including steroidogenic acute regulatory protein (StAR), whose expression is tissue-specific, or the recently identified Niemann Pick C1 protein (2Liscum L. Klansek J.J. Curr. Opin. Lipidol. 1998; 9: 131-135Crossref PubMed Scopus (97) Google Scholar, 3Cruz J.C. Sugii S., Yu, C. Chang T.Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). MLN64 cDNA was identified from a breast cancer-derived metastatic lymph node cDNA library by differential hybridization using malignant (metastatic lymph node) versus nonmalignant (breast fibroadenoma and normal lymph node) tissues. Chromosomal mapping showed that the MLN64 gene is located in the q12-q21 region of the long arm of chromosome 17 (4). This region is altered in 20–30% of breast cancers, the most common modification being the amplification of the proto-oncogene c-erb B-2 (5Fukushige S. Matsubara K. Yoshida M. Sasaki M. Suzuki T. Semba K. Toyoshima K. Yamamoto T. Mol. Cell. Biol. 1986; 6: 955-958Crossref PubMed Scopus (278) Google Scholar, 6Slamon D.J. Godolphin W. Jones L.A. Holt J.A. Wong S.G. Keith D.E. Levin W.J. Stuart S.G. Udove J. Ullrich A. Press M.F. Science. 1989; 244: 707-712Crossref PubMed Scopus (6289) Google Scholar, 7Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Science. 1987; 235: 177-182Crossref PubMed Scopus (10014) Google Scholar). The proto-oncogene c-erb B-2 is a marker of poor prognosis and tumor aggressiveness in breast cancers; its overexpression in tumors has been correlated to hormone therapy failure (8Ross J.S. Fletcher J.A. Stem Cells. 1998; 16: 413-428Crossref PubMed Scopus (615) Google Scholar). In breast cancers, an invariable coamplification and consequent overexpression ofMLN64 and c-erb B-2 in 22.5% of the cases tested (98 cases) was observed, suggesting that overexpression ofMLN64 could be of clinical relevance for breast cancer development and/or progression (9Bieche I. Tomasetto C. Regnier C.H. Moog-Lutz C. Rio M.C. Lidereau R. Cancer Res. 1996; 56: 3886-3890PubMed Google Scholar). MLN64 cDNA encodes for a protein of 445 residues containing four potential transmembrane regions at its amino-terminal part. In addition, MLN64 shares a conserved COOH-terminal region with StAR called the StAR homology domain (SHD) (10Moog-Lutz C. Tomasetto C. Regnier C.H. Wendling C. Lutz Y. Muller D. Chenard M.P. Basset P. Rio M.C. Int. J. Cancer. 1997; 71: 183-191Crossref PubMed Scopus (117) Google Scholar). Recently, a larger domain, including the SHD, has been defined as the StAR-related lipid transfer (START) domain (20Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (450) Google Scholar). A wide variety of proteins involved in different cell processes possess a START domain, such as the phosphatidylcholine transfer protein, the signal-transducing protein p122-RhoGAP, or a putative acyl-CoA thioesterase. Interestingly, it has been shown that mutations in the StAR gene, which lead to COOH-terminal truncated proteins in which the SHD domain is deleted, are responsible for congenital adrenal hyperplasia, a disease characterized by severely impaired steroidogenesis (12Lin D. Sugawara T. Strauss J.F. Clark B.J. Stocco D.M. Saenger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (874) Google Scholar, 13Bose H.S. Sugawara T. Strauss J.F. Miller W.L. N. Engl. J. Med. 1996; 335: 1870-1878Crossref PubMed Scopus (534) Google Scholar, 14Arakane F. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain D. Stocco D.M. Miller W.L. Strauss J.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (254) Google Scholar). The functional relationship between MLN64 and StAR was previously investigated. It was shown that, like StAR, MLN64 can enhance steroidogenesis in an in vitro assay. Removal of the SHD domain resulted in the complete loss of steroidogenic activity, while removal of the NH2-terminal region of MLN64 increased this activity (15Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8462-8467Crossref PubMed Scopus (205) Google Scholar). StAR is a mitochondrial protein that regulates the acute production of steroids in the adrenal glands and gonads in response to corticotropin and luteinizing hormone, respectively. StAR regulates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol from the outer to the inner mitochondrial membrane, where it is converted into pregnenolone (16Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (942) Google Scholar). Investigations into the mechanism of action of StAR have shown that StAR is a sterol transfer protein that acts directly on the mitochondria (17King S.R. Ronen-Fuhrmann T. Timberg R. Clark B.J. Orly J. Stocco D.M. Endocrinology. 1995; 136: 5165-5176Crossref PubMed Google Scholar, 18Arakane F. Kallen C.B. Watari H. Foster J.A. Sepuri N.B. Pain D. Stayrook S.E. Lewis M. Gerton G.L. Strauss J.F. J. Biol. Chem. 1998; 273: 16339-16345Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In addition, StAR has been shown to be a sterol transfer protein in vitro (19Kallen C.B. Billheimer J.T. Summers S.A. Stayrook S.E. Lewis M. Strauss J.F. J. Biol. Chem. 1998; 273: 26285-26288Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Recently, the three-dimensional structure of the START domain of MLN64 was solved, and its ability to bind cholesterol at an equimolar ratio was reported (20Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (450) Google Scholar). MLN64 is likely to be involved in cholesterol transport, and cholesterol is the precursor of all steroid hormones. Since c-erb B-2 and MLN64 are coamplified and overexpressed in breast cancer, a role for MLN64 in facilitating intratumoral biosynthesis of steroid hormones can be postulated, which may relate to the hormonal resistance of part of the c-erb B-2-expressing tumors. This study was aimed at identifying the exact subcellular localization of MLN64 to define where MLN64 is likely to play a key role during cholesterol trafficking. The MCF7 human breast cancer and the SK-OV-3 human ovarian cancer cell lines were provided by the American Type Culture Collection (ATCC, Manassas, VA) and routinely maintained in our laboratory and cultured as recommended. The Chinese hamster ovary (CHO) cell line was a kind gift of Dr. L. Liscum (Tufts University, Boston, MA). The NPC2 fibroblast strain was obtained from case 16 in Vanier et al. (21Vanier M.T. Duthel S. Rodriguez-Lafrasse C. Pentchev P. Carstea E.D. Am. J. Hum. Genet. 1996; 58: 118-125PubMed Google Scholar). Human lipoprotein-deficient serum and human LDL were prepared in the laboratory as described previously (21Vanier M.T. Duthel S. Rodriguez-Lafrasse C. Pentchev P. Carstea E.D. Am. J. Hum. Genet. 1996; 58: 118-125PubMed Google Scholar). To produce stable cell lines expressing MLN64, a 1.5-kilobase Bam HI fragment corresponding to the open reading frame of the human MLN64 cDNA (GenBankTMaccession number X80198) was cloned into the Bam HI site of the pCMVneo vector (22Baker S.J. Markowitz S. Fearon E.R. Willson J.K. Vogelstein B. Science. 1990; 249: 912-915Crossref PubMed Scopus (1610) Google Scholar), thus generating the pCMVneo-MLN64 plasmid. To map the domain responsible for the sorting of MLN64, NH2-terminal deletion mutants of pSG5 MLN64 (10Moog-Lutz C. Tomasetto C. Regnier C.H. Wendling C. Lutz Y. Muller D. Chenard M.P. Basset P. Rio M.C. Int. J. Cancer. 1997; 71: 183-191Crossref PubMed Scopus (117) Google Scholar) were constructed. pSG5 MLN64-(30–445) was obtained in two steps. An intermediate plasmid was constructed by creating two in-frameNhe I sites at amino acid positions 2 and 28 by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and the synthetic oligonucleotides 1 (5′-GGGGCCCACC AGGATGGCTA GCCTGCCCAG GGAGCTGA) and 2 (5′-GGGCTCCTCA CTGTCCGCTA GCCAGAGCCT CTCCTCGC). Sequences corresponding to amino acids 2–29 were then excised by a Nhe I digest, and the remaining plasmid was recircularized to produce pSG5 MLN64-(30–445). pSG5 MLN64-(47–445) and pSG5 MLN64-(54–445) were obtained in a similar way. For pSG5 MLN64-(47–445), the two in-frame Nhe I sites at amino acid positions 2 and 45 were obtained by site-directed mutagenesis using the synthetic oligonucleotides 1 and 3 (5′-GCCTGAGAAG CGAAGGGCTA GCTCTGATGT CCGCCGCAC). For pSG5 MLN64-(54–445), the two in-frame Nhe I sites at amino acid positions 2 and 52 were obtained by site-directed mutagenesis using the synthetic oligonucleotides 1 and 4 (5′-CTCTGATGTC CGCCGCGCTA GCTGTCTCTT CGTCACCT). pSG5 MLN64-(1–218) was obtained by insertion of an in-frame stop codon at amino acid position 219 by site-directed mutagenesis using the following oligonucleotide: 5′-GCAGGGTCTG ACTAGTAATC AGATGAAG. pSG5 MLN64-(1–171) was obtained by insertion of an in-frame stop codon at amino acid position 172 by site-directed mutagenesis using the following oligonucleotide: 5′-GGTTCCTTGA CTTTTAAATC CTACCCCAGG. pSG5 MLN64-(1–145) was obtained by insertion of an in-frame stop codon at amino acid position 146 by site-directed mutagenesis using the following oligonucleotide: 5′-CTCTGAGCTG CTTTAAAAAG GGGCATTTG. pSG5 MLN64 LL1 was constructed by mutating Leu61-Leu62 to Ala61-Ser62 by site-directed mutagenesis using the following synthetic oligonucleotide: 5′-ACTTCGTCACC TTCGACGCTA GCTTCATCTC CCTGCTC. Similarly, pSG5 MLN64 LL2 was constructed by mutating Leu66-Leu67 to Ala66-Ser67 (5′-CCTGCTCTTC ATCTCCGCTA GCTGGATCAT CGAACTG), pSG5 MLN64 LL3 by mutating Leu109-Leu110 to Ala109-Ser110 (5′-CTTCCGCTTC TCTGGAGCTA GCCTAGGCTA TGCCGTGC), pSG5 MLN64 LL4 by mutating Leu144-Leu145 to Ala144-Ser145 (5′-GGTCATCCTC TCTGAGGCTA GCAGCAAAGG GGCATTTG), pSG5 MLN64 LL5 (and Y3) by mutating Tyr152-Leu153 to Ala152-Ser153 (5′-CAAAGGGGCA TTTGGCGCTA GCCTCCCCAT CGTCTCTT), pSG5 MLN64 Y1 by mutating Tyr89 to Val89 (5′-GCAGGAGATC ATCCAGGTTA ACTTTAAAAC TTCC), pSG5 MLN64 Y2 by mutating Gly112-Tyr113 to Ala112-Ser113 (5′-CTCTGGACTG CTCCTAGCTA GCGCCGTGCT GCAGCTCC). The pCR3.1 NPC1 expression plasmid was a kind gift of Dr. E. Ikonen (National Public Health Institute, Helsinki, Finland). MCF7 cells were transfected by calcium phosphate coprecipitation with either the pCMVneo-MLN64 expression vector or the pCMVneo vector, both linearized by Hin dIII. The medium was changed after 20 h, and on the following day, the selection was begun in the same medium supplemented with 400 μg/liter G418 (Life Technologies, Inc.). After 2 weeks of selection, resistant clones were subcloned, and expression of MLN64 was assessed by Western blot as described previously (10Moog-Lutz C. Tomasetto C. Regnier C.H. Wendling C. Lutz Y. Muller D. Chenard M.P. Basset P. Rio M.C. Int. J. Cancer. 1997; 71: 183-191Crossref PubMed Scopus (117) Google Scholar). The rabbit polyclonal 605 (pAbMLN64-Ct) and mouse monoclonal 2BE2F4 (mAbMLN64-Ct) antibodies were raised against the synthetic peptide HSAKPPTHKYVRGENG corresponding to residues 369–384 of human MLN64 as described previously (10Moog-Lutz C. Tomasetto C. Regnier C.H. Wendling C. Lutz Y. Muller D. Chenard M.P. Basset P. Rio M.C. Int. J. Cancer. 1997; 71: 183-191Crossref PubMed Scopus (117) Google Scholar, 23Schreiber V. Moog-Lutz C. Regnier C.H. Chenard M.P. Boeuf H. Vonesch J.L. Tomasetto C. Rio M.C. Mol. Med. 1998; 4: 675-687Crossref PubMed Google Scholar). In addition, a rabbit polyclonal antibody 1611 (pAbMLN64-Nt) was raised against the peptide MSKLPRELTRDLERSLPAV corresponding to residues 1–19 of human MLN64. The 6C4 monoclonal anti-LBPA antibody was described previously (24Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar). The M1G8 monoclonal anti-cathepsin D antibody was a generous gift from Dr. M. Garcia and Prof. H. Rochefort (INSERM U148, Montpellier, France). The monoclonal anti-CD63 antibody was purchased from Chemicon (Temecula, CA). The rabbit polyclonal anti-NPC1 antibody was a kind gift of Dr. E. Ikonen (National Public Health Institute, Helsinki, Finland). Cy3-conjugated affinity-purified donkey anti-mouse IgG- and Cy2-conjugated affinity-purified goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Biotin-conjugated donkey anti-mouse antibody was purchased from Vector Laboratories (Burlingame, CA). SK-OV-3 and MCF7 cells were grown to 70% confluence on glass coverslips. After washing with phosphate-buffered saline (PBS), cells were fixed 10 min at room temperature in 4% paraformaldehyde in PBS and permeabilized for 10 min with 0.1% Triton X-100 in PBS. After blocking in 1% bovine serum albumin in PBS, cells were incubated at room temperature with the primary antibodies, either pAbMLN64-Ct or mAbMLN64-Ct together with anti-cathepsin D M1G8, anti-LBPA 6C4, anti-CD63, or anti-NPC1. Cells were washed three times in PBS and incubated 1 h with Cy3- and Cy2-conjugated secondary antibodies (1:400). Cells were washed three times in PBS, and in some cases, nuclei were counterstained with Hoechst-33258 dye. Slides were mounted in Aqua Poly/Mount (Polysciences Inc., Warrington, PA). Observations were made with a confocal microscope (Leica TCS4D; Heidelberg, Germany) or with a fluorescence microscope (Leica DMLB 30T). MCF7 cells were grown on glass coverslips to 70% confluence. After washing with PBS, cells were fixed 45 min at room temperature in 2.5% glutaraldehyde in PBS followed by a 15-min incubation in 1% sodium borohydrure in PBS. Cells were then permeabilized for 20 min with 0.1% saponin in PBS. After blocking for 30 min in 5% normal donkey serum in PBS, cells were incubated overnight at 4 °C with mAbMLN64-Ct antibody (1:1000 dilution). After three washes with PBS, cells were incubated 1.5 h with biotin-conjugated donkey anti-mouse antibody. Cells were then processed for immunoperoxidase labeling using the Vectastain Elite ABC standard kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions, which was followed by incubation with 0.0125% diaminobenzidine and 0.005% H2O2 in 0.05 m Tris buffer, pH 7.6. Cells were postfixed with 2.5% glutaraldehyde in PBS and then with 2% OsO4 for 30 min. They were then dehydrated with graded concentrations of ethanol and conventionally embedded in epoxy resin. Glass coverslips were dissolved in hydrofluoric acid, and ultrathin sections were observed under a transmission electron microscope (Hitachi 7500, Japan). MCF7 cells, on glass coverslips, were washed three times with Dulbecco's modified Eagle's medium without serum. For endocytosis of antibodies, cells were incubated for 1 h with either anti-cathepsin D M1G8, mAbMLN64-Ct, or pAbMLN64-Nt antibodies. Antibodies were used at the same concentration as for immunofluorescence. Antibodies were microinjected into the cell cytoplasm together with lysine fixable dextran fluorescein 40000 MW (Molecular Probes, Inc., Eugene, OR) as described previously (25Dierich A. Gaub M.P. Le Pennec J.P. Astinotti D. Chambon P. EMBO J. 1987; 6: 2305-2312Crossref PubMed Scopus (41) Google Scholar). Microinjections were followed by an incubation of the cells for 1 h. Antibodies were used 10 times more concentrated than for immunofluorescence. Detection of either internalized or microinjected antibodies was performed by incubating fixed cells with the secondary antibody as described above (see “Immunocytofluorescence”). Niemann-Pick C2 fibroblasts were grown on glass coverslips to 70% confluence and were transfected with the expression vector pSG5 MLN64 with FuGENE6 transfection reagent (Roche Molecular Biochemicals). After transfection, cells were cultured in medium supplemented with 5% lipoprotein-deficient serum for 24 h and then changed to medium supplemented with 5% lipoprotein-deficient serum and 50 μg/ml human LDL for an additional 24 h of culture. Cells were then processed for immunofluorescence to identify transfected cells with mAbMLN64-Ct anti-MLN64 as described above (see “Immunocytofluorescence”) except that permeabilization with Triton X-100 was omitted. Staining of free cholesterol was performed after fixation using 50 μg/ml filipin (Sigma) for 30 min. To assess the subcellular localization of the MLN64 protein, we performed indirect immunofluorescence experiments using two cancer-derived cell lines. A human ovarian cancer cell line, SK-OV-3, that overexpresses MLN64 at both the RNA and protein levels and a human breast cancer cell line, MCF7, that does not express detectable levels of MLN64 mRNA were chosen (4Tomasetto C. Regnier C. Moog-Lutz C. Mattei M.G. Chenard M.P. Lidereau R. Basset P. Rio M.C. Genomics. 1995; 28: 367-376Crossref PubMed Scopus (228) Google Scholar, 10Moog-Lutz C. Tomasetto C. Regnier C.H. Wendling C. Lutz Y. Muller D. Chenard M.P. Basset P. Rio M.C. Int. J. Cancer. 1997; 71: 183-191Crossref PubMed Scopus (117) Google Scholar). A punctuate cytoplasmic staining was observed using an anti-MLN64 antibody in the SK-OV-3 cells (Fig.1 A, a), whereas no protein expression could be detected in MCF7 cells (Fig. 1 A,c). MCF7 cells overexpressing MLN64 (MCF7/MLN64) showed a similar punctuate staining as observed for SK-OV-3 cells (Fig.1 A, b). To identify the subcellular structures where the MLN64 protein resides, MCF7/MLN64 cells were colabeled with antibodies directed against known organelle-resident proteins or lipids. Double staining experiments using MLN64 and cathepsin D or LampI, two markers of both endosomes and lysosomes (26Laurent-Matha V. Farnoud M.R. Lucas A. Rougeot C. Garcia M. Rochefort H. J. Cell Sci. 1998; 111: 2539-2549PubMed Google Scholar, 24Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar), showed a large overlap of both signals, the relative quantity of both proteins being variable from one vesicle to another (Fig. 1 B,a–c, and data not shown). Double labeling experiments with MLN64 and CD63, a marker for all types of endosomes (27Metzelaar M.J. Wijngaard P.L. Peters P.J. Sixma J.J. Nieuwenhuis H.K. Clevers H.C. J. Biol. Chem. 1991; 266: 3239-3245Abstract Full Text PDF PubMed Google Scholar), showed also a large overlap of both signals, with the relative quantity of both proteins again being variable from one vesicle to another (Fig.1 B, d–f). Colocalization of MLN64 and LBPA, a lipid restricted to late endosomes (24Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar), showed that both signals completely overlapped (Fig. 1 B, g–i). Thus, among the endosome/lysosome vesicles, MLN64 appeared to specifically reside in the late endosomes. In addition, we noted that the immunocytofluorescence staining of MLN64 appeared as a ring on most of the endosomes (Fig. 1 B, g–i). When the cells are costained with anti-LBPA, a lipid exclusively located in late endosomes (24Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar), MLN64 staining surrounded LBPA-stained spheres (Fig.1 B, i). To identify endosomal addressing signals, we analyzed the MLN64 protein sequence for known motifs. MLN64 possesses four putative transmembrane domains (10Moog-Lutz C. Tomasetto C. Regnier C.H. Wendling C. Lutz Y. Muller D. Chenard M.P. Basset P. Rio M.C. Int. J. Cancer. 1997; 71: 183-191Crossref PubMed Scopus (117) Google Scholar). As a control of the functionality of these transmembrane domains, we decided to analyze potential N-glycosylation sites, which have been shown to direct luminal proteins to endosomes/lysosomes (28Kornfeld S. Annu. Rev. Biochem. 1992; 61: 307-330Crossref PubMed Scopus (936) Google Scholar). MLN64 contains two potential sites of N-glycosylation at positions 219 and 311; we performed site-directed mutagenesis of these amino acids (Asn219 → Ala219 and Asn311→ Ser311) (Table I). The localization of mutated proteins was compared with a marker of late endosomes (data not shown). Neither single mutants nor the double mutant have a modified subcellular localization. These data indicate that putative N-glycosylation of MLN64 are not involved in the sorting of MLN64, which strongly suggests that the transmembrane domains of the protein are functional. The contribution of another sorting signal typical of endosomal transmembrane protein was therefore tested. The GYXXZ motif (where Z represents an aliphatic or aromatic residue) is responsible for the sorting of the Lamp proteins to lysosome and endosome membranes (29Hunziker W. Geuze H.J. Bioessays. 1996; 18: 379-389Crossref PubMed Scopus (238) Google Scholar). MLN64 contains one such motif at positions 112–116. Site-directed mutagenesis of this motif did not alter the endosomal localization of the protein (TableI). Therefore, this motif is not involved in the sorting of MLN64. Since common signals were not responsible for the addressing of MLN64, we decided to map the MLN64 endosomal sorting domain using recurrent deletions in the NH2 and COOH termini of the protein.Table ISequences and positions of the potential N-glycosylation sites and GYXXZ type sorting motif of MLN64MutagenesisLocalization of the mutant proteinAsn219 → AlaEndosomesAsn311 → SerEndosomesAsn219 → Ala, Asn311 → SerEndosomesGY113AVL → ASAVLEndosomesMutagenesis of these sites on the cDNA of MLN64 cloned in the pSG5 expression vector and the subcellular localization of the resulting mutant proteins are indicated. Open table in a new tab Mutagenesis of these sites on the cDNA of MLN64 cloned in the pSG5 expression vector and the subcellular localization of the resulting mutant proteins are indicated. Three NH2-terminal deletions were constructed by site-directed mutagenesis in the eukaryotic expression vector pSG5 MLN64 (Fig. 2 A). Subcellular localization was assayed by transient transfection in MCF7 cells followed by immunofluorescence using pAbMLN64-Ct. The localization of mutated proteins was compared with a marker of late endosomes (data not shown). Deletions of the NH2-terminal part to position 29 or 46 (pSG5 MLN64-(30–445) and pSG5 MLN64-(47–445)) showed a localization (Fig. 2 B, b) similar to the wild-type protein (Fig. 2 B, a). Further NH2-terminal deletion (pSG5 MLN64-(54–445)) involving the transmembrane domains of the protein led to a mislocalization of the protein, which then exhibited a reticular pattern characteristic of the endoplasmic reticulum (Fig. 2 B, c). Similarly, three COOH-terminal deletions were constructed (Fig. 2 A), and subcellular localization was assayed using pAbMLN64-Nt. The localization of mutated proteins was compared with a marker of late endosomes (data not shown). When the COOH-terminal part of the protein is deleted from amino acids 219 or 172 to 445 (pSG5 MLN64-(1–218), pSG5 MLN64-(1–171)), the protein remained in endosomes (Fig.2 B, d and e). Further deletion (pSG5 MLN64-(1–145)) including the transmembrane helices-containing domain led to a modified localization of the protein, which then resided in the Golgi apparatus (Fig. 2 B, f). NH2- and COOH-terminal deletions showed that the minimal domain for sorting to the endosome was included within amino acids 47–171 of the MLN64 protein. Further deletions of the protein would include the transmembrane helices-containing domain and could therefore modify the overall structure of MLN64 and its anchorage to the membrane. Thus, we decided to perform point mutations within the sequence located from residue 47 to 172 to identify amino acids involved in the sorting. Most of the membrane proteins located in the endosome/lysosome exhibit dileucine or tyrosine type addressing signals (30Kirchhausen T. Bonifacino J.S. Riezman H. Curr. Opin. Cell Biol. 1997; 9: 488-495Crossref PubMed Scopus (352) Google Scholar). We therefore decided to systematically mutate tyrosine and dileucine motifs within the minimal region necessary to target the protein to endosomes. Five dileucine motifs (LL) are present in this region of the protein (Fig.3 A, LL1–LL5). Mutations of four of them (LL1 and LL3–LL5) did not change the localization of the protein (Fig. 3 B, a; data not shown) when compared with a marker of late endosomes (data not shown). In contrast, mutation of LL2 located at positions 66 and 67 led to a mislocalization of the protein to the endoplasmic reticulum (Fig. 3 B,b). Three different tyrosines (Y1–Y3) are present between amino acids 47 and 172. Y2 is included in the previously studied GYXXZ motif (Table I). Mutations of Y2 or Y3 did not change the subcellular localization of MLN64 (Fig. 3 B,c; data not shown) when compared with a marker of late endosomes (data not shown). On the contrary, the mutation of Y1 (residue 89) modified the subcellular localization of the protein, which then resided in the endoplasmic reticulum (Fig. 3 B,d). Thus, two motifs, the dileucine motif 66–67 and the tyrosine 89 are important for proper addressing of MLN64 to late endosomes. The immunocytofluorescence staining of MLN64 (Fig.1 B, g–i) suggested that MLN64 localizes to the limiting membrane of endosomes. To confirm this localization of MLN64, we performed immunoelectron microscopy on MCF7/MLN64 cells using the mAbMLN6" @default.
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