Cholesterol-binding molecules MLN64 and ORP1L mark distinct late endosomes with transporters ABCA3 and NPC1
2013; Elsevier BV; Volume: 54; Issue: 8 Linguagem: Inglês
10.1194/jlr.m037325
ISSN1539-7262
AutoresRik van der Kant, Ilse Zondervan, Lennert Janssen, Jacques Neefjes,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoCholesterol is an essential lipid in eukaryotic cells and is present in membranes of all intracellular compartments. A major source for cellular cholesterol is internalized lipoprotein particles that are transported toward acidic late endosomes (LE) and lysosomes. Here the lipoprotein particles are hydrolyzed, and free cholesterol is redistributed to other organelles. The LE can contain over half of the cellular cholesterol and, as a major sorting station, can contain many cholesterol-binding proteins from the ABCA, STARD, and ORP families. Here, we show that metastatic lymph node 64 (MLN64, STARD3) and oxysterol-binding protein-related protein 1L (ORP1L) define two subpopulations of LE. MLN64 is present on a LE containing the cholesterol transporter ABCA3, whereas ORP1L localizes to another population of LE containing Niemann Pick type C1 (NPC1), a cholesterol exporter. Endocytosed cargo passes through MLN64/ABCA3-positive compartments before it reaches ORP1L/NPC1-positive LE. The MLN64/ABCA3 compartments cycle between LE and plasma membrane and frequently contact "later" ORP1L/NPC1-containing LE. We propose two stages of cholesterol handling in late endosomal compartments: first, cholesterol enters MLN64/ABCA3-positive compartments from where it can be recycled to the plasma membrane, and later, cholesterol enters ORP1L/NPC1 endosomes that mediate cholesterol export to the endoplasmic reticulum. Cholesterol is an essential lipid in eukaryotic cells and is present in membranes of all intracellular compartments. A major source for cellular cholesterol is internalized lipoprotein particles that are transported toward acidic late endosomes (LE) and lysosomes. Here the lipoprotein particles are hydrolyzed, and free cholesterol is redistributed to other organelles. The LE can contain over half of the cellular cholesterol and, as a major sorting station, can contain many cholesterol-binding proteins from the ABCA, STARD, and ORP families. Here, we show that metastatic lymph node 64 (MLN64, STARD3) and oxysterol-binding protein-related protein 1L (ORP1L) define two subpopulations of LE. MLN64 is present on a LE containing the cholesterol transporter ABCA3, whereas ORP1L localizes to another population of LE containing Niemann Pick type C1 (NPC1), a cholesterol exporter. Endocytosed cargo passes through MLN64/ABCA3-positive compartments before it reaches ORP1L/NPC1-positive LE. The MLN64/ABCA3 compartments cycle between LE and plasma membrane and frequently contact "later" ORP1L/NPC1-containing LE. We propose two stages of cholesterol handling in late endosomal compartments: first, cholesterol enters MLN64/ABCA3-positive compartments from where it can be recycled to the plasma membrane, and later, cholesterol enters ORP1L/NPC1 endosomes that mediate cholesterol export to the endoplasmic reticulum. Cholesterol is a key component of cellular membranes and is essential for the regulation of membrane rigidity, cellular compartmentalization, enzyme function, and the production of steroid hormones (1Ikonen E. Cellular cholesterol trafficking and compartmentalization.Nat. Rev. Mol. Cell Biol. 2008; 9: 125-138Crossref PubMed Scopus (996) Google Scholar). A major source of cellular cholesterol is the endocytosis of lipoproteins from the cell surface and subsequent processing of these particles in late endosomal (LE) and lysosomal compartments. At the plasma membrane of cells, low-density lipoprotein receptors (LDLR) internalize lipoprotein particles by clathrin-coated endocytosis. These lipoprotein particles, including VLDL and LDL, are a major source of cholesterol in most cells. After initial endocytosis, the lipoproteins are transported toward acid LE compartments where cholesteryl esters are hydrolized by acid lipases to generate free unesterified cholesterol (1Ikonen E. Cellular cholesterol trafficking and compartmentalization.Nat. Rev. Mol. Cell Biol. 2008; 9: 125-138Crossref PubMed Scopus (996) Google Scholar). This free cholesterol can subsequently be transferred to other compartments, such as recycling endosomes, the endoplasmic reticulum (ER), the plasma membrane (PM), and mitochondria (1Ikonen E. Cellular cholesterol trafficking and compartmentalization.Nat. Rev. Mol. Cell Biol. 2008; 9: 125-138Crossref PubMed Scopus (996) Google Scholar, 2Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. Intracellular trafficking of cholesterol monitored with a cyclodextrin.J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 3Lange Y. Ye J. Chin J. The fate of cholesterol exiting lysosomes.J. Biol. Chem. 1997; 272: 17018-17022Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar–4Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss 3rd, J.F. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.J. Biol. Chem. 2002; 277: 33300-33310Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Late (multivesicular) endosomes can contain over half of the total cellular cholesterol content (5Mobius W. van Donselaar E. Ohno-Iwashita Y. Shimada Y. Heijnen H.F. Slot J.W. Geuze H.J. Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway.Traffic. 2003; 4: 222-231Crossref PubMed Scopus (346) Google Scholar), and many cholesterol-binding proteins localize to these compartments to control the flow of intercellular cholesterol. How the functions of these proteins relate to each other and how they communicate to distribute the LE cholesterol to other compartments is unclear. Metastatic lymph node 64 (MLN64), also known as STARD3, is a lipid-binding protein from the steroidogenic acute regulatory protein (StAR)-related lipid transfer domain (START) protein family with two functional domains: a C-terminal domain containing the START domain that projects into the cytoplasm and an N-terminal 4-transmembrane domain (6Alpy F. Stoeckel M.E. Dierich A. Escola J.M. Wendling C. Chenard M.P. Vanier M.T. Gruenberg J. Tomasetto C. Rio M.C. The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein.J. Biol. Chem. 2001; 276: 4261-4269Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). MLN64 is reported to be involved in the egress of cholesterol from LE to mitochondria (4Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss 3rd, J.F. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.J. Biol. Chem. 2002; 277: 33300-33310Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 7Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and it regulates late endosomal tethering and fusion dynamics involving actin (8Holtta-Vuori M. Alpy F. Tanhuanpaa K. Jokitalo E. Mutka A.L. Ikonen E. MLN64 is involved in actin-mediated dynamics of late endocytic organelles.Mol. Biol. Cell. 2005; 16: 3873-3886Crossref PubMed Scopus (62) Google Scholar). Another cholesterol-binding protein that controls LE dynamics is the oxysterol-binding protein-related protein 1L (ORP1L). ORP1L is recruited to LE membranes by binding to the small GTPase RAB7, and it is part of a tripartite complex of RAB7, RAB7-interacting lysosomal protein (RILP), and ORP1L that regulates recruitment of the dynein motor and the homotypic fusion and vacuole protein sorting (HOPS) complex to late endosomes and thereby minus-end transport and tethering (9Johansson M. Rocha N. Zwart W. Jordens I. Janssen L. Kuijl C Olkkonen V.M. Neefjes J. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor betalll spectrin.J. Cell Biol. 2007; 176: 459-471Crossref PubMed Scopus (352) Google Scholar, 10Rocha N. Kuijl C. van der Kant R. Janssen L. Houben D. Janssen H. Zwart W. Neefjes J. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.J. Cell Biol. 2009; 185: 1209-1225Crossref PubMed Scopus (457) Google Scholar, 11Johansson M. Lehto M. Tanhuanpaa K. Cover T.L. Olkkonen V.M. The oxysterol-binding protein homologue ORP1L interacts with Rab7 and alters functional properties of late endocytic compartments.Mol. Biol. Cell. 2005; 16: 5480-5492Crossref PubMed Scopus (165) Google Scholar–12van der Kant, R., Fish, A., Janssen, L., Janssen, H., Krom, S., Ho, N., Brummelkamp, T., Carette, J., Rocha, N., Neefjes, J. Late endosomal transport and tethering are coupled processes controlled by RILP and the cholesterol sensor ORP1L. J. Cell Sci. Epub ahead of print. http://www.ncbi.nlm.nih.gov/pubmed/23729732.Google Scholar). Although ORP1L is probably not directly involved in shuttling cholesterol out of the LE, it is able to sense the amount of cholesterol on the cytosolic face of the LE-limiting membrane and induces contact sites between LE and the ER to control binding of the dynein motor and the HOPS complex to RAB7-RILP (10Rocha N. Kuijl C. van der Kant R. Janssen L. Houben D. Janssen H. Zwart W. Neefjes J. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.J. Cell Biol. 2009; 185: 1209-1225Crossref PubMed Scopus (457) Google Scholar, 12van der Kant, R., Fish, A., Janssen, L., Janssen, H., Krom, S., Ho, N., Brummelkamp, T., Carette, J., Rocha, N., Neefjes, J. Late endosomal transport and tethering are coupled processes controlled by RILP and the cholesterol sensor ORP1L. J. Cell Sci. Epub ahead of print. http://www.ncbi.nlm.nih.gov/pubmed/23729732.Google Scholar). Cholesterol levels thus control positioning and tethering of LE. Another protein associating with the RAB7-RILP complex that controls LE transport is neuronal ceroid lipofuscinosis protein CLN3. When mutated, this protein causes classical juvenile onset neuronal ceroid lipofuscinosis, a fatal inherited neurodegenerative lysosomal storage disorder in which lipopigments accumulate in lysosomes (13Uusi-Rauva K. Kyttala A. van der Kant R. Vesa J. Tanhuanpää K. Neefjes J. Olkonnen V.M. Jalanko A. Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments.Cell. Mol. Life Sci. 2012; 69: 2075-2089Crossref PubMed Scopus (61) Google Scholar). Proteins of the ATP binding cassette class A (ABCA) proteins have recently been shown to be present in LE compartments due to a conserved targeting motif (14Beers M.F. Hawkins A. Shuman H. Zhao M. Newitt J.L. Maguire J.A. Ding W. Mulugeta S. A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments.J. Lipid Res. 2011; 52: 1471-1482Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). ABCA proteins are transporters that use ATP to drive translocation of various substrates, including lipids, across membranes. ABCA3 probably functions as lipid pump for the translocation of phospholipids and cholesterol into lysosomal-like organelles (lamellar bodies) (15Cheong N. Zhang H. Madesh M. Zhao M. Yu K. Dodia C. Fisher A.B. Savani R.C. Shuman H. ABCA3 is critical for lamellar body biogenesis in vivo.J. Biol. Chem. 2007; 282: 23811-23817Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar).The different cholesterol-binding proteins on late endosomes are likely involved in different steps of cholesterol transport and handling, and different diseases are associated with these molecules. Mutations in ABCA3 cause fatal respiratory distress syndrome in newborns, probably due to a defect in lamellar body biogenesis where exocytosis is required for the formation of the lipid surface-lining layer of the alveolar epithelium of lungs (15Cheong N. Zhang H. Madesh M. Zhao M. Yu K. Dodia C. Fisher A.B. Savani R.C. Shuman H. ABCA3 is critical for lamellar body biogenesis in vivo.J. Biol. Chem. 2007; 282: 23811-23817Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Cholesterol egress from the LE into the ER requires proteins such as Niemann Pick type C protein 1 (NPC1) and NPC2, which transfer cholesterol from the lumen of the LE into the LE-limiting membrane (16Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Crossref PubMed Scopus (698) Google Scholar, 17Mukherjee S. Maxfield F.R. Lipid and cholesterol trafficking in NPC.Biochim. Biophys. Acta. 2004; 1685: 28-37Crossref PubMed Scopus (166) Google Scholar). Mutations in the Niemann Pick type C proteins cause a fatal, neurodegenerative disorder in which cholesterol accumulates in LE and disturbs transport when the altered cholesterol content is sensed by ORP1L (10Rocha N. Kuijl C. van der Kant R. Janssen L. Houben D. Janssen H. Zwart W. Neefjes J. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.J. Cell Biol. 2009; 185: 1209-1225Crossref PubMed Scopus (457) Google Scholar). How cholesterol is transferred from the LE to the ER is debated. Cholesterol could be directly transferred from the LE to the ER (18Underwood K.W. Andemariam B. McWilliams G.L. Liscum L. Quantitative analysis of hydrophobic amine inhibition of intracellular cholesterol transport.J. Lipid Res. 1996; 37: 1556-1568Abstract Full Text PDF PubMed Google Scholar) possibly mediated by an ER-resident protein of the oxysterol-binding protein family (ORP5) (19Du X. Kumar J. Ferguson C. Schulz T.A. Ong Y.S. Hong W. Prinz W.A. Parton R.G. Brown A.J. Yang H. A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking.J. Cell Biol. 2011; 192: 121-135Crossref PubMed Scopus (223) Google Scholar). However, only about 30% LE cholesterol is directly transported from LE to the ER, whereas 70% passes through the plasma membrane prior to esterification in the ER (2Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. Intracellular trafficking of cholesterol monitored with a cyclodextrin.J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). LE cholesterol is rapidly moved toward the plasma membrane, whereas reesterification in the ER shows a 0.5–1 h lag, arguing against direct LE to ER movement of cholesterol (3Lange Y. Ye J. Chin J. The fate of cholesterol exiting lysosomes.J. Biol. Chem. 1997; 272: 17018-17022Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). MLN64 has been shown to egress cholesterol from LE to mitochondria even in the absence of functional Niemann-Pick type C1 proteins (4Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss 3rd, J.F. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.J. Biol. Chem. 2002; 277: 33300-33310Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 7Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Apart from these findings, little is known about the exact localization and cross-talk between the different cholesterol sensing and transferring proteins on the LE. Here, we address the location of MLN64 in relation to ORP1L. We show that MLN64 and ORP1L define two distinct LE compartments. Whereas MLN64 is present in "early" ABCA3-positive LE compartments, ORP1L defines a population of "late" late endosomes labeled by the cholesterol transporter NPC1. The subcompartments possibly reflect sites where LE cholesterol is handled differently to ensure proper cellular homeostasis of this essential and complex hydrophobic molecule. Rabbit anti-GFP and rabbit anti-mRFP antibodies were generated in-house using purified His-mRFP and His-GFP recombinant proteins, respectively. The rabbit-anti MLN64 serum was generated in-house using purified His-START domain of human MLN64. Other antibodies used were anti-NPC1 (Novus Biologicals), rabbit anti-ORP1L (gift from V. Olkkonen, National Public Health Institute, Helsinki, Finland), mouse anti-CD63 (20Vennegoor C. Rumke P. Circulating melanoma-associated antigen detected by monoclonal antibody NKI/C-3.Cancer Immunol. Immunother. 1986; 23: 93-100Crossref PubMed Scopus (19) Google Scholar), mouse monoclonal anti-EEA1 (BD Biosciences), anti-HA (Roche), anti-EEA1 (BD Biosciences), anti-M6PR (Affinity Bioregeants), and anti-NPC1 (BD Biosciences). MelJuSo cells were cultured in Iscove's modified Dulbecco's medium (IMDM; Invitrogen) supplemented with 8% FCS. SKBR3 cells were cultured in Dulbeco's modified Eagle medium (DMEM) supplemented with 8% FCS in a 5% CO2-humidified culture hood at 37°C. Transfected cells were fixed 24 h posttransfection with 4% formaldehyde in PBS for 30 min and permeabilized for 5 min with 0.05% Triton X-100 in PBS at room temperature. Nonspecific binding of antibodies was blocked by 0.5% BSA in PBS for 40 min, after which cells were incubated with primary antibodies in 0.5% BSA in PBS for 1 h at room temperature. Bound primary antibodies were visualized with Alexa-Fluor secondary antibody conjugates (Invitrogen). Cells were mounted in Vectashield mounting medium (Vector Laboratories). For methanol fixation, cells were fixed for 5 min in ice-cold methanol, subsequently blocked by 0.5% BSA in PBS for 40 min, and treated similar to the formaldehyde-fixed samples. All specimens were analyzed by confocal laser-scanning microscopes (TCS-SP1, TCS-SP2, or AOBS; Leica) equipped with HCX Plan-Apochromat 63× NA 1.32 and HCX Plan-Apochromat lbd.bl 63× NA 1.4 oil-corrected objective lenses (Leica). The acquisition software used was LCS (Leica). RILP and ORP1L constructs have been described previously (9Johansson M. Rocha N. Zwart W. Jordens I. Janssen L. Kuijl C Olkkonen V.M. Neefjes J. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor betalll spectrin.J. Cell Biol. 2007; 176: 459-471Crossref PubMed Scopus (352) Google Scholar, 10Rocha N. Kuijl C. van der Kant R. Janssen L. Houben D. Janssen H. Zwart W. Neefjes J. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.J. Cell Biol. 2009; 185: 1209-1225Crossref PubMed Scopus (457) Google Scholar, 21Jordens I. Fernandez-Borja M. Marsman M. Dusseljee S. Janssen L. Calafat J. Janssen H. Wubbolts R. Neefjes J. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.Curr. Biol. 2001; 11: 1680-1685Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). YFP-MLN-64 was a kind gift from Dr. F. Alpy (Département de Pahtologie Moléculaire, Insitut de Genetique et de Biologie Moleculaire et Cellulaire, Communauté Urbaine de Strasbourg, France). YFP-MLN64 M307R, N311D was generated using site-directed mutagenesis and was sequence verified. Cells transfected with ORP1L, ΔORD, ΔORDPHDPHD, MLN64, or combinations of these were incubated at 37°C with Alexa647-conjugated ovalbumin (Molecular Probes) or Dil-LDL (Molecular Probes, final concentration 20 μg/ml) in serum-free medium for 20 min. After washing, cells were cultured, fixed after various culturing times, and then analyzed as described. To measure MLN64 accumulation at the plasma membrane, a region of interest (vector) was drawn over cells and a histogram showing the intensity over the vector was generated in ImageJ. For the other figures, the Manders M1 and M2 coefficients (the percentage of channel A that colocalizes with channel B and reverse) were calculated using the JaCoP plugin in ImageJ. To study the endogenous localization of MLN64, we generated a rabbit anti-serum antibody against the C-terminal START domain of human MLN64. This anti-serum recognized MLN64 by WB (Fig. 1A); human MLN64 has a predicted molecular weight of 49 kDa. Expression levels of MLN64 are reported to be similar in most tissues, with the exception of higher expression in lung and blood cells (8Holtta-Vuori M. Alpy F. Tanhuanpaa K. Jokitalo E. Mutka A.L. Ikonen E. MLN64 is involved in actin-mediated dynamics of late endocytic organelles.Mol. Biol. Cell. 2005; 16: 3873-3886Crossref PubMed Scopus (62) Google Scholar, 22Wu C. Orozco C. Boyer J. Leglise M. Goodale J. Batalov S. Hodge C.L. Haase J. Janes J. Huss 3rd, J.W. Su A.I. BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources.Genome Biol. 2009; 10: R130Crossref PubMed Scopus (1093) Google Scholar). We were unable to detect significant endogenous MLN64 in HeLa and MelJuSo cells, but we could detect endogenous MLN64 in the breast cancer cell line SKBR3 in which MLN64 is amplified. To study the localization of MLN64, we fixed untransfected and MLN64-YFP-transfected SKBR3 cells with paraformaldehyde (PFA), permeabilized cells with Triton-X100, and stained for MLN64 using our antibody. Our antibody recognized high levels of MLN64 in MLN64-YFP-transfected cells (Fig. 1B). In untransfected cells, endogenous MLN64 showed a vesicular staining pattern in addition to a nuclear background staining. Vesicular staining was not observed when cells were stained with preimmune serum (supplementary Fig. I). Previous studies employing tagged MLN64 revealed that MLN64 is a late endosomal protein (4Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss 3rd, J.F. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.J. Biol. Chem. 2002; 277: 33300-33310Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 6Alpy F. Stoeckel M.E. Dierich A. Escola J.M. Wendling C. Chenard M.P. Vanier M.T. Gruenberg J. Tomasetto C. Rio M.C. The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein.J. Biol. Chem. 2001; 276: 4261-4269Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 7Charman M. Kennedy B.E. Osborne N. Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein.J. Lipid Res. 2010; 51: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar–8Holtta-Vuori M. Alpy F. Tanhuanpaa K. Jokitalo E. Mutka A.L. Ikonen E. MLN64 is involved in actin-mediated dynamics of late endocytic organelles.Mol. Biol. Cell. 2005; 16: 3873-3886Crossref PubMed Scopus (62) Google Scholar).We confirmed the localization of endogenous MLN64 at CD63-marked late endosomes (Fig. 1C). MLN64 was absent from early endosomes (EEA1 labeled) or mannose-6-phosphate-receptor (M6PR)-positive vesicles (Fig. 1C). Fixation and permeabilization can affect epitope accessibility (23Schnell U. Dijk F. Sjollema K.A. Giepmans B.N. Immunolabeling artifacts and the need for live-cell imaging.Nat. Methods. 2012; 9: 152-158Crossref PubMed Scopus (324) Google Scholar). As an alternative for PFA/Triton-X100 fixation, methanol can also be used. Under these conditions, we again observed a vesicular staining of MLN64 that colocalized with CD63. In addition, we detected staining for MLN64 at the plasma membrane (Fig. 1D). Our data suggest that endogenous MLN64 is present at the plasma membrane and in late endosomes. Although lysosomes are the supposedly end-stage for most cargo internalized from the plasma membrane, many lysosomal proteins are found at the plasma membrane as the result of late endosomal-plasma membrane fusion. These include LE markers, such as CD63 and CD83, which are usually rapidly recycled back to late endosomes by targeting signals in their cytoplasmic tail (24Pols M.S. Klumperman J. Trafficking and function of the tetraspanin CD63.Exp. Cell Res. 2009; 315: 1584-1592Crossref PubMed Scopus (456) Google Scholar, 25Klein E. Koch S. Borm B. Neumann J. Herzog V. Koch N. Bieber T. CD83 localization in a recycling compartment of immature human monocyte-derived dendritic cells.Int. Immunol. 2005; 17: 477-487Crossref PubMed Scopus (29) Google Scholar). When we expressed YFP-tagged MLN64 at high levels, we observed a small fraction at the plasma membrane (Fig. 2A). To further test that MLN64 cycles between the plasma membrane and LE, we blocked endocytosis by expression of dominant-negative (DN) dynamin (26Lee A. Frank D.W. Marks M.S. Lemmon M.A. Dominant-negative inhibition of receptor-mediated endocytosis by a dynamin-1 mutant with a defective pleckstrin homology domain.Curr. Biol. 1999; 9: 261-264Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). MLN64 accumulated at the plasma membrane when endocytosis was blocked (Fig. 2B). MLN64 is proposed to regulate late endosomal dynamics. Silencing of MLN64 results in dispersion of late endosomes and a delay in degradation of endosomal cargo (8Holtta-Vuori M. Alpy F. Tanhuanpaa K. Jokitalo E. Mutka A.L. Ikonen E. MLN64 is involved in actin-mediated dynamics of late endocytic organelles.Mol. Biol. Cell. 2005; 16: 3873-3886Crossref PubMed Scopus (62) Google Scholar). Another cholesterol-binding protein that controls late endosomal dynamics is ORP1L. ORP1L regulates late endosomal transport by recruiting the ER protein VAP-A, which removes dynein from LE (10Rocha N. Kuijl C. van der Kant R. Janssen L. Houben D. Janssen H. Zwart W. Neefjes J. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.J. Cell Biol. 2009; 185: 1209-1225Crossref PubMed Scopus (457) Google Scholar). ORP1L also regulates multivesicular body formation (27Kobuna H. Inoue T. Shibata M. Gengyo-Ando K. Yamamoto A. Mitani S. Arai H. Multivesicular body formation requires OSBP-related proteins and cholesterol.PLoS Genet. 2010; 6: (pii: e1001055)Crossref PubMed Scopus (48) Google Scholar). As both ORP1L and MLN64 localize to late endosomes, we hypothesized that they could act in assembly to control cholesterol-dependent processes at LE. However, we observed that only a minor fraction of ORP1L and MLN64 colocalized on the same vesicles, whereas the majority of ORP1L and MLN64 localized to different compartments (Fig. 3A). ORP1L was located on (and clustered) small CD63-positive LE, whereas MLN64 was located on other, enlarged LE compartments that also contained the LE marker CD63. In fact, these cholesterol-sensing proteins subdivided the late endosomes. MLN64 and ORP1L vesicles were located adjacent to each other, while colocalization was observed on only a few vesicles. To further study the dynamics of the ORP1L and MLN64 vesicles, we performed live-cell imaging on cells overexpressing ORP1L and MLN64 (supplementary Movie I and Fig. 3B). Again, we observed mutual exclusion of ORP1L and MLN64 on vesicles that closely localized. Vesicles with ORP1L moved together with vesicles containing MLN64 over long distances in the periphery of cells, indicating some sort of interaction or tethering between these vesicles. To further verify these findings, we expressed ORP1L in cells and stained for endogenous MLN64 or expressed MLN64 while staining for ORP1L (supplementary Fig. II). Again a predominant mutual exclusion of closely adjacent vesicles of both types was observed. ORP1L is part of a tripartite complex of RAB7-RILP-ORP1L. As ORP1L and MLN64 are on different vesicles, we wondered whether RILP and MLN64 are also mutually exclusive. Indeed, whereas ORP1L and RILP localized to same vesicles (Fig. 3C, upper panel), RILP and MLN64 localized to different late endosomal subpopulations (Fig. 3C, lower panel). Together, our data suggest that MLN64 and RAB7-RILP-ORP1L localize to distinct late endosomal subpopulations. ORP1L and MLN64 both contain a cholesterol-sensing domain that can bind cholesterol at the cytosolic site of the late endosomal-limiting membrane. To test whether these domains are important for the mutual exclusion of MLN64 and ORP1L within the late endosomal compartment, we expressed proteins with mutations in these domains. The START domain of MLN64 requires two amino acids (M307 and N311) for cholesterol binding; a MLN64(M307R, N311D) mutation has been reported to be deficient in cholesterol binding (8Holtta-Vuori M. Alpy F. Tanhuanpaa K. Jokitalo E. Mutka A.L. Ikonen E. MLN64 is involved in actin-mediated dynamics of late endocytic organelles.Mol. Biol. Cell. 2005; 16: 3873-3886Crossref PubMed Scopus (62) Google Scholar). We verified the late endosomal location of a MLN64(M307R, N311D)-YFP construct (Fig. 4A). Expression of this construct with ORP1L again yielded mutual exclusion of both proteins within late endosomes (Fig. 4B, upper panel), suggesting that cholesterol binding by the MLN64 START domain is not essential for spatial segregation of MLN64 and ORP1L. We then tested whether the cholesterol-binding ORD domain of ORP1L is involved by coexpressing a truncation mutant of ORP1L missing the ORD domain (ORP1L-ΔORD) and MLN64. Again a predominant exclusion of both proteins within LE could be observed (Fig. 4B, middle panel). However, in about 20% of cells, we observed enlarged vesicles in the periphery of cells that were positive for both MLN64 and ORP1L-ΔORD. In addition, under these conditions of ORP1L-ΔORD expression, the MLN64-positive LE clustered (Fig. 4B and supplementary Fig. III). ORP1L has two conformations on the late endosomal membrane: the ORP1L-ΔORD mutant reflects
Referência(s)