A Proteomic Analysis of Lysosomal Integral Membrane Proteins Reveals the Diverse Composition of the Organelle

Lysosomes are endocytic subcellular compartments that contribute to the degradation and recycling of cellular material. Using highly purified rat liver tritosomes (Triton WR1339-filled lysosomes) and an ion exchange chromatography/LC-tandem MS-based protein/peptide separation and identification procedure, we characterized the major integral membrane protein complement of this organelle. While many of the 215 proteins we identified have been previously associated with lysosomes and endosomes, others have been associated with the endoplasmic reticulum, Golgi, cytosol, plasma membrane, and lipid rafts. At least 20 proteins were identified as unknown cDNAs that have no orthologues of known function, and 35 proteins were identified that function in protein and vesicle trafficking. This latter group includes multiple Rab and SNARE proteins as well as ubiquitin. Defining the roles of these proteins in the lysosomal membrane will assist in elucidating novel lysosomal functions involved in cellular homeostasis and pathways that are affected in various disease processes. Lysosomes are endocytic subcellular compartments that contribute to the degradation and recycling of cellular material. Using highly purified rat liver tritosomes (Triton WR1339-filled lysosomes) and an ion exchange chromatography/LC-tandem MS-based protein/peptide separation and identification procedure, we characterized the major integral membrane protein complement of this organelle. While many of the 215 proteins we identified have been previously associated with lysosomes and endosomes, others have been associated with the endoplasmic reticulum, Golgi, cytosol, plasma membrane, and lipid rafts. At least 20 proteins were identified as unknown cDNAs that have no orthologues of known function, and 35 proteins were identified that function in protein and vesicle trafficking. This latter group includes multiple Rab and SNARE proteins as well as ubiquitin. Defining the roles of these proteins in the lysosomal membrane will assist in elucidating novel lysosomal functions involved in cellular homeostasis and pathways that are affected in various disease processes. Lysosomes are single membrane-bound subcellular organelles that are thought to be the chief degradatory compartments in the endosomal pathway. They house a diverse complement of hydrolases that function optimally at acidic pH values. Most resident hydrolytic enzymes are localized to the luminal compartment and are largely soluble glycoproteins capable of hydrolyzing all the major macromolecules of the cell. The simpler molecules arising as products of these digestive events are translocated from the intralysosomal compartment across the membrane and released into the cytoplasm for reutilization. Substances for digestion are acquired by the lysosomes via a series of processes including endocytosis, phagocytosis, and autophagy. The pivotal role lysosomes play in cell metabolism is manifested by the occurrence of at least 40 enzymopathies (1Meikle P.J. Hopwood J.J. Clague A.E. Carey W.F. Prevalence of lysosomal storage disorders..J. Am. Med. Assoc. 1999; 281: 249-254Google Scholar) affecting the catabolism of neutral, phospho-, and glycolipids; complex carbohydrates; and proteins where indigestible macromolecular intermediates become stored inside the organelle, altering the homeostasis of the cell and leading to a disease state. More recently, lysosomes have been found to be highly dynamic organelles involved in regulated secretion (2Jaiswal J.K. Andrews N.W. Simon S.M. Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells..J. Cell Biol. 2002; 159: 625-635Google Scholar), repair of damaged plasma membrane (3Reddy A. Caler E.V. Andrews N.W. Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes..Cell. 2001; 106: 157-169Google Scholar), and formation of osteoclast ruffled border (4Stenbeck G. Formation and function of the ruffled border in osteoclasts..Semin. Cell Dev. Biol. 2002; 13: 285-292Google Scholar). Classical lysosomes are considered to be part of a family of lysosome-related organelles (for a review, see Ref. 5Dell’Angelica E.C. Mullins C. Caplan S. Bonifacino J.S. Lysosome-related organelles..FASEB J. 2000; 14: 1265-1278Google Scholar) that have a range of cell-specific functions, e.g. melanosomes, platelet dense granules, and azurophilic granules. In addition to the storage diseases, lysosomes have been implicated in a wide range of other human pathologies such as Alzheimer’s disease (6Pasternak S.H. Bagshaw R.D. Guiral M. Zhang S. Ackerley C.A. Pak B.J. Callahan J.W. Mahuran D.J. Presenilin-1, nicastrin, amyloid precursor protein, and γ-secretase activity are co-localized in the lysosomal membrane..J. Biol. Chem. 2003; 278: 26687-26694Google Scholar, 7Pasternak S.H. Callahan J.W. Mahuran D.J. The role of the endosomal/ lysosomal system in amyloid-β production and the pathophysiology of Alzheimer’s disease: reexamining the spatial paradox from a lysosomal perspective..J. Alzheimer’s Dis. 2004; 6: 53-65Google Scholar), autoimmune diseases, and resistance to infectious disease, cancer, and drugs. The roles played by lysosomes in these areas remain poorly defined largely due to a lack of in-depth knowledge concerning the composition and topologies of their integral membrane proteins and the identity of other cytosolic proteins that may form permanent and/or transient macromolecular complexes to facilitate membrane-membrane interactions and fusion events. While molecular characterization of lysosomal diseases involving the luminal, i.e. soluble, enzymes is well advanced, only a few defects associated with lysosomal membrane proteins, such as Danon disease (LAMP-2) 1The abbreviations used are: LAMP, lysosome-associated membrane protein; LIMP, lysosomal integral membrane protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; MS/MS, tandem MS; ER, endoplasmic reticulum; NPC1, Niemann-Pick type C1; MGC, Mammalian Gene Collection; V-ATPase, vacuolar-type H+-translocating ATPase; VAMP, vesicle-associated membrane protein; EMP70, endomembrane protein of 70 kDa; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; CREG, cellular repressor of E1A-stimulated genes; Gl, grey-lethal. (8Nishino I. Fu J. Tanji K. Yamada T. Shimojo S. Koori T. Mora M. Riggs J.E. Oh S.J. Koga Y. Sue C.M. Yamamoto A. Murakami N. Shanske S. Byrne E. Bonilla E. Nonaka I. DiMauro S. Hirano M. Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease)..Nature. 2000; 406: 906-910Google Scholar), Niemann-Pick C (NPC1) (9Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie E.J. Dwyer N.K. Neufeld E.B. Chang T.Y. Liscum L. Strauss J.F. Ohno K. Zeigler M. Carmi R. Sokol J. Markie D. O’Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle D.A. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis..Science. 1997; 277: 228-231Google Scholar), cystinosis (cystinosin) (10Town M. Jean G. Cherqui S. Attard M. Forestier L. Whitmore S.A. Callen D.F. Gribouval O. Broyer M. Bates G.P. van’t Hoff W. Antignac C. A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis..Nat. Genet. 1998; 18: 319-324Google Scholar), and Salla disease (sialin) (11Verheijen F.W. Verbeek E. Aula N. Beerens C.E. Havelaar A.C. Joosse M. Peltonen L. Aula P. Galjaard H. van der Spek P.J. Mancini G.M. A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases..Nat. Genet. 1999; 23: 462-465Google Scholar), have been identified. The lysosomal membrane is critical for maintenance of cell homeostasis as it provides the communication link between the degradative milieu of the lysosome and the cytosol. Aside from well known lysosomal membrane proteins, i.e. the LAMPs and LIMPs, it can be predicted from physiological uptake and flux studies that multiple classes of transporter proteins exist. The goal of our present study to the protein composition of the integral membrane of the This by subcellular and protein separation with protein of the for a of lysosomes is the to the to and in While many have been used for lysosomal we of the and WR1339-filled rat liver lysosomes as by H. P. J.W. S. C. The separation of and lysosomes from the of with and of Cell Biol. Scholar). tritosomes are considered lysosomes as a of (Triton tritosomes lysosomal (6Pasternak S.H. Bagshaw R.D. Guiral M. Zhang S. Ackerley C.A. Pak B.J. Callahan J.W. Mahuran D.J. Presenilin-1, nicastrin, amyloid precursor protein, and γ-secretase activity are co-localized in the lysosomal membrane..J. Biol. Chem. 2003; 278: 26687-26694Google Scholar, H. P. J.W. S. C. The separation of and lysosomes from the of with and of Cell Biol. Scholar, R.D. S.H. Mahuran D.J. Callahan J.W. is a resident lysosomal membrane 2003; Scholar, J. of the membrane proteins of rat liver and J. Scholar) and to lysosomes in endosomal and fusion R. M. S. by rat between and Scholar). In this study we identified 215 proteins in the integral membrane protein of the many previously associated with the lysosome and that are only identified as a membrane that a complex protein composition and the that the lysosome is a highly dynamic organelle. The of WR1339-filled lysosomes been previously (6Pasternak S.H. Bagshaw R.D. Guiral M. Zhang S. Ackerley C.A. Pak B.J. Callahan J.W. Mahuran D.J. Presenilin-1, nicastrin, amyloid precursor protein, and γ-secretase activity are co-localized in the lysosomal membrane..J. Biol. Chem. 2003; 278: 26687-26694Google Scholar, H. P. J.W. S. C. The separation of and lysosomes from the of with and of Cell Biol. Scholar, R.D. S.H. Mahuran D.J. Callahan J.W. is a resident lysosomal membrane 2003; Scholar). from were an of the of to in a were and at for to a A by the at for The in a of and and at for lysosomes were from the with and at for present in all of were for to H. P. J.W. S. C. The separation of and lysosomes from the of with and of Cell Biol. Scholar). the of and Scholar). The tritosomes were as previously (6Pasternak S.H. Bagshaw R.D. Guiral M. Zhang S. Ackerley C.A. Pak B.J. Callahan J.W. Mahuran D.J. Presenilin-1, nicastrin, amyloid precursor protein, and γ-secretase activity are co-localized in the lysosomal membrane..J. Biol. Chem. 2003; 278: 26687-26694Google Scholar). tritosomes were in pH and to with and at A membrane by for at at and the The membrane with pH and as to a soluble The membrane the integral membrane The integral membrane in and 40 The at for to of to a of and for in the at The protein pH of the and the were to of The protein at for at to a exchange in were with a of and in The protein were and the were in of proteins the of the of Scholar). The proteins were by in The were for in in a and into were for digestion with as previously R.D. Callahan J.W. Mahuran D.J. of protein with for of 2000; Scholar). were to separation a with a in with a were into for this were a of and a at of The were ion the for protein were by and proteins that as by were by of that were identified with have that an series for a and have of and In and were found as well as of the from A of identified proteins by to protein a of all the identified of protein from purified tritosomes and the rat liver were by and to Primary used were by J. and and from with and have been characterized as lysosomes H. P. J.W. S. C. The separation of and lysosomes from the of with and of Cell Biol. Scholar, J. of the membrane proteins of rat liver and J. Scholar, R. M. S. by rat between and Scholar, J.J. for and in a vesicle in lysosome of rat S. A. Scholar). have previously the of lysosomal and in our of and the of from and S.H. Bagshaw R.D. Guiral M. Zhang S. Ackerley C.A. Pak B.J. Callahan J.W. Mahuran D.J. Presenilin-1, nicastrin, amyloid precursor protein, and γ-secretase activity are co-localized in the lysosomal membrane..J. Biol. Chem. 2003; 278: 26687-26694Google and R.D. S.H. Mahuran D.J. Callahan J.W. is a resident lysosomal membrane 2003; and see of tritosomes that all of the are WR1339-filled lysosomes dense and no other The of these lysosomes have for acid activity as of by and an of the dense and the of that are acid is no membrane that is for acid The of organelles and the and of the with of and physiological WR1339-filled lysosomes an as a of a into to a in a of proteins identified. of tritosomes to the soluble proteins, proteins were by with Y. S. of by of to endoplasmic Cell Biol. Scholar) in an i.e. the integral membrane of of the in due to and in the membrane proteins, e.g. A. K. C. H. A. of in lysosomal 2002; Scholar), the integral membrane in and and at pH The integral membrane proteins were by ion exchange a The goal of the by to proteins and to their from highly acidic proteins, e.g. the LAMPs (for see Y. P. At the acidic functions for lysosomal membrane Cell Biol. 2003; 13: and S.A. M. P. A. I. of the lysosomal membrane..J. Cell Biol. Scholar). we the pH of the system that these and other proteins with see to the these of the protein The proteins from were by in that were into and for and the were and identified by the of were for in the identification of 215 proteins by in the lysosomal integral membrane protein were into their proteins predicted in the from cDNAs were as a be found that the function of the protein into the and protein as it a family identified from the lysosomal integral membrane protein acid complex type to gene to to factor to factor protein protein protein protein cytosolic protein cytosolic protein cytosolic protein protein to protein to protein protein to protein to to protein to protein and Cell cell protein protein rat type membrane to to protein to to gene to to to protein C anion resistance associated protein ion to to protein family to to protein to protein, with at least of to to to to protein family family family anion and lipid protein to protein to to associated protein to repressor of integral membrane protein acid protein for protein amyloid to cellular repressor of E1A-stimulated protein to to protein to to to to to to to protein; associated protein to protein to protein to protein to protein to to to to for for and protein protein protein protein to to a to to type membrane protein; to exchange factor to to to to to to to to to protein in a new were identified in that to their molecular with such as known e.g. to be found with an of and found the molecular that our protein separation and identification processes were of the composition of the membrane, we the of protein molecular in to predicted from of the Mammalian Gene The that our is with to protein be that the of proteins of identified in the present study is predicted from the that membrane proteins a of the lysosomal membrane proteins in our we used a of protein of the of by the molecular of the The of proteins in of this are in type in I. and as (for a review, see Ref. Y. P. At the acidic functions for lysosomal membrane Cell Biol. 2003; 13: Scholar) and and lysosomal acid the lipid and R. and are major integral proteins of lipid 2001; Scholar, M. of lipid for S. A. 2003; Scholar) were found in in the lysosomal membrane A. K. C. H. A. of in lysosomal 2002; Scholar). proteins found in were nicastrin, and and the gene is we have found the the identified for are to the of acid of S.H. G. Gu S.A. S.J. M. L. is the of a with an Scholar). identified the membrane-bound form of is a type protein localized to the to in including the lysosomal membrane D.J. J. J. and of rat by with Biol. Chem. Scholar). tritosomes were by for and the protein we found that and were in A and of includes the of a disease at this by soluble with a new S. Y. R. N. K. M. N. K. Y. of by Alzheimer’s Biol. Chem. 2003; 278: Scholar). found other in the lysosomal membrane with The of and the of other in these that a previously role for such proteins may in the lysosomal have previously the of and of the γ-secretase as well as acid γ-secretase activity in the lysosomal membrane (6Pasternak S.H. Bagshaw R.D. Guiral M. Zhang S. Ackerley C.A. Pak B.J. Callahan J.W. Mahuran D.J. Presenilin-1, nicastrin, amyloid precursor protein, and γ-secretase activity are co-localized in the lysosomal membrane..J. Biol. Chem. 2003; 278: 26687-26694Google Scholar). as a major lysosomal in this with the of and we from other of this e.g. nicastrin, no other of the γ-secretase found in a study of lysosome-related organelle, T. Y. K. J. J. H. J. D.F. E. of identification of novel 2003; proteins from and gene factor protein and cell type to anion and lipid integral membrane protein protein for protein and protein protein protein protein of proteins and used for in a new proteins of the lysosomal integral membrane were found to have and This includes proteins involved in and proteins in the is a of proteins in and is of the cytosolic of the of the the of in a study of vesicles M.A. J.S. D. A. J. S. E. M. R. A. J.J. characterization of membrane Biol. Chem. 2001; Scholar), and we this to other and and and the of of these proteins is it been that is for the membrane of the protein family C. of and the integral membrane protein Cell Biol. 2004; 6: Scholar, R. S. of the to the and the membrane protein Cell Biol. 2004; 6: Scholar). identified in in the molecular range of to and in the integral membrane as a to other of proteins by of the C of to a of the protein, by the of the molecules to the ubiquitin. of is the for the addition of formation (for a review, see Ref. and Biol. 2001; Scholar). is as a for cytosolic of membrane proteins at the plasma membrane been to be for their to and degradation in the lysosome K. S. S. I. I. of is for their and Cell Biol. 2003; Scholar, Y. K. M. Y. G. J. Y. of is by Biol. Chem. 2003; 278: Scholar). we the that in the present In with the major role of the lysosome in molecules of degradative we identified proteins that an endomembrane protein of 70 may have membrane functions E. of a membrane protein that is highly 1998; Scholar), proteins of the family the transporter and the uptake transporter and the transporter and of the the cholesterol transporter and of the vacuolar were Aside from the V-ATPase, of the identified were that these are proteins in the lysosomal a of the proteins identified from studies of the J. R. S. J.F. S. E. R. C. M. The into Cell Biol. 2001; Scholar) and T. Y. K. J. J. H. J. D.F. E. of identification of novel 2003; Scholar) and it with of the 215 proteins from our present proteins in our lysosomal integral membrane were found in and/or such as and of the V-ATPase, anion and were to all these The proteins and were found in and other Rab proteins, and were in as well as and In our we identified a of other ER, cytosol, and proteins as metabolism in in the lysosomal membrane protein these proteins in of the of of these proteins have been to be associated with and/or and are from of the in lysosomes and these organelles been previously that membrane may from membrane, and the of the to with a in the of proteins J. R. S. J.F. S. E. R. C. M. The into Cell Biol. 2001; Scholar, E. S. C. E. O. J. J.J. M. is a of into 2002; Scholar). In with of of proteins that the membrane protein is is present at a This that membrane and associated proteins may be involved in lysosomal and are at of the organelle. The of the and the identification of cellular proteins at the to their in function their This is the and of the in lysosomes are in of the are to by lysosomes this by the of a of lysosomes in rat of highly purified organelles to be in a single tritosomes are lysosomes a of their protein composition may be considered and i.e. are a of the of the of The 215 proteins to our knowledge the of proteins identified from a membrane of the lysosome to A study identified proteins from a soluble lysosomal that to a A. A. S. J. of human to and 2002; Scholar). lysosomal membrane proteins are to the via this as by the highly of their of ion exchange and by digestion and provides a of protein is and can be used for protein from multiple complex protein the of lipid proteins in the lysosomal are membrane in cholesterol and that have the to and proteins for various cell events. They have been to SNARE proteins involved in vesicle fusion The and proteins that vesicle fusion are localized in lipid present Biol. Chem. 2002; 277: Scholar) and have been found associated with endosomal M. S. C. L. van der and of J. 2002; Scholar), lysosomal A. K. C. H. A. of in lysosomal 2002; Scholar), and J.F. S. J. S. M. lipid Biol. Chem. 2001; Scholar). a in the of lipid in with their into J.F. S. J. S. M. lipid Biol. Chem. 2001; this is in with our present of an of lipid in the lysosomal membrane major and functions for this organelle. with this we have identified the rat of protein, a of the endosomal of the mitogen-activated protein kinase and is known to to identified in this the cytosolic of W. I. D. A. A. A novel protein with the mitogen-activated protein kinase a Cell Biol. 2001; Scholar), a for the endosomal of D. W. of the complex to is mediated by and for 2002; Scholar). cellular repressor of E1A-stimulated identified a study of lysosomal proteins A. A. S. M. S. J. a human of lysosomal 2000; Scholar). is a that of E. R. M. G. The of human 2000; Scholar) and cell in a factor A. G. The cell the factor 2003; Scholar). identified a for to with lysosomes of by 2003; Scholar). identified were and in a complex that endosomes, and this complex is for fusion events with other W. C. D. S. R. A SNARE complex fusion of of SNARE and J. 2000; Scholar) and lysosomes G. M. E.J. D.A. is localized to with and is for Biol. 2000; Scholar). these a role for the lysosomal membrane in communication and events. A well function of the lysosomal membrane been the of the products of degradation to the for by the cell. few proteins to these in the lysosome have been identified. A group of proteins that we have identified in this are predicted to have functions, e.g. the in the proteins can be for molecular function with to the lysosomal membrane and luminal of lysosomes is well identification of proteins in lysosomes and their organelles may are a many well known proteins of the lysosomal membrane identified in this study were identified in other of the and to to the processes of lysosome and maintenance from our of the been implicated in a form of human N. N. M. M. A. A. J. in and Med. 2003; Scholar). In of this gene causes a in of in H. a new in the house Scholar) and a in by osteoclast N. N. L. R. J. The a in osteoclast 2001; Scholar), i.e. this protein the function of lysosomes (for see G. Formation and function of the ruffled border in osteoclasts..Semin. Cell Dev. Biol. 2002; 13: 285-292Google and E.J. G.M. Biol. 2002; Scholar). The to the lysosomal membrane of protein and a of other proteins with no previously function will in elucidating their this it is that the protein composition of the lysosomal membrane is by from Golgi, ER, and plasma membrane this the identification in the integral membrane of e.g. D.J. J. J. and of rat by with Biol. Chem. Scholar) and other of have been to have (for a review, see Ref. of 2002; Scholar). proteins were e.g. and no and plasma membrane proteins, e.g. and were of proteins and to the lysosome from and the is well a of cytoplasm and organelles for protein to the lysosome LAMP-2 as a Y. G. A. D. R. J. K. P. of and cardiomyopathy in 2000; 406: Scholar). and cytosolic proteins such as and have been found to be associated with T. K. R. I. K. M. E. degradation of a cytosolic Biol. Chem. 1999; Scholar, M. and characterization of from rat J. 1998; Scholar, T. D. E. Membrane of endoplasmic in vacuolar from rat Biol. Chem. Scholar). In the lysosome is thought to be the of the endocytic and to have a degradative for luminal our lysosomal it that the membrane is a diverse that membrane and protein from a of subcellular and and membrane the of of the proteins of the lysosomal membrane is of a highly complex organelle. The diverse of proteins in the lysosomal membrane be into to for endosomal compartments S. Membrane in the and endocytic 2003; Scholar) that are by at least Rab proteins P. L. of vesicle from to the in cells..J. Cell Biol. 2002; Scholar). we identified in the lysosomal membrane, it to be of these Rab proteins is involved in the of protein and in the lysosomal and Zhang for and

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.