Tumor protein D54 binds intracellular nanovesicles via an extended amphipathic region

Abstract

Tumor protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54, and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical (e.g., COPI and COPII) transport vesicles. Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by four amphipathic helices (AH1-4), which could fold upon binding to lipid membranes. Limited proteolysis, CD spectroscopy, tryptophan fluorescence, and cysteine mutagenesis coupled to covalent binding of a membrane-sensitive probe showed that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. Furthermore, site-directed mutagenesis indicated that AH2 and AH3 have a predominant role in TPD54 binding to membranes both in cells and using model liposomes. We found that AH3 has the physicochemical features of an amphipathic lipid packing sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Accordingly, we observed that binding of TPD54 to liposomes is very sensitive to membrane curvature and lipid unsaturation. We conclude that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and ALPS-independent mechanisms. Tumor protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54, and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical (e.g., COPI and COPII) transport vesicles. Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by four amphipathic helices (AH1-4), which could fold upon binding to lipid membranes. Limited proteolysis, CD spectroscopy, tryptophan fluorescence, and cysteine mutagenesis coupled to covalent binding of a membrane-sensitive probe showed that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. Furthermore, site-directed mutagenesis indicated that AH2 and AH3 have a predominant role in TPD54 binding to membranes both in cells and using model liposomes. We found that AH3 has the physicochemical features of an amphipathic lipid packing sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Accordingly, we observed that binding of TPD54 to liposomes is very sensitive to membrane curvature and lipid unsaturation. We conclude that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and ALPS-independent mechanisms. In cells, transport vesicles are produced from membrane-bound organelles by vesicular coat complexes, which self-assemble at the organelle surface into spherical shells to promote the budding of coated vesicles (1Bonifacino J.S. Glick B.S. The mechanisms of vesicle budding and fusion.Cell. 2004; 116: 153-166Abstract Full Text Full Text PDF PubMed Scopus (1256) Google Scholar, 2Hurley J.H. Boura E. Carlson L.-A. Róźycki B. Membrane budding.Cell. 2010; 143: 875-887Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The best characterized protein coats are COPII, COPI, AP1-clathrin, and AP2-clathrin, which promote vesicle budding from the endoplasmic reticulum, Golgi apparatus (GA), trans-Golgi network (TGN), and plasma membrane, respectively. These different coats and the corresponding classes of vesicles have been abundantly studied over the last decades, notably as regards to the mechanisms of coat assembly, coat structure, vesicular content in cargoes, and associated diseases (3Dell'Angelica E.C. Bonifacino J.S. Coatopathies: genetic disorders of protein coats.Annu. Rev. Cell Dev. Biol. 2019; 35: 131-168Crossref PubMed Scopus (29) Google Scholar, 4Lee M.C.S. Miller E.A. Goldberg J. Orci L. Schekman R. Bi-directional protein transport between the ER and Golgi.Annu. Rev. Cell Dev. Biol. 2004; 20: 87-123Crossref PubMed Scopus (687) Google Scholar). Investigations by electron microscopy both in situ and from reconstituted systems indicate that these vesicles have a well-defined diameter, which is in the range of 80 nm (5Barlowe C. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum.Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1025) Google Scholar, 6Malhotra V. Serafini T.A. Orci L. Shepherd J.C. Rothman J.E. Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack.Cell. 1989; 58: 329-336Abstract Full Text PDF PubMed Scopus (319) Google Scholar). Although protein coats can adapt their size to form bigger vesicles for the packaging of large cargoes (7McCaughey J. Stephens D.J. ER-to-golgi transport: a sizeable problem.Trends Cell Biol. 2019; 29: 940-953Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 8Hutchings J. Zanetti G. 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Surprisingly, a new class of transport vesicles has been recently identified through cellular investigations of tumor protein D54 (TPD54) (11Larocque G. La-Borde P.J. Clarke N.I. Carter N.J. Royle S.J. Tumor protein D54 defines a new class of intracellular transport vesicles.J. Cell Biol. 2019; 219e201812044Google Scholar). TPD54 is a small (206 aa, 22.2 kDa) protein that belongs to a family of four proteins: TPD52, TPD53 (or TPD52L1), TPD54 (or TPD52L2), and TPD55 (or TPD52L3) (12Boutros R. Fanayan S. Shehata The tumor protein 2004; PubMed Scopus Google Scholar). These proteins are in cells which is to the microscopy that TPD54 with membrane-bound and (11Larocque G. La-Borde P.J. Clarke N.I. Carter N.J. Royle S.J. Tumor protein D54 defines a new class of intracellular transport vesicles.J. Cell Biol. 2019; 219e201812044Google Scholar). In a large of TPD54 is found in the is suggests that the cytosolic of TPD54 to very small which could by microscopy (11Larocque G. La-Borde P.J. Clarke N.I. Carter N.J. Royle S.J. Tumor protein D54 defines a new class of intracellular transport vesicles.J. Cell Biol. 2019; 219e201812044Google Scholar). TPD54 is to the by a the of vesicles 30 at the of that these vesicles are involved in membrane of TPD54 a Golgi as as a in the of a model TPD54 vesicles proteins, which are small proteins involved in vesicular as as proteins, which promote vesicle (11Larocque G. La-Borde P.J. Clarke N.I. Carter N.J. Royle S.J. Tumor protein D54 defines a new class of intracellular transport vesicles.J. Cell Biol. 2019; 219e201812044Google Scholar). Here, we that TPD54 recognizes membranes through the of amphipathic helices an amphipathic lipid packing sensor (ALPS) are amphipathic of to that are characterized by the of small and by the of and by the of bulky A. B. The of amphipathic PubMed Scopus Google Scholar, G. B. helices and membrane 2010; PubMed Scopus Google Scholar, G. R. T. B. amphipathic for membrane Biol. PubMed Scopus Google Scholar, R. H. S. B. at the of the Golgi that vesicles to size and lipid Scopus Google Scholar, J. G. B. B. to membrane curvature through the of a lipid packing sensor J. PubMed Scopus Google Scholar). are fold as at the surface of membranes lipid packing G. R. T. B. amphipathic for membrane Biol. PubMed Scopus Google Scholar, S. L. R. G. C. et lipid packing sensor with J. Full Text Full Text PDF PubMed Scopus Google Scholar, N. Membrane of and the amphipathic lipid packing sensor (ALPS) protein PubMed Scopus Google Scholar, L. R. S. B. et in packing to that by J. Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. H. B. R. of membrane curvature and lipid packing and protein 2014; PubMed Scopus Google Scholar). are in several proteins that with membranes B. of membrane curvature Rev. PubMed Scopus Google Scholar). These the COPI coat and J. G. B. B. to membrane curvature through the of a lipid packing sensor J. PubMed Scopus Google Scholar, B. G. S. J. et sensor to the of to membrane PubMed Scopus Google Scholar, B. B. B. an lipid membranes and J. 2010; 29: PubMed Scopus Google Scholar, T. H. of a amphipathic that to in the early PubMed Scopus Google Scholar, membrane curvature and for vesicular Cell Biol. PubMed Scopus Google and as the G. R. T. B. amphipathic for membrane Biol. PubMed Scopus Google Scholar, R. H. S. B. at the of the Golgi that vesicles to size and lipid Scopus Google Scholar, G. V. B. of and lipid membranes by a PubMed Scopus Google Scholar, of vesicle and binding is for in of the Biol. PubMed Scopus Google Scholar), the L. R. I. A. et of a membrane of the in membrane Cell Biol. 2010; PubMed Scopus Google Scholar, R. C. The membranes a PubMed Scopus Google and L. A. A. et membrane curvature by an amphipathic lipid packing sensor PubMed Scopus Google Scholar). In are in the protein J. S. B. B. G. mechanism to a lipid between PubMed Scopus Google Scholar, V. binding mechanism of amphipathic sensor motif, from B. PubMed Scopus Google Scholar), the protein L. G. J.H. Schekman R. of by the class Biol. 2019; PubMed Scopus Google Scholar, A. and membrane curvature by Sci. S. A. PubMed Scopus Google Scholar, S. L. J. et of and Scopus Google Scholar), the B. J. J. S. B. and of membrane lipid by J. PubMed Scopus Google and of the G. R. T. B. amphipathic for membrane Biol. PubMed Scopus Google Scholar, flexibility and of the membrane PubMed Scopus Google Scholar). We that TPD54 a for and lipid membranes in as characterized In cells, the of TPD54 with membranes. in between TPD54 and the which small transport vesicles through an motif, the of for the of small transport vesicles. In with binding of TPD54 to small liposomes is accompanied by a large structural that not to the to the of the coiled-coil region. the organization of TPD54 (206 as from several and (11Larocque G. La-Borde P.J. Clarke N.I. Carter N.J. Royle S.J. Tumor protein D54 defines a new class of intracellular transport vesicles.J. Cell Biol. 2019; 219e201812044Google Scholar). for the of that the protein is in four with different to The and last of the protein and are to The a to The is at the between and TPD54 contains a coiled-coil which from to and which has been to to and between protein The role of the coiled-coil in by PubMed Scopus Google Scholar). The recently J. T. A. et protein for the PubMed Scopus Google with with an between and corresponding to the other are as with We the R. B. G. a to with PubMed Scopus Google to the of in the and and identified TPD54 as of the proteins an G. R. T. B. amphipathic for membrane Biol. PubMed Scopus Google Scholar). The of TPD54 the TPD54 with we observed that other and of the motif, an amphipathic are to as AH2 and with AH3 corresponding to the which for from a to an in the of a membrane and are in both and in to which have a of four and G. R. T. B. amphipathic for membrane Biol. PubMed Scopus Google Scholar, J. G. B. B. to membrane curvature through the of a lipid packing sensor J. PubMed Scopus Google Scholar). as as are not in which that a their the organization of TPD54 with both a coiled-coil and amphipathic to the of the a very coiled-coil as a small transport vesicles through R. H. S. B. at the of the Golgi that vesicles to size and lipid Scopus Google Scholar, G. V. B. of and lipid membranes by a PubMed Scopus Google Scholar, S. Membrane The of vesicle to the Golgi is in the coiled-coil 2014; PubMed Scopus Google Scholar). We TPD54 from a form by a binding of the between and TPD54 can with lipid we TPD54 with liposomes and the by the of an with of through nm by and through nm which are not in cells, form lipid membranes that are very to the binding of to the of their which C. C. R. L. R. et promote the membrane of by PubMed Google Scholar). TPD54 to nm liposomes to liposomes and to nm liposomes. TPD54 is to with membranes and binding is very sensitive to both membrane curvature and lipid structural changes the binding of TPD54 to we we TPD54 with and the over by and of with We that TPD54 showed with an to 30 than the which the from the between TPD54 and with for and to binding to features of the TPD54 A. G. binding of membrane Sci. S. A. PubMed Scopus Google Scholar). we observed that TPD54 a of which to a the and In the of and to the of a with an size the identified as to and to in the of and and In with the analysis suggests that a small of which to the coiled-coil is the of TPD54 with liposomes an important of TPD54 from both and a with an at to into smaller of of to by that the aa, which are of the In the of the coiled-coil at early and into smaller In the of analysis of the to the that to the with to the of a which observed in the of liposomes and which both the coiled-coil and the four observed for showed a in as to the and the of the that the of small vesicles by TPD54 to changes in the protein to which to the of the coiled-coil region. the that the that from in the of small liposomes contains the four we CD to changes in the TPD54 to small liposomes. In the CD of TPD54 at and nm, which are of of these of the CD using the C. analysis and for 29: PubMed Scopus Google suggests that the content in from in to in the of liposomes. that the coiled-coil of TPD54 is to is in in suggests the of the of to of these The TPD54 of the coiled-coil contains tryptophan and other tryptophan is in the TPD54 tryptophan is very sensitive to changes and from in the of we the of TPD54 in the and in the of liposomes. in binding of TPD54 to liposomes a large tryptophan which accompanied by a the that binding of TPD54 to membranes to a large of the protein structure, the the tryptophan to R. B. G. a to with PubMed Scopus Google Scholar), and to the of the and tryptophan to the membrane upon TPD54 binding to small we We liposomes with of the lipid The of with the of to a tryptophan in lipid membranes B. S. of the into membrane upon to PubMed Scopus Google Scholar, J. B. J. et in as an to protein and at membrane 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). of TPD54 to liposomes a observed with liposomes These that to the membrane is in the TPD54 liposomes not with the TPD54 in a we cysteine which we with the membrane-sensitive probe We which the structural in the coiled-coil AH2 AH3 In showed binding to liposomes than to we observed between the we their in the of liposomes. the and AH3 we observed a large to in upon the of liposomes liposomes and observed in the of large liposomes In TPD54 in which the probe is of the showed TPD54 in which the probe is of the showed a in the of liposomes liposomes. the of the cysteine which are in in with the and that the and AH3 to the membrane TPD54 to membranes. we the of AH3 and to the of TPD54. we which with in cells and the of that the of these with the lipid We four and which a in the of the showed a In with a (11Larocque G. La-Borde P.J. Clarke N.I. Carter N.J. Royle S.J. Tumor protein D54 defines a new class of intracellular transport vesicles.J. Cell Biol. 2019; 219e201812044Google Scholar), between a cytosolic a and in the The to the with Golgi the the in the of the the the Golgi of TPD54 and we the in that is the motif, intracellular and and et showed that the of TPD54 as by microscopy is measurements and analysis of the in the to vesicular (11Larocque G. La-Borde P.J. Clarke N.I. Carter N.J. Royle S.J. Tumor protein D54 defines a new class of intracellular transport vesicles.J. Cell Biol. 2019; 219e201812044Google Scholar). We measurements of in the the and the and from these as as from the with which in the range of to The which could to than observed with that rarely in the in which to in the very to that of and than in the These and indicate that the of a to the of the protein to which identified as the of the and of mutagenesis of to and the cellular a role of AH2 and AH3 (ALPS) in TPD54 the role of these in the of we with TPD54 we a in the of for for with the of cysteine for AH2 and for and In we a both the and a in with of liposomes showed that a in for liposomes as to a in for liposomes as to In the of and of a with in both AH3 and showed a as the AH3 These that AH2 and AH3 are involved in TPD54 binding to membranes. we using liposomes of We observed binding of to liposomes by and nm, binding to liposomes liposomes 30 and nm, binding and binding with liposomes the of for liposomes by of to and size to both membrane curvature and lipid is a of we the of TPD54 with other the of and the of and showed binding to small liposomes not to in the nm We of the lipid which the membrane binding of has V. J. B. et and are membrane curvature vesicle Cell Biol. PubMed Scopus Google Scholar). a the binding of which could observed with liposomes. the indicate that TPD54 contains AH2 and The which to an motif, the to for the of TPD54 for and membranes. of that the recognizes vesicles through microscopy showed that motif, to the Golgi of cells in which the of the between vesicles and of vesicle and binding is for in of the Biol. PubMed Scopus Google Scholar). the of an in the of small transport vesicles by we and TPD54 a of transport which to in their We the of and TPD54 of overexpressed protein the small size of transport vesicles and the organization of the we analysis using microscopy in to we of TPD54 and of using the from the proteins not both the Golgi In the of overexpressed not with that of the from a that in the of to an coiled-coil R. H. S. B. at the of the Golgi that vesicles to size and lipid Scopus Google from the of We that and TPD54 not the cellular both the in of and TPD54 and the of their amphipathic we in which we the of with the of TPD54 a both AH2 and AH3 These in with the of R. H. S. B. at the of the Golgi that vesicles to size and lipid Scopus Google Scholar, H. H. B. of the with and other and of the membrane 2014; PubMed Scopus Google Scholar), which the suggests that AH2 and AH3 of important for membrane are not in a different protein the of TPD54 is as as that of and showed that the of the of to membrane binding R. H. S. B. at the of the Golgi that vesicles to size and lipid Scopus Google Scholar). The that the protein TPD54 intracellular vesicles that are smaller than transport vesicles 60 nm in as a that the of transport vesicles in cells (11Larocque G. La-Borde P.J. Clarke N.I. Carter N.J. Royle S.J. Tumor protein D54 defines a new class of intracellular transport vesicles.J. Cell Biol. 2019; 219e201812044Google Scholar). The which an analysis of TPD54 with cellular a for the capture of small vesicles by TPD54. CD and indicate that is for coiled-coil region. binding to small TPD54 a structural which the of the that is from to and which with a in In the of the coiled-coil and from the the that upon membrane AH2 and AH3 of which to and to a to membrane in the of AH2 AH3 the of TPD54 for small liposomes by and the membrane of the protein in cells. TPD54 binds to lipid membranes in a curvature-dependent and lipid manner. We not binding of TPD54 liposomes with a diameter 60 nm using the lipid which large in membranes C. C. R. L. R. et promote the membrane of by PubMed Google Scholar). liposomes of which is the abundant in cellular TPD54 binding the diameter is 60 The of TPD54 to membrane curvature and lipid with to as binding the lipid Although we that TPD54 is sensitive to that not the of TPD54 to both membrane curvature and lipid TPD54 to other proteins as to A. B. The of amphipathic PubMed Scopus Google Scholar, G. B. helices and membrane 2010; PubMed Scopus Google Scholar, B. of membrane curvature Rev. PubMed Scopus Google Scholar). the of TPD54 has a curvature different from other for the capture of nanovesicles with a diameter of 30 nm as to vesicles with a diameter of 60 to 80 nm, The are measurements with liposomes by through by by electron microscopy and measurements of produced by a PubMed Scopus Google Scholar, of liposomes of size by through PubMed Scopus Google Scholar), these liposomes that an size the curvature of other These microscopy to binding of protein liposomes J. et membranes amphipathic helices and protein Biol. PubMed Scopus Google Scholar, J.E. et curvature by amphipathic a using and Biol. Full Text Full Text PDF PubMed Scopus Google from vesicles B. B. B. an lipid membranes and J. 2010; 29: PubMed Scopus Google Scholar, A. T. of and proteins in membrane curvature J. Full Text Full Text PDF PubMed Scopus Google Scholar). The that of TPD54 is not to and that the of TPD54 from that of the which recognizes transport vesicles through an motif, that other with the to TPD54 and the to different of vesicles. In the of both the and the very are involved in the capture of different vesicles S. The coiled-coil proteins capture different of transport Biol. PubMed Scopus Google Scholar). a recent the of and the of suggests that to and a of and are involved in intracellular nanovesicle the mechanism is not G. D.J. C. La-Borde P.J. et nanovesicles during Cell Biol. PubMed Scopus Google Scholar). with the of with different in the of small vesicles by TPD54. recent in investigations that changes in can to in the amphipathic can for other L. A. L. et to vesicles in Full Text Full Text PDF PubMed Scopus Google Scholar). the of and other in TPD54 a to a class of transport vesicles. the amphipathic of TPD54 to capture intracellular nanovesicles through and investigations their structural investigations and of mutagenesis coupled to (e.g., and electron to the of these proteins at the membrane surface J. R. of membrane-bound from site-directed and Sci. S. A. PubMed Scopus Google Scholar, J. of the upon membrane binding of to the membrane Biol. 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). We that four of TPD54 to to lipid membrane as in the to their to the the of an in TPD54 a for the mechanism of capture of very small transport vesicles by We the of in TPD54 using the from the T. of protein from Scopus Google Scholar). the of several B. a of 2010; PubMed Scopus Google Scholar). is an G. of protein with and of PubMed Scopus Google Scholar). using a network G. A. S. J. et with of PubMed Scopus Google Scholar). We the for the of coiled-coil A. J. from protein PubMed Scopus Google and for the of TPD54 J. T. A. et protein for the PubMed Scopus Google Scholar). The TPD54 from by The TPD54 into a at the and that the and the with the The and the of the by We TPD54 as a in in with at by the of at for at in a at in 50 and of and the by at for 30 in a The protein from the using at a of for TPD54 from the by in the 50 of the between and which the at the of the TPD54 by a in and in and at Purification of has been J. G. B. B. to membrane curvature through the of a lipid packing sensor J. PubMed Scopus Google Scholar, B. G. S. J. et sensor to the of to membrane PubMed Scopus Google Scholar). from and and with the G. R. T. B. amphipathic for membrane Biol. PubMed Scopus Google Scholar). of TPD54 with using a of in 50 The and of to the of the cysteine which found to have to coiled-coil probe with and by The of protein by at as and in a The lipid in and the and to promote the of liposomes followed by through of size and 30 the the using a probe The size and by using a size small liposomes have to the of and TPD54 for in 50 at in a The with with and with an of with and a of at in a for and of and 50 and by using in with liposomes at a of to membrane binding of TPD54. at the indicated and the by The by with and by The by coupled to a with a using a The at analysis a at a of The and both B. The by the in as probe and The in the to range with the to analysis with and protein with The protein and with liposomes at in a with an of CD at in a CD at a of 50 from to and for the which the in the of of the of using the CD analysis and in a using a with a and at fluorescence, at nm and from to nm tryptophan fluorescence, at nm and from to nm between TPD54 and by tryptophan at nm and the of at nm over The 50 fluorescence, TPD54 followed by liposomes of size and tryptophan fluorescence, TPD54 followed by liposomes of size and and for the corresponding that a in the of cells from in with and in a at using to the the cells with for at Golgi using for using a using the of and at the we the Golgi from the in the we the corresponding to the to a of at the Golgi and a of the in the the we the in and the with a and using and in the and respectively. these using a proteins with a using are the and the and contains The that have of with the of We and for and by the A. and B. A. A. S. A. S. 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