Endocytosed Transferrin Receptors Recycle via Distinct Dynamin and Phosphatidylinositol 3-Kinase-dependent Pathways

Abstract

Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1G273D-overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin-1G273D on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity. Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1G273D-overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin-1G273D on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity. Membrane proteins that are endocytosed via clathrin-coated vesicles are first targeted to early endosomes (EE). 1The abbreviations used are: EE, early endosomes; RE, recycling endosomes; Tf, transferrin; TfR, transferrin receptor; dynts, temperature-sensitive dynamin mutant; dynwt, wild type dynamin-1; PI, phosphatidylinositol; WM, wortmannin; LY, LY294002; HRP, horseradish peroxidase; BFA, brefeldin A. 1The abbreviations used are: EE, early endosomes; RE, recycling endosomes; Tf, transferrin; TfR, transferrin receptor; dynts, temperature-sensitive dynamin mutant; dynwt, wild type dynamin-1; PI, phosphatidylinositol; WM, wortmannin; LY, LY294002; HRP, horseradish peroxidase; BFA, brefeldin A. From there, they can be transported either to other intracellular compartments, including late endosomes, lysosomes, and the trans-Golgi network, or recycle to the plasma membrane (1Marsh M. McMahon H.T. Science. 1999; 285: 215-220Google Scholar). Transferrin (Tf) and the transferrin receptor (TfR) have extensively been used as prototype markers for the recycling pathway(s). Tf remains associated with its receptor after endocytosis and dissociates only after recycling of the complex to the plasma membrane. Endocytosed Tf·TfR is diverged from lysosome-targeted proteins in EE with a t½ of <2–3 min (2Stoorvogel W. Geuze H.J. Strous G.J. J. Cell Biol. 1987; 104: 1261-1268Google Scholar, 3Dunn K.W. McGraw T.E. Maxfield F.R. J. Cell Biol. 1989; 109: 3303-3314Google Scholar) and recycles to the plasma membrane with at½ of 10–15 min (see Ref. 4Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Google Scholar and references therein). The recycling pathway involves recycling endosomes (RE), a network of tubular membranes, which in some cell types, but not all, is concentrated in the perinuclear area as the so-called recycling compartment (5Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Google Scholar, 6Tooze J. Hollinshead M. J. Cell Biol. 1991; 115: 635-653Google Scholar). A significant fraction of endocytosed TfR seems, however, to bypass RE and recycle directly from EE (4Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Google Scholar, 7Hopkins C.R. Trowbridge I.S. J. Cell Biol. 1983; 97: 508-521Google Scholar, 8Presley J.F. Mayor S. Dunn K.W. Johnson L.S. Mcgraw T.E. Maxfield F.R. J. Cell Biol. 1993; 122: 1231-1241Google Scholar, 9Hopkins C.R. Gibson A. Shipman M. Strickland D.K. Trowbridge I.S. J. Cell Biol. 1994; 125: 1265-1274Google Scholar, 10Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Google Scholar, 11Sheff D. Pelletier L. O'Connell C.B. Warren G. Mellman I. J. Cell Biol. 2002; 156: 797-804Google Scholar). The functional significance of this bifurcation into two distinct recycling pathways and the mechanistic relationship between EE and RE are poorly understood (4Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Google Scholar, 7Hopkins C.R. Trowbridge I.S. J. Cell Biol. 1983; 97: 508-521Google Scholar, 9Hopkins C.R. Gibson A. Shipman M. Strickland D.K. Trowbridge I.S. J. Cell Biol. 1994; 125: 1265-1274Google Scholar, 11Sheff D. Pelletier L. O'Connell C.B. Warren G. Mellman I. J. Cell Biol. 2002; 156: 797-804Google Scholar, 12Geuze H.J. Slot J.W. Schwartz A.L. J. Cell Biol. 1987; 104: 1715-1723Google Scholar, 13Sönnichsen B. De Renzis S. Nielsen E. Rietdorf J. Zerial M. J. Cell Biol. 2000; 149: 901-914Google Scholar). In polarized cells, however, RE are instrumental for sorting TfR away from apical destined receptors and selective transfer to the basolateral plasma membrane (14Futter C.E. Gibson A. Allchin E.H. Maxwell S. Ruddock L.J. Odorizzi G. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1998; 141: 611-623Google Scholar, 15Gibson A. Futter C.E. Maxwell S. Allchin E.H. Shipman M. Kraehenbuhl J.P. Domingo D. Odorizzi G. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1998; 143: 81-94Google Scholar, 16Sheff D.R. Daro E.A. Hull M. Mellman I. J. Cell Biol. 1999; 145: 123-139Google Scholar). Endosomal tubules generate clathrin-coated vesicles (17Stoorvogel W. Oorschot V. Geuze H.J. J. Cell Biol. 1996; 132: 21-33Google Scholar) that in polarized cells have been proposed to carry TfR to the basolateral plasma membrane (14Futter C.E. Gibson A. Allchin E.H. Maxwell S. Ruddock L.J. Odorizzi G. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1998; 141: 611-623Google Scholar). For nonpolarized cells, we recently demonstrated that a significant fraction of endocytosed Tf is recycled to the plasma membrane by clathrin-coated vesicles that bud from RE in a dynamin-dependent manner (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). Cells overexpressing dynamin-1G273D (dynts), a temperature-sensitive dynamin mutant, accumulated dynts-carrying clathrin-coated buds on RE and retained ∼30% of endocytosed Tf in this compartment at the nonpermissive temperature. Whether TfR is actively or passively incorporated into RE-derived clathrin-coated vesicles remains, however, unclear (4Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Google Scholar, 8Presley J.F. Mayor S. Dunn K.W. Johnson L.S. Mcgraw T.E. Maxfield F.R. J. Cell Biol. 1993; 122: 1231-1241Google Scholar, 18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar, 19Presley J.F. Mayor S. Mcgraw T.E. Dunn K.W. Maxfield F.R. J. Biol. Chem. 1997; 272: 13929-13936Google Scholar). The distinction between EE and RE is mainly based on their morphology, the flow of cargo molecules, and their spatial distribution within cells. The organizational complexity of the endosomal system is illustrated further by the distribution of Rab GTPases that regulate endocytosis and recycling. Rab5 regulates transfer from the plasma membrane to EE, whereas Rab4 (16Sheff D.R. Daro E.A. Hull M. Mellman I. J. Cell Biol. 1999; 145: 123-139Google Scholar, 20van der Sluijs P. Hull M. Webster P. Male P. Goud B. Mellman I. Cell. 1992; 70: 729-740Google Scholar) and Rab11 (21Ren M.D. Xu G.X. Zeng J.B. Delemoschiarandini C. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6187-6192Google Scholar, 22Ullrich O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Google Scholar, 23Wilcke M. Johannes L. Galli T. Mayau V. Goud B. Salamero J. J. Cell Biol. 2000; 151: 1207-1220Google Scholar) are implicated in TfR recycling. Rab11 is primarily found in association with RE and the trans-Golgi network and regulates trafficking through these compartments (22Ullrich O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Google Scholar, 23Wilcke M. Johannes L. Galli T. Mayau V. Goud B. Salamero J. J. Cell Biol. 2000; 151: 1207-1220Google Scholar, 24Trischler M. Stoorvogel W. Ullrich C. J. Cell Sci. 1999; Scholar). on the other is found on EE and RE, but remains unclear which recycling pathway is by this P. Mol. Biol. 1999; Scholar). and Rab11 are into distinct within the B. De Renzis S. Nielsen E. Rietdorf J. Zerial M. J. Cell Biol. 2000; 149: 901-914Google Scholar, 24Trischler M. Stoorvogel W. Ullrich C. J. Cell Sci. 1999; that EE and RE be as distinct is by the that RE to be directly from EE D. Pelletier L. O'Connell C.B. Warren G. Mellman I. J. Cell Biol. 2002; 156: 797-804Google Scholar). are for a of including membrane 1998; Scholar, A. Curr. Opin. Cell Biol. 13: Scholar). The 3-kinase and LY294002 the of Tf recycling G. C. A. Proc. Natl. Acad. Sci. U. S. A. 1995; Scholar, C. Mcgraw T.E. J. Biol. Chem. 1996; Scholar, M. D. S. J. Cell Biol. 1996; 132: Scholar, D.J. W. T. M. Mol. Biol. Cell. 1996; 7: Scholar, L. M. M. T. J. Cell Biol. 2002; Scholar, A. J.P. as did that the of of the U. J. A. J. Cell Biol. 1998; 143: Scholar). of 3-kinase by the receptor the recycling of TfR and other receptors 1995; Scholar, K.W. J. Scholar). was that but not recycling of the TfR and the receptor L. M. M. T. J. Cell Biol. 2002; Scholar). Collectively, these data indicate that 3-kinase is in a recycling In the we provide for two TfR recycling pathways. One of these involves EE and RE and on clathrin-coated vesicles and dynamin whereas the other pathway bypasses RE and requires 3-kinase activity. cells M. Proc. Natl. Acad. Sci. U. S. A. 1992; Scholar) and cells with of wild type or T. der J. Cell Biol. 1995; Scholar) were by and as (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). and cells were on and for min in with Tf to in the or of at either or was at as (17Stoorvogel W. Oorschot V. Geuze H.J. J. Cell Biol. 1996; 132: 21-33Google Scholar) (see and cells were either directly min at in in or for min at in the or of and were by in for min in and and with by Tf through EE and RE, cells were first with for at after which plasma was at Cells were with Tf in the or of for at was and cells were either directly or for min at in the or of and were and in and by a was as (17Stoorvogel W. Oorschot V. Geuze H.J. J. Cell Biol. 1996; 132: 21-33Google Scholar, 18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar) with cells were on with and in the of horseradish Tf for at was the min of was by of and dynamin was were as (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). Cells were first from Tf by by a in in a at cells were in for min at in the or of was endocytosed at in the or of as was by at was at by at and and was as of plasma The data were for as in in which of Tf was Recycling of was as (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). In cells were with at or in the or of LY, or brefeldin A The cells were at and plasma was as cells were at in the or of LY, WM, or to and the was was the from the cells by at The data were for as in in which of Tf was Cells that a (dynts), but not cells that wild type retained ∼30% of endocytosed in RE as a of of clathrin-coated vesicles from this compartment (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). a TfR recycling pathway that requires 3-kinase activity. been that the 3-kinase the of TfR recycling C. Mcgraw T.E. J. Biol. Chem. 1996; Scholar, M. D. S. J. Cell Biol. 1996; 132: Scholar, D.J. W. T. M. Mol. Biol. Cell. 1996; 7: Scholar, L. M. M. T. J. Cell Biol. 2002; Scholar, A. J.P. Scholar, 1995; Scholar). at but and at J. Biol. Chem. 1997; 272: Scholar, S. S. I. E. T. M. J. Biol. Chem. 1992; Scholar). is in and In the unrelated is a of 3-kinase and in J. Biol. Chem. 1994; Scholar). the role of 3-kinase on the distribution of TfR, and were to for at with either in the or of or was at and the cells were and for a for of with EE and the of into EE were not by or M. T. P. and W. For cells in the of LY, the of endocytosed was from EE to a through these In the of LY, however, endocytosed accumulated in compartments a from For cells in the of LY, the of endocytosed in RE, as by their perinuclear tubular and of whereas only was in EE (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). distribution is with the that as as recycling from RE were inhibited by at (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). did not with the of RE or with the of in tubular perinuclear RE in cells cells accumulated Tf at RE, of the of LY, that from EE to RE was not by the of Tf through EE cells were with at a that is for Tf by and cells but not from EE (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar) (see Ref. M.D. Xu G.X. Zeng J.B. Delemoschiarandini C. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6187-6192Google Scholar and references therein). for at in the or of LY, cell was at The cells were either directly after or for or min at in the or of and cells were and for at to EE in and cells In the of within min at was of EE in and cells a cells recycled with the to RE cells retained in RE after a did not with the of EE at and the of this compartment was in and and cells and by at that the from EE requires 3-kinase activity. Tf through RE requires 3-kinase we in which cells were with two of Tf Cells were first with to for at was at and the cells were at with either in the or of to EE Cell was at and the cells were either directly or at in the or of and after the EE were by the of and vesicles to EE with only and RE by only RE were but not in the perinuclear In the of recycled of the Tf within a at In the of LY, however, endosomes retained whereas RE were that the from EE, but not from RE, was In cells, of and in perinuclear RE after or min of M. T. P. and W. in the or of were was by Collectively, these data indicate that 3-kinase the of Tf from EE but not to and from the of 3-kinase on in we (17Stoorvogel W. Oorschot V. Geuze H.J. J. Cell Biol. 1996; 132: 21-33Google Scholar) cells were directly on and in the of for min at to endosomes with activity. was either or the min of the cells were at in the of that through and is in endosomes. this endosomes were and with The cells were with with and with (17Stoorvogel W. Oorschot V. Geuze H.J. J. Cell Biol. 1996; 132: 21-33Google Scholar, 18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). with the the of endosomal and whereas the of the tubular RE did not were for tubular RE buds that were with and Ref. 18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). buds in the of and and with the that Tf recycling from RE was to 3-kinase by as in were for and in the and in the of tubular endosomal buds for and the effects of and on TfR cells were with of was inhibited by but not by was with but to (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google In TfR was on dynamin but not on 3-kinase activity. was first endocytosed and accumulated in EE at either in the or of plasma was at The cells were at in the or of to recycling of with (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google cells retained ∼30% of at In the of LY, the of recycling by was and did not by first Recycling by cells was inhibited by LY, with intracellular after a The inhibitory effects of and were were was by A recycling pathway in which and inhibited the effects of and on recycling. min to inhibitory cells are to T. der J. Cell Biol. 1995; Scholar). in from EE to RE have been by LY, have at RE to this we the of the was at but the and cells recycled with cells that did not the that the of on EE was of the was after min of at recycling was and with with and did not the at the a recycling In the Tf is recycled by two with on 3-kinase and the other on dynamin In this with of the pathways into the The inhibitory effects of and recycling however, that at these the pathways of to a The of recycling by cells of after a or that was not from the pathway into the dynamin-dependent pathway ∼30% of endocytosed was retained by cells (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar) and not into the the of the Tf recycling pathway for (17Stoorvogel W. Oorschot V. Geuze H.J. J. Cell Biol. 1996; 132: 21-33Google Scholar, 18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google and have not inhibitory effects on Tf recycling. The mechanistic between and is that the of on endosomes, whereas with and accumulated clathrin-coated buds on endosomes. cells to recycle ∼30% of endocytosed after min of In cells recycled in the of BFA, a recycling One of these data is that in cells was in clathrin-coated whereas in cells be into the In this and can be to recycling synergistically. was were for the of and The effects of and on cells were to on cells that of did not the 3-kinase These data are with two distinct recycling pathways on the can for of the of on the of Tf from RE we of the that at cells accumulated Tf in RE was to the of through EE, and inhibited from RE (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). at cells were at from and at For cells, the inhibitory of recycling at was at with as a of a in EE after at retained only ∼30% of endocytosed in the of LY, its in RE after at The of in this is a of the with which inhibitory the at after at (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar, T. der J. Cell Biol. 1995; Scholar). inhibitory on recycling by cells after at that 3-kinase is not for from RE to the plasma membrane. In we demonstrated that cells to recycle ∼30% of endocytosed Tf at the nonpermissive to with the formation of clathrin-coated vesicles from RE (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). did not at that to the fraction of TfR that recycled via this A of these data was that TfR recycle via a pathway and that by was on the however, we that are two recycling pathways. this is the first in which two TfR recycling pathways have been by A fraction of endocytosed TfR after into EE, via RE in a process that involves the of dynamin-dependent clathrin-coated vesicles from this fraction is recycled directly from EE in a 3-kinase are in of two recycling pathways a 3-kinase and dynamin at distinct cells accumulated endocytosed Tf in RE, whereas cells retained Tf in EE (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar) and did not with Tf from EE to RE and and or with from RE to the plasma membrane and did not affect the of Tf from EE in cells, RE were in and accumulated clathrin-coated whereas EE were (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). the other did not the of RE, whereas the of EE was and the inhibitory of on Tf recycling by cells was the drug was directly after or after or min of and that Tf was not to compartments other EE as a of the did not the of Tf at the the that have of Tf at RE, for the to the cells were to and a of clathrin-coated bud formation on RE (17Stoorvogel W. Oorschot V. Geuze H.J. J. Cell Biol. 1996; 132: 21-33Google only a on the and on the of Tf recycling. The is that the recycling pathway from RE is by BFA, Tf is into the recycling In this and have inhibitory effects on and this was of RE in Tf recycling been demonstrated (4Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Google C.R. Trowbridge I.S. J. Cell Biol. 1983; 97: 508-521Google Scholar, 9Hopkins C.R. Gibson A. Shipman M. Strickland D.K. Trowbridge I.S. J. Cell Biol. 1994; 125: 1265-1274Google Scholar, 12Geuze H.J. Slot J.W. Schwartz A.L. J. Cell Biol. 1987; 104: 1715-1723Google but for a second pathway that bypasses RE been In nonpolarized cells, a recycling pathway been with M. Maxfield F.R. J. Biol. Chem. 2000; Scholar) but been for recycling Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar) and for Tf in E.H. J. Biol. Chem. 1983; Scholar) and cells D. Pelletier L. O'Connell C.B. Warren G. Mellman I. J. Cell Biol. 2002; 156: 797-804Google Scholar). and D. Pelletier L. O'Connell C.B. Warren G. Mellman I. J. Cell Biol. 2002; 156: 797-804Google Scholar) recently Tf recycling data from cells in a that two recycling pathways. In this of endocytosed Tf recycled via RE, and the other recycled directly from EE with a The of Tf through polarized cells were into a with two distinct recycling pathways (16Sheff D.R. Daro E.A. Hull M. Mellman I. J. Cell Biol. 1999; 145: 123-139Google Scholar). was recently demonstrated in cells that with recycling of TfR and the whereas a recycling was L. M. M. T. J. Cell Biol. 2002; Scholar). data are with these that in cells the of the two pathways were retained of endocytosed Tf, but the recycling of the other was (18van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Google Scholar). can be for the of Tf recycling through dynamin-dependent and pathways are was that Tf recycling by cells was by min in the of and did not with first The of Tf from RE in cells is with a perinuclear of RE in cells with and cells. RE are not to EE in to EE, concentrated in the perinuclear The at which RE in the perinuclear area however, between cell types, and the between EE and RE is poorly One is that EE and RE are distinct compartments that are by (5Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Google Scholar, Maxfield F.R. J. Cell Biol. 1995; Scholar). RE be formed as tubular of EE, from which they as a and to the perinuclear with this recently been demonstrated that RE can be formed in the cell D. Pelletier L. O'Connell C.B. Warren G. Mellman I. J. Cell Biol. 2002; 156: 797-804Google Scholar, 13Sönnichsen B. De Renzis S. Nielsen E. Rietdorf J. Zerial M. J. Cell Biol. 2000; 149: 901-914Google Scholar) and that Rab5 and markers of EE and RE, are in distinct within the B. De Renzis S. Nielsen E. Rietdorf J. Zerial M. J. 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Cell Biol. 1998; 143: Scholar). of 3-kinase by the receptor the of recycling of TfR and other receptors 1995; Scholar, K.W. J. Scholar, L.J. J. Biol. Chem. 1984; Scholar, S. J. Biol. Chem. Scholar). is inhibited by and is in a and association of with EE V. J. S. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). is a Rab5 for to S. Zerial M. 1999; Scholar, M. M. Zerial M. J. Biol. Chem. 2000; Scholar) and with EE through the of its with phosphatidylinositol in some 3-kinase the association of with endosomes V. J. S. Proc. Natl. Acad. Sci. U. S. A. 1997; is found to be to these and be in the of 3-kinase A. J.P. Scholar, C.E. Hopkins C.R. J. Cell Biol. Scholar, A. S. A. J. C. Zerial M. 1998; Scholar, A. E. J. W. L. D. S. J. Biol. Chem. 2002; Scholar, Scholar) can be by a membrane association of Scholar). The in can the membrane association of 3-kinase is inhibited A. S. A. J. C. Zerial M. 1998; Scholar). The formation of vesicles within is on and with the formation of vesicles by 3-kinase is of the of EE J. Cell Biol. 1995; Scholar, C.E. Hopkins C.R. J. Cell Biol. Scholar, M. J. S. J. Curr. Biol. 1999; Scholar). EE however, as a of with the recycling of membrane from EE C. Mcgraw T.E. J. Biol. Chem. 1996; Scholar, A. J.P. Scholar, C.E. Hopkins C.R. J. Cell Biol. Scholar). of TfR in EE and of this compartment in to or LY, as demonstrated in this are with these The only TfR trafficking that was by was from EE to the plasma membrane. for a pathway from EE to the plasma the of this pathway remains vesicles that directly from EE to the plasma membrane be vesicles have not been recycling through of EE with the plasma membrane. In a have been demonstrated to directly with the plasma in the of their into the W. Geuze H.J. G. 2002; Scholar). this we can only on the of the that are for TfR recycling. that 3-kinase for their to endosomes or proteins A. Cell Biol. Scholar, J. 2000; Scholar, D.J. A. J. Scholar). as for other that be for this that recycling are poorly to the complexity and of the system in which pathways The that two TfR recycling pathways can be to these further at the for and S. L. for the cell are to for of the