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Phosphoinositide 3-Kinases and Their FYVE Domain-containing Effectors as Regulators of Vacuolar/Lysosomal Membrane Trafficking Pathways

1999; Elsevier BV; Volume: 274; Issue: 14 Linguagem: Inglês

10.1074/jbc.274.14.9129

ISSN

1083-351X

Autores

Andrew E. Wurmser, Jonathan D. Gary, Scott D. Emr,

Tópico(s)

Retinal Development and Disorders

Resumo

phosphatidylinositol phosphoinositide SNAP (soluble NSF (N-ethylmaleimide-sensitive factor)) receptor carboxypeptidase Y early endosome antigen 1 retrieval sequence-alkaline phosphatase Kinases that phosphorylate phosphatidylinositol (PtdIns)1 at specific position(s) of the inositol ring play critical regulatory roles in a diverse array of cellular functions including growth, differentiation, apoptosis, and cytoskeletal rearrangement (1Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1292) Google Scholar, 2Rameh L.E. Chen C.S. Cantley L.C. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 3Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (909) Google Scholar). The downstream effects of phosphoinositide (PI) kinases (e.g. p110 PI 3-kinase) are carried out by phosphorylated derivatives of PtdIns, which serve as second messengers that recruit effector proteins to specific subcellular localizations and/or influence their activity (4Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar,5Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (826) Google Scholar). In addition to the classical signaling roles of lipid kinases at the plasma membrane, the activity of these kinases is also required for membrane trafficking along the secretory and endocytic pathways (6De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (659) Google Scholar). Vesicle-mediated delivery within the cell entails: (i) the formation and packaging of cargo into transport intermediates (vesicles), a process requiring the activity of coat proteins; (ii) the docking/fusion of such transport intermediates with the appropriate target organelle, which depends upon SNARE proteins and Rab GTPases; and (iii) the recycling of transport components (e.g.receptors and SNARES) (7Burd C.G. Babst M. Emr S.D. Semin. Cell Dev. Biol. 1998; 9: 527-533Crossref PubMed Scopus (42) Google Scholar, 8Novick P. Brennwald P. Cell. 1993; 75: 597-601Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 9Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). The lipid composition of vesicular transport intermediates is also critical, and roles for phosphoinositides, phosphatidic acid, and lysobisphosphatidic acid have been documented (6De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (659) Google Scholar, 10Randazzo P.A. Kahn R.A. J. Biol. Chem. 1994; 269: 10758-10763Abstract Full Text PDF PubMed Google Scholar, 11Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (647) Google Scholar). In particular, phosphoinositides, which can be modified at specific sites of the inositol ring, either singly or in combination, represent versatile molecules through which the cell generates distinct second messengers. Here we review recent progress in yeast and mammalian systems, which has converged to clarify the function of 3-phosphoinositides in membrane trafficking.Requirement for PtdIns-3-P in Vesicular TrafficThe importance of PtdIns-3-P in vesicular transport was first revealed during the study of Golgi to vacuole/lysosome transport in yeast (12Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (802) Google Scholar). The yeast vacuole, an acidified organelle that contains active hydrolytic enzymes, is the functional analog of the mammalian lysosome (13Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Crossref PubMed Google Scholar). Newly synthesized hydrolases and cargo destined for degradation by these hydrolases are transported to the vacuole along several specialized trafficking pathways (14Vida T.A. Huyer G. Emr S.D. J. Cell Biol. 1993; 121: 1245-1256Crossref PubMed Scopus (124) Google Scholar, 15Cowles C.R. Snyder W.B. Burd C.G. Emr S.D. EMBO J. 1997; 16: 2769-2782Crossref PubMed Scopus (174) Google Scholar, 16Wendland B. Emr S.D. Riezman H. Curr. Opin. Cell Biol. 1998; 10: 513-522Crossref PubMed Scopus (150) Google Scholar, 17Klionsky D.J. J. Biol. Chem. 1998; 273: 10807-10810Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The carboxypeptidase Y (CPY) pathway, the best characterized of these, mediates the delivery of CPY and other resident vacuolar proteins from the late Golgi to an intermediate endosomal compartment, which ultimately fuses with the vacuole (14Vida T.A. Huyer G. Emr S.D. J. Cell Biol. 1993; 121: 1245-1256Crossref PubMed Scopus (124) Google Scholar, 18Futter C.E. Pearse A. Hewlett L.J. Hopkins C.R. J. Cell Biol. 1996; 132: 1011-1023Crossref PubMed Scopus (433) Google Scholar). This sequence of transport steps requires the function of more than 40 gene products designated Vps, forvacuolar protein sorting (19Rothman J.H. Stevens T.H. Cell. 1986; 47: 1041-1051Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 20Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (723) Google Scholar, 21Rothman J.H. Howald I. Stevens T.H. EMBO J. 1989; 8: 2057-2065Crossref PubMed Scopus (175) Google Scholar, 22Banta L.M. Robinson J.S. Klionsky D.J. Emr S.D. J. Cell Biol. 1988; 107: 1369-1383Crossref PubMed Scopus (299) Google Scholar). One of these genes, VPS34, encodes a PtdIns 3-kinase (12Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (802) Google Scholar,23Herman P.K. Emr S.D. Mol. Cell. Biol. 1990; 10: 6742-6754Crossref PubMed Scopus (353) Google Scholar). In vitro studies revealed that Vps34p specifically phosphorylates PtdIns but not PtdIns-4-P or PtdIns-4,5-P2 at the D-3 position of the inositol ring (12Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (802) Google Scholar). Point mutations within highly conserved amino acid motifs of the Vps34p kinase domain deplete cells of PtdIns-3-P in vivo and result in the missorting and secretion of CPY (12Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (802) Google Scholar). These results suggest a role for PtdIns-3-P in the sorting of proteins to the yeast vacuole.3′-Phosphorylated phosphoinositides also appear to play important roles in membrane trafficking to the mammalian lysosome. The fungal metabolite wortmannin, a potent inhibitor of PI 3-kinases, blocks homotypic endosome fusion in vitro and impairs the transport of cathepsin D and internalized platelet-derived growth factor receptor to the lysosome in vivo (24Brown W.J. DeWald D.B. Emr S.D. Plutner H. Balch W.E. J. Cell Biol. 1995; 130: 781-796Crossref PubMed Scopus (251) Google Scholar, 25Davidson H.W. J. Cell Biol. 1995; 130: 797-805Crossref PubMed Scopus (184) Google Scholar, 26Shpetner H. Joly M. Hartley D. Corvera S. J. Cell Biol. 1996; 132: 595-605Crossref PubMed Scopus (157) Google Scholar). A human homolog of the yeast Vps34 PtdIns 3-kinase has been cloned and found to be sensitive to nanomolar levels of wortmannin (27Volinia S. Dhand R. Vanhaesebroeck B. MacDougall L.K. Stein R. Zvelebil M.J. Domin J. Panaretou C. Waterfield M.D. EMBO J. 1995; 14: 3339-3348Crossref PubMed Scopus (306) Google Scholar). This suggests that human Vps34p confers wortmannin sensitivity upon lysosomal trafficking in mammalian cells, although a role for p110 PI 3-kinase in the early stages of endocytosis is also likely (28Joly M. Kazlauskas A. Fay F.S. Corvera S. Science. 1994; 263: 684-687Crossref PubMed Scopus (252) Google Scholar). Interestingly, the block in homotypic endosome fusion caused by wortmannin can be overcome by the overexpression of Rab5 in its active GTP-bound form (29Li G. D'Souza-Schorey C. Barbieri M.A. Roberts R.L. Klippel A. Williams L.T. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211Crossref PubMed Scopus (246) Google Scholar), suggesting a link between Rab5 function and PI 3-kinase activity.PtdIns-3-P Transits Golgi/Endosomal Compartments to the VacuoleIn yeast, Vps34p is recruited from the cytosol to the Golgi and/or endosome by a membrane-associated serine/threonine protein kinase, Vps15p (30Herman P.K. Stack J.H. DeModena J.A. Emr S.D. Cell. 1991; 64: 425-437Abstract Full Text PDF PubMed Scopus (155) Google Scholar, 31Stack J.H. Herman P.K. Schu P.V. Emr S.D. EMBO J. 1993; 12: 2195-2204Crossref PubMed Scopus (259) Google Scholar, 32Stack J.H. DeWald D.B. Takegawa K. Emr S.D. J. Cell Biol. 1995; 129: 321-334Crossref PubMed Scopus (197) Google Scholar). Interactions between Vps15p and Vps34p, which are dependent on Vps15p protein kinase activity, serve both to localize Vps34p to its substrate and stimulate its PtdIns 3-kinase activity >10-fold (32Stack J.H. DeWald D.B. Takegawa K. Emr S.D. J. Cell Biol. 1995; 129: 321-334Crossref PubMed Scopus (197) Google Scholar). Inactivation of the Vps15p protein kinase results in severe decreases in cellular levels of PtdIns-3-P and the missorting of vacuolar hydrolases (30Herman P.K. Stack J.H. DeModena J.A. Emr S.D. Cell. 1991; 64: 425-437Abstract Full Text PDF PubMed Scopus (155) Google Scholar, 32Stack J.H. DeWald D.B. Takegawa K. Emr S.D. J. Cell Biol. 1995; 129: 321-334Crossref PubMed Scopus (197) Google Scholar). Thus, Vps15p functions as an upstream regulator of Vps34p. Up-regulation of the mammalian isoform of Vps34p is likely to occur by a similar mechanism. p150, a human homolog of yeast Vps15p, has been identified as a protein kinase that directly interacts with human Vps34 and stimulates its PtdIns 3-kinase activity (33Panaretou C. Domin J. Cockcroft S. Waterfield M.D. J. Biol. Chem. 1997; 272: 2477-2485Crossref PubMed Scopus (183) Google Scholar). In yeast, subcellular fractionation data localize Vps15p to a Golgi/endosome-enriched fraction, suggesting a functional role for PtdIns-3-P in membrane trafficking between the Golgi and endosome (30Herman P.K. Stack J.H. DeModena J.A. Emr S.D. Cell. 1991; 64: 425-437Abstract Full Text PDF PubMed Scopus (155) Google Scholar,31Stack J.H. Herman P.K. Schu P.V. Emr S.D. EMBO J. 1993; 12: 2195-2204Crossref PubMed Scopus (259) Google Scholar).Termination or modification of signals mediated by phosphoinositides have classically been attributed to the action of cytoplasmic phospholipases and phosphatases. For example, in mammalian cells PtdIns-4,5-P2 is cleaved to distinct second messengers by phospholipase C in response to tyrosine kinase and G-protein-coupled receptor activation (34Rhee S.G. Trends Biochem. Sci. 1991; 16: 297-301Abstract Full Text PDF PubMed Scopus (192) Google Scholar, 35Sternweis P.C. Smrcka A.V. Trends Biochem. Sci. 1992; 17: 502-506Abstract Full Text PDF PubMed Scopus (174) Google Scholar). PtdIns-4,5-P2 turnover is also mediated by Type II 5-phosphatases like synaptojanin and OCRL (36McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar,37Attree O. Olivos I.M. Okabe I. Bailey L.C. Nelson D.L. Lewis R.A. McInnes R.R. Nussbaum R.L. Nature. 1992; 358: 239-242Crossref PubMed Scopus (403) Google Scholar). Similarly, cytoplasmic phosphatases may carry out the turnover of 3-phosphoinositides as proteins exhibiting phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (e.g. SHIPs) and PtdIns-3-P 3-phosphatase activity have been identified (38Woscholski R. Parker P.J. Trends Biochem. Sci. 1997; 22: 427-431Abstract Full Text PDF PubMed Scopus (70) Google Scholar) and purified (39Caldwell K.K. Lips D.L. Bansal V.S. Majerus P.W. J. Biol. Chem. 1991; 266: 18378-18386Abstract Full Text PDF PubMed Google Scholar), respectively.In yeast, dramatic decreases in the cellular levels of PtdIns-3-P occur within minutes after inactivation of Vps34p (32Stack J.H. DeWald D.B. Takegawa K. Emr S.D. J. Cell Biol. 1995; 129: 321-334Crossref PubMed Scopus (197) Google Scholar), the result of an arrest in PtdIns-3-P synthesis and the continued function of a PtdIns-3-P turnover pathway. Unlike other phosphoinositides, however, PtdIns-3-P may not be degraded within the cytoplasm of the cell. Interestingly, yeast mutants compromised for vacuolar hydrolase activity exhibit severalfold increases in PtdIns-3-P levels in vivo, indicating that the consumption of PtdIns-3-P requires the activity of lumenal vacuolar hydrolases (40Wurmser A.E. Emr S.D. EMBO J. 1998; 17: 4930-4942Crossref PubMed Scopus (127) Google Scholar). Cellular levels of PtdIns-4-P and PtdIns-4,5-P2 are not affected in hydrolase-deficient strains (40Wurmser A.E. Emr S.D. EMBO J. 1998; 17: 4930-4942Crossref PubMed Scopus (127) Google Scholar). Indeed, the vacuole/lysosome contains candidate lipases and phosphatases (41Sandhoff K. Kolter T. Trends Cell Biol. 1996; 6: 98-103Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 42Kaneko Y. Toh-e A. Oshima Y. Mol. Cell. Biol. 1982; 2: 127-137Crossref PubMed Scopus (71) Google Scholar), which may function in the turnover of PtdIns-3-P but not other phosphoinositides.An intact vacuolar transport pathway is also required to deliver PtdIns-3-P from its site of synthesis at the Golgi/endosome to the vacuole. Impairing endosome-to-vacuole transport through the deletion of VAM3 (vacuolar t-SNARE) (43Wada Y. Nakamura N. Ohsumi Y. Hirata A. J. Cell Sci. 1997; 110: 1299-1306Crossref PubMed Google Scholar) or YPT7 (Rab GTPase) (44Wichmann H. Hengst L. Gallwitz D. Cell. 1992; 71: 1131-1142Abstract Full Text PDF PubMed Scopus (202) Google Scholar) causes severalfold increases in PtdIns-3-P, presumably by blocking delivery of vacuole-bound PtdIns-3-P that accumulates in a prevacuolar endosomal compartment (40Wurmser A.E. Emr S.D. EMBO J. 1998; 17: 4930-4942Crossref PubMed Scopus (127) Google Scholar).Vps15p/Vps34p-mediated synthesis of PtdIns-3-P occurs in the cytoplasmic leaflet of the Golgi/endosome membrane. A mechanism is therefore required to overcome the separation between PtdIns-3-P and the vacuolar hydrolases that are required to degrade it. Morphological studies have shown that mutants which stabilize PtdIns-3-P levels also accumulate lumenal vesicles within the endosome or vacuole (40Wurmser A.E. Emr S.D. EMBO J. 1998; 17: 4930-4942Crossref PubMed Scopus (127) Google Scholar, 45Darsow T. Rieder S.E. Emr S.D. J. Cell Biol. 1997; 138: 517-529Crossref PubMed Scopus (295) Google Scholar). It is likely that a significant pool of PtdIns-3-P is sorted into these vesicles, which invaginate into the endosome or vacuole. These vesicles, together with PtdIns-3-P, are then degraded by hydrolases in the vacuole lumen (see Fig. 1). Thus, the turnover mechanism for the bulk of PtdIns-3-P is distinct from that of other phosphoinositides. In addition, because PtdIns-3-P is present in endosomal and vacuolar membranes, PtdIns-3-P has the capacity to regulate not only Golgi-to-endosome transport but endosome and possibly vacuole function as well. This is especially likely because the progression of internalized cargo along the endocytic pathway is compromised upon inactivation of Vps34p, consistent with a role for PtdIns-3-P in endosome function (40Wurmser A.E. Emr S.D. EMBO J. 1998; 17: 4930-4942Crossref PubMed Scopus (127) Google Scholar, 46Munn A.L. Riezman H. J. Cell Biol. 1994; 127: 373-386Crossref PubMed Scopus (229) Google Scholar). Thus, the data predict the existence of Golgi, endosomal, and, possibly, vacuolar effectors functioning downstream of the Vps34 PtdIns 3-kinase.FYVE Domain-containing Proteins as Effectors of Vps34 PtdIns 3-Kinase SignalingSeveral proteins have been postulated to function as downstream effectors of PtdIns 3-kinases (47Patki V. Virbasius J. Lane W.S. Toh B.H. Shpetner H.S. Corvera S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7326-7330Crossref PubMed Scopus (202) Google Scholar, 48Rapoport I. Miyazaki M. Boll W. Duckworth B. Cantley L.C. Shoelson S. Kirchhausen T. EMBO J. 1997; 16: 2240-2250Crossref PubMed Scopus (182) Google Scholar). One of these, the mammalian EEA1 (early endosome antigen 1) protein, is required for homotypic endosome-endosome fusion in vitro (49Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar). Critical to the involvement of EEA1 in this process is its ability to associate with endosomal membranes (49Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar), an interaction that is inhibited by wortmannin (47Patki V. Virbasius J. Lane W.S. Toh B.H. Shpetner H.S. Corvera S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7326-7330Crossref PubMed Scopus (202) Google Scholar). EEA1 thus represents a potential PtdIns-3-P-binding protein.Recent studies have addressed the capacity of EEA1 to directly bind PtdIns-3-P. In vitro, recombinant EEA1 cosediments with liposomes containing PtdIns-3-P but not PtdIns phosphorylated at other positions of the inositol ring (50Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 51Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Crossref PubMed Scopus (440) Google Scholar, 52Patki V. Lawe D.C. Corvera S. Virbasius J.V. Chawla A. Nature. 1998; 394: 433-434Crossref PubMed Scopus (249) Google Scholar). The lipid binding activity is attributable to a cysteine-rich sequence motif encoded by the C terminus of EEA1, referred to as the FYVE (Fab1,YGLO23, Vps27, and EEA1) domain (53Mu F.T. Callaghan J.M. Steele-Mortimer O. Stenmark H. Parton R.G. Campbell P.L. McCluskey J. Yeo J.P. Tock E.P. Toh B.H. J. Biol. Chem. 1995; 270: 13503-13511Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 54Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). FYVE domains coordinate 2 Zn2+ ions via 8 cysteine/histidine residues spaced in a specific manner (CX 2CX 9–39CX 1–3(C/H)X 2–3CX 2CX 4–48CX 2C) (54Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). This protein motif also contains a basic amino acid patch adjacent to the 3rd cysteine residue (50Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 54Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), which is critical for binding of acidic PtdIns-3-P (50Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). In addition to binding PtdIns-3-Pin vitro, Aequorea victoria green fluorescent protein coupled to the EEA1-FYVE domain localizes to endosomal and vacuolar compartments in yeast (50Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). This localization is dependent on Vps34 PtdIns 3-kinase activity, demonstrating that the FYVE domain is sufficient to mediate membrane association in vivo (50Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Conversely, deletion of the FYVE domain of EEA1 (54Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) or the treatment of cells with wortmannin (47Patki V. Virbasius J. Lane W.S. Toh B.H. Shpetner H.S. Corvera S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7326-7330Crossref PubMed Scopus (202) Google Scholar) disrupts the endosomal association of this protein.As mentioned above, overexpression of Rab5 rescues wortmannin-induced inhibition of homotypic endosome fusion (29Li G. D'Souza-Schorey C. Barbieri M.A. Roberts R.L. Klippel A. Williams L.T. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211Crossref PubMed Scopus (246) Google Scholar), suggesting a possible link between PI 3-kinase signaling and Rab5. Consistent with this, overexpression of Rab5 in its active, GTP-bound state is sufficient to restore endosomal association of EEA1 (49Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar). In fact, in addition to binding PtdIns-3-P, EEA1 acts as an effector of Rab5 as it directly interacts with Rab5-GTP (49Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar). Therefore, by defining EEA1 as a PtdIns-3-P-binding protein in vitro and through in vivo studies in yeast, a molecular mechanism for wortmannin inhibition of endosomal trafficking events in mammalian cells has been resolved.FYVE domains are not unique to EEA1 but present in the mammalian Hrs and yeast Vac1, Vps27, and Fab1 proteins (53Mu F.T. Callaghan J.M. Steele-Mortimer O. Stenmark H. Parton R.G. Campbell P.L. McCluskey J. Yeo J.P. Tock E.P. Toh B.H. J. Biol. Chem. 1995; 270: 13503-13511Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 54Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Like the FYVE domain of EEA1, the FYVE domains of these proteins also bind PtdIns-3-P (50Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar,51Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Crossref PubMed Scopus (440) Google Scholar). Thus, FYVE domains function as modular PtdIns-3-P binding motifs. Moreover, these FYVE domain-containing proteins play important roles in membrane trafficking events of the secretory and endocytic pathways. Vac1p, the yeast ortholog of EEA1 (47Patki V. Virbasius J. Lane W.S. Toh B.H. Shpetner H.S. Corvera S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7326-7330Crossref PubMed Scopus (202) Google Scholar, 55Weisman L.S. Wickner W. J. Biol. Chem. 1992; 267: 618-623Abstract Full Text PDF PubMed Google Scholar), functions as a multivalent regulatory protein that interacts with the yeast Rab5 GTPase (Vps21p), the endosomal t-SNARE, Pep12p, and the Sec1p homolog, Vps45p (56Horazdovsky B.F. Busch G.R. Emr S.D. EMBO J. 1994; 13: 1297-1309Crossref PubMed Scopus (168) Google Scholar, 57Singer-Kruger B. Stenmark H. Dusterhoft A. Philippsen P. Yoo J.S. Gallwitz D. Zerial M. J. Cell Biol. 1994; 125: 283-298Crossref PubMed Scopus (183) Google Scholar, 58Burd C.G. Peterson M. Cowles C.R. Emr S.D. Mol. Biol. Cell. 1997; 8: 1089-1104Crossref PubMed Scopus (140) Google Scholar, 59Peterson M.R. Burd C.G. Emr S.D. Curr. Biol. 1999; (in press)Google Scholar, 60Cowles C.R. Emr S.D. Horazdovsky B.F. J. Cell Sci. 1994; 107: 3449-3459PubMed Google Scholar). PtdIns-3-P, together with the GTP-bound Vps21p, may regulate the ordered series of biochemical interactions between Vac1p and these other proteins, which together are required to ensure the high fidelity of vesicle (CPY-containing) docking/fusion with the endosome (58Burd C.G. Peterson M. Cowles C.R. Emr S.D. Mol. Biol. Cell. 1997; 8: 1089-1104Crossref PubMed Scopus (140) Google Scholar). Vps27p, the likely yeast counterpart of mammalian Hrs (61Komada M. Masaki R. Yamamoto A. Kitamura N. J. Biol. Chem. 1997; 272: 20538-20544Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), mediates the maturation of endosomes (e.g. receptor recycling, multivesicular body formation), a process required for endosome fusion with the vacuole (18Futter C.E. Pearse A. Hewlett L.J. Hopkins C.R. J. Cell Biol. 1996; 132: 1011-1023Crossref PubMed Scopus (433) Google Scholar, 62Murphy R.F. Trends Cell Biol. 1991; 1: 77-82Abstract Full Text PDF PubMed Scopus (127) Google Scholar). An intact FYVE domain is required for Vps27p function (63Piper R.C. Cooper A.A. Yang H. Stevens T.H. J. Cell Biol. 1995; 131: 603-617Crossref PubMed Scopus (340) Google Scholar), underscoring a role for PtdIns-3-P in endosomal maturation. The FYVE domain protein Fab1 regulates a third, distinct membrane trafficking event. Within 10 min after inactivation of Fab1p a 2.5-fold enlargement of the vacuole occurs (64Yamamoto A. DeWald D.B. Boronenkov I.V. Anderson R.A. Emr S.D. Koshland D. Mol. Biol. Cell. 1995; 6: 525-539Crossref PubMed Scopus (233) Google Scholar, 65Gary J.D. Wurmser A.E. Bonangelino C.J. Weisman L.S. Emr S.D. J. Cell Biol. 1998; 143: 65-79Crossref PubMed Scopus (339) Google Scholar), suggesting a role for Fab1p in vacuolar membrane efflux/degradation. Point mutations within the FYVE domain of Fab1p also result in enlarged vacuole phenotypes, 2J. D. Gary and S. D. Emr, unpublished observations. revealing that this subregion may be essential for Fab1p localization or activity (see below). These results define the Vps34 PtdIns 3-kinase as a regulatory kinase that modulates multiple downstream effectors which act at distinct stages of membrane trafficking to and from the vacuole. Two additional FYVE domain-containing open reading frames are present within the yeast genome and whereas a cellular function has yet to be assigned to these proteins, they also promise to be downstream effectors of Vps34p.Conversion of PtdIns-3-P, a Signal for Anterograde Traffic to the Vacuole, to PtdIns-3,5-P2, a Signaling Lipid Required for Vacuole Membrane HomeostasisPtdIns-3,5-P2 is a newly identified phosphoinositide, discovered both in yeast and higher eukaryotic cells (66Whiteford C.C. Brearley C.A. Ulug E.T. Biochem. J. 1997; 323: 597-601Crossref PubMed Scopus (127) Google Scholar, 67Dove S.K. Cooke F.T. Douglas M.R. Sayers L.G. Parker P.J. Michell R.H. Nature. 1997; 390: 187-192Crossref PubMed Scopus (389) Google Scholar). This lipid is synthesized directly from a preexisting pool of PtdIns-3-P, indicating the existence of a PtdIns-3-P 5-kinase (66Whiteford C.C. Brearley C.A. Ulug E.T. Biochem. J. 1997; 323: 597-601Crossref PubMed Scopus (127) Google Scholar, 67Dove S.K. 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Cell Biol. 1998; 143: 65-79Crossref PubMed Scopus (339) Google Scholar). This analysis led to the identification of a new subgroup defined by Fab1p (65Gary J.D. Wurmser A.E. Bonangelino C.J. Weisman L.S. Emr S.D. J. Cell Biol. 1998; 143: 65-79Crossref PubMed Scopus (339) Google Scholar), a protein essential to the maintenance of normal vacuole morphology (64Yamamoto A. DeWald D.B. Boronenkov I.V. Anderson R.A. Emr S.D. Koshland D. Mol. Biol. Cell. 1995; 6: 525-539Crossref PubMed Scopus (233) Google Scholar). The fact that the C-terminal kinase domain of Fab1p diverges from lipid kinases with known activities suggested that Fab1p could have a distinct substrate specificity (65Gary J.D. Wurmser A.E. Bonangelino C.J. Weisman L.S. Emr S.D. J. Cell Biol. 1998; 143: 65-79Crossref PubMed Scopus (339) Google Scholar). 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