proteins can associate with the rarer PtdIns 3,4,5-trisphosphate. (PtdIns(3,4,5)P3) lipid, ... Another minor PI 3-kinase product, PtdIns 3-phosphate. (PtdIns(3)P) ...
4175
Journal of Cell Science 112, 4175-4183 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0677
COMMENTARY FYVE-finger proteins – effectors of an inositol lipid Harald Stenmark1,* and Rein Aasland2 1Dept of Biochemistry, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway and 2Dept of Molecular Biology, University of Bergen, N-5020 Bergen, Norway
*Author for correspondence
Published on WWW 17 November 1999
SUMMARY The binding of cytosolic proteins to specific intracellular membranes containing phosphorylated derivatives of phosphatidylinositol (PtdIns) is a common theme in vital cellular processes, such as cytoskeletal function, receptor signalling and membrane trafficking. Recently, several potential effectors of the phosphoinositide 3-kinase product PtdIns 3-phosphate (PtdIns(3)P) have emerged through the observation that a conserved zinc-finger-like domain, the FYVE-finger, binds specifically to this lipid. Here we review
current knowledge about the structural basis for the FYVE-PtdIns(3)P interaction, its role in membrane recruitment of proteins and the functions of FYVE-finger proteins in membrane trafficking and other cellular processes.
INTRODUCTION
THE FYVE-FINGER – A CONSERVED PTDINS(3)PBINDING DOMAIN
Many biochemical processes in the cell occur at the interface between the cytosol and an intracellular membrane. Vesicular trafficking, signal transduction and actin-regulated membrane rearrangements are examples of processes that require the recruitment of cytosolic proteins to a specific membrane in a reversible and regulated manner. Such a recruitment is in part accomplished through the binding of cytosolic proteins to the cytoplasmic domains of transmembrane proteins or to the GTP-bound forms of membrane-associated small GTPases. However, an additional mechanism is becoming increasingly evident: many proteins become reversibly localised to membranes through interaction with specific lipid headgroups. Phosphoinositides (PIs), derivatives of phosphatidylinositol (PtdIns) that are phosphorylated at the 3-, 4- or 5-positions of the inositol headgroup, play a central role in this context. The differentially phosphorylated PdtIns headgroups are recognised by specific subsets of cytosolic effector proteins. For instance, many proteins containing pleckstrin homology (PH) domains bind to the abundant phosphoinositide, PtdIns 4,5-bisphosphate (PtdIns(4,5)P2), whereas other PH domain proteins can associate with the rarer PtdIns 3,4,5-trisphosphate (PtdIns(3,4,5)P3) lipid, which is produced by agonist-activated phosphoinositide 3-kinases (PI 3-kinases) (Rameh and Cantley, 1999). Another minor PI 3-kinase product, PtdIns 3-phosphate (PtdIns(3)P), regulates membrane trafficking in yeast and higher eukaryotes, but until recently its protein effectors were not known. Here, we discuss the function of proteins containing a novel zinc finger termed the FYVE-finger, which was recently found to bind specifically to PtdIns(3)P.
Key words: Membrane traffic, Phosphoinositide, PI 3-kinase, Signal transduction, Zinc finger
A cysteine-rich domain reminiscent of a zinc finger is involved in the membrane localisation of the autoantigen EEA1, and this motif is present in several other proteins. We dubbed it the FYVE-finger for Fab1p, YOTB, Vac1p and EEA1 (Stenmark et al., 1996). The FYVE finger has eight conserved cysteines, which coordinate two Zn2+ ions in a ‘cross-braced’ topology (Schwabe and Klug, 1994; Stenmark et al., 1996). In addition, it contains several other conserved residues, most prominently an R(R/K)HHCRxCG motif surrounding the third and fourth cysteine residues (positions 33-41 in Fig. 1). Several hydrophobic positions are also conserved among the FYVE fingers, as is an arginine residue at position 74. Some FYVEfinger proteins deviate from this consensus at a few positions. Recently, the biochemical function of the FYVE finger was revealed when three groups demonstrated that several FYVEfinger proteins bind specifically to PtdIns(3)P (Burd and Emr, 1998; Gaullier et al., 1998; Patki et al., 1998). The highly conserved features of the FYVE fingers strongly suggest that they have very similar structures and that they all bind to PtdIns(3)P or a similar ligand. Several proteins, such as Rabphilin-3A and Rim (see Fig. 1, lower part), have FYVE-related domains that lack several of the conserved features of FYVE fingers, most notably the basic R(R/K)HHCR motif and the arginine residue at position 74. Furthermore, they contain a conserved glycine residue at position 53 instead of that at position 41. The lack of the basic patch indicates that these domains do not bind to PtdIns(3)P, and we have experimentally verified this in the case of
4176 H. Stenmark and R. Aasland FYVE fingers Vps27p structure FYVE consensus HsEEA1 CeT10G3.5 Hs-ae54a12 Hs-zd64b11 ScYGL023p ScVps27p Hs-zt93d07_A Hs-zt93d07_B CeC07G1.5 HsHRS CeT23B5.2 HsKIAA0993 Hs-ym48d08 HsL33C6p CeF22G12.4 MmAnkhzn CeF01F1.6 ScPib1p HsKIAA0647 HsKIAA0371 ScVac1p_A CeYLN2 Hs-ys04h11 CeYW91 CeYOTB Hs-wd13b12 HsKIAA0321 Hs-tj23f01 HsFGD1 MmFGD2 MmFGD3 RnFRABIN ScVac1p_B CeD1022.7 HsSARA HsKIAA0305 CeC28C12.1 ScFab1p MmPIKfyve CeCAA91791.1
helix b2 b3 b4 b1 w# d C% C F@%% r+hhCr CG ##C cs % % %r#C C# # lnrkwaednevqncma--cgkg-fsvt---- vrrhhcrqcgnifca----ecsaknalt---- ---psskkpvrvcdacfndlq ssrkwlddaeaincte--cgkv-fslt---- vrkhhcrvcgkiycn----pcssksvri---- ---asaknpvracntcftdsq qghawlkddeathcrq--ceke-fsis---- rrkhhcrncghifcn----tcssnelal---- ---psypkpvrvcdschtlll qglvwlkdkeathckl--ceke-fsls---- krkhhcrncgeifcn----acsdnelpl---- ---psspkpvrvcdschalli tkdhwipdskrnscry--chkp-ftlw---- erkhhcrhcgdifcq----dhlrhwlyl ggingggtlckicddclveye tpadwi---dsdacmi--cskk-fsll---- nrkhhcrscggvfcq----ehssnsipl---- -pdlgiyepvrvcdscfedyd apaywrpnsqilscnk--cats-fkdn---- dtkhhcracgegfcd----scssktrpv---- pervwgpapvrvcdncyearn rpaywvpdheilhchn--crke-fsik---- lskhhcracgqgfcd----ecshdrrav---- -psrgwdhpvrvcfncnkkpg vapewa---dgpecyr--crsv-fsvf---- trkhhcracgqifcd----kcssrelal---- -pqfgiekevrvcetcyekkv rapdwv---daeechr--crvq-fgvm---- trkhhcracgqifcg----kcsskysti---- -pkfgiekevrvcepcyeqln radhwvqdvtrqrcdd--cehk-ftla---- drkhhcrncgqifcs----tcsrfeshi---- -trmnisrpvrvcrkcfqrlq aadhwvkdeggdscsg--csvr-fslt---- errhhcrncgqlfcq----kcsrfqsei---- -krlkisspvrvcqncyynlq eepqwvpdkecrrcmq--cdak-fdfl---- trkhhcrrcgkcfcd----rccsqkvpl---- -rrmcfvdpvrqcaecalvsl dppewvpdeacgfcta--ckap-ftvi---- rrkhhcrscgkifcs----rcsshsapl---- -prygqvkpvrvcthcymfhv aeprws---dgdtcd---cgar-fslt---- srkhhcrhcgrhvcs----kcsettmpi---- -akygeekrvrvcdvcahvis keppwc---dgsncye--ctak-fgvt---- trkhhcrhcgrllch----kcstkeipi---- -ikfdlnkpvrvcnicfdvlt qevrwqwdddvdqcsn--cdts-farv---- kvkphclhcgrifcm----nclkdtvps---- ---gpnhrpanvckvchtlln nlarwqadeeahscfq--cktn-fsfl---- vrrhhcrccgrifcs----sctenfvny knsdvesppyrtcnecydnll evtrwvpdhmashcyn--cdce-fwla---- krrhhcrncgnvfca----gcchlklpi---- -pdqqlydpvlvcnscyehiq emtrwlpdhlaahcya--cdsa-fwla---- srkhhcrncgnvfcs----sccnqkvpv---- -psqqlfepsrvckscysslh tvtpwrddrsvlfcni--csep-fgll---- lrkhhcrlcgmvvcddanrncsneisig ddllhipisirlcshcidmlf etpewk---tsdccqk--cnqpffwnl lrqhhcrtcgsavcg----sccdnwtty---- -ppmgyetkiricndcnarmk ktpewl---dsdscqk--cdqpffwnf lrqhhcrkcgkavcg----kcsskrssn---- -ppmgfefevrvcdschegit pnttwe--gesghcay--ckke-fnkl sdrqhhcrncgrvvce----dcsknrfsv---- -ieegksvqkracdscydsmh haavwvpdgeavkcmv--cgktqfnlv---- qrrhhcrncgrvvcg----acssrtfri---- --dnvhkkpvrvcdhcfdsls haavwvpdseatvcmr--cqkakftpv---- nrrhhcr?cg? vvcg----pcsekrfll---- --psqsskpvr?cdfcyddl ? arhqwvpdetesicmv--ccrehftmf---- nrrhhcrrcgrlvcs----scstkkmvv---- --egcrenparvcdqcysycn raprwirdnevtmcmk--ckep-fnalt--- rrrhhcracgyvvcw----kcsdykaql---- --eydggklskvckdcyqiis raptpirekevtmcmr--cqep-fnsit--- krrhhckacghvvcg----kcsefrarl---- --vydnnrsnrvctdcyvalh rapqwvrdkmvtmcmr--cqep-fnalt--- rrrhhcracgyvvca----kcsdyrael---- --kydsnrpnrvcltcytflt sssktrrdkekpgcks--cget-fnsit--- krryrcklcgevicr----kcsefkaen---- ------skqsrvcrecfleep raprwirdnevtmcmk--ckes-fnalt--- rrrhhcracghvvcw----kcsdykaql---- --eydggrlnkvckdcyqims krshwekfkkgksccht-cgrt-lnnn----igaincrkcgklycr----rhlpnmikl aqydprngkwyncchdcfvtkp tspywipd---secpn--cml--ftii---- trrhhcracgrvlcg----sccnekafl- qeegkklqavrvckpcsamla vapvwvpdsqapncmk--cear-ftft---- krrhhcracgkvfca----sccslkckl---- --lymdrkearvcvichsvlm kqptwvpdseapncmn--cqvk-ftft---- krrhhcracgkvfcg----vccnrkckl---- ---qylekearvcvvcyetis ikpvwlpdnisneclmegcste-fnii---- nrrhhcrdcgwlick----fck-gqapl---- --skydftkqnvcsecfdrhy skeywmkdesskecfs--cgkt-fntf---- rrkhhcricgqifcs----sctllidgd---- --rfgchakmrvcyncyehad lkqywmpdsqckecyd--csek-fttf---- rrrhhcrlcgqifcs----rccnqeipg---- -kfmgytgdlractycrkial .....mpdstgrecyq--ceer-fttf----rrrhhcrlcgqifca----kccsshidg-----aalgymgelrlcdycarkvq
[1345-1410] [1098-1163] est est [ 445- 527] [ 166- 230] est est [ 189- 253] [ 156- 220] [ 856- 923] [ 285- 352] est [1037 1100] [1115-1179] [ 337- 402] [ 10- 88] [ 928- 995] [1112-1179] [ 208- 297] [ 282- 357] est [ 786- 856] [ 145- 212] est [ 808- 875] est [ 724- 790] [ 451- 518] [ 521- 584] [ 552- 619] [ 64- 135] 7 [ 534- 601] [ 590- 656] [ 740- 805] [ 691- 758] [ 233- 299] [ 162- 229] [ 15 ankyrin repeats
Part of multiprotein complex?
T23B5.2 YLN2
WD repeats (implicated in protein-protein interactions). The N-terminus of T23B5.2 has similarity to beige/ChediakHigashi syndrome (lysosomal disorder) protein
YW91
N-terminal region with similarity to myotubularins, including a putative dual-specificity phosphatase domain
?
C28C12.10 YOTB?
GEF domain; PH domains flank the FYVE finger
GEF for Cdc42, regulation of actin cytoskeleton
Large (1542-residue) protein
?
D1022.7
Smad-binding domain; D1022.7 is an AKAP
Mediator of TGFβ signalling. Membrane recruitment of PKA
DEP-domain, chaperonindomain, PI 5-kinase domain
PI 5-kinase, formation of multivesicular endosomes or membrane recycling from vacuole/lysosome
N-terminal VHS-domain, coiledcoil region implicated in STAMbinding
Endosome-lysosome/vacuole trafficking. Formation of multivesicular endosomes. Signal transduction? Regulated exocytosis?
KIAA00321 SARA‡ KIAA0305
Structural features N-terminal C2H2 finger, long coiled-coil regions, Rab5 binding domains
CAA91791.1
Fab1p
C07G1.5
Vps27p
Endosome-lysosome transport?
*The significance of a reported β-1-4-galactosyltransferase activity (Uehara and Muramatsu, 1997) of the murine homologue of human KIAA0993 is unclear, given that the protein tested consists only of WD repeats and the FYVE finger. This is probably a partial cDNA, like KIAA0993, whose full-length version might have a structure similar to that of CeT23B5.2. ‡The FYVE-finger protein, NSP (Meckelein et al., 1998) appears to be a splice variant of SARA. This protein was reported to be homologous to serine proteases, but the similarity is very limited, and there is no experimental evidence to support this idea.
represented in Fig. 1 by the mouse or rat homologues owing to the lack of complete human sequence. In C. elegans, we found 11 FYVE-finger sequences. In addition to CeYPT7 (Fig. 1), the nematode has several more-divergent FYVE-related domains that we did not analyse further. Only two FYVEfinger sequences identified so far, yeast Vac1p and the human EST zt93d07, contain two FYVE fingers. By contrast, in the cases of other domains that interact with small structural features (e.g. phosphotyrosine) more than one copy is often present. The multicellular organisms appear to have expanded their repertoire of FYVE-finger proteins. Assuming that most of the human genome was formed by two duplications (Spring, 1997), we expect that mammals will have between 30 and 40 FYVE finger proteins, and that we have already seen most of the different types of mammalian FYVE-finger proteins. Even though not all mammalian proteins have been identified, there is a remarkable correspondence between the FYVE-finger proteins found in mammals and those in C. elegans. On the basis of the available data, we can thus divide the FYVE-finger proteins into nine groups that comprise proteins with highly different structures and functions (Table 1 and Fig. 3). While some are rod-like molecules involved in membrane tethering, others contain kinase, phosphatase, and
GDP/GTP exchange factor (GEF) domains. Note that several FYVE-finger proteins contain PH domains which presumably bind to phosphoinositides other than PtdIns(3)P, and various domains implicated in protein-protein interactions. This indicates that FYVE-finger proteins, in addition to their PtdIns(3)P interaction, might associate with a variety of phosphoinositide and protein ligands. Although the functions of most FYVE-finger proteins are not known, the different groups of FYVE-finger proteins evidently participate in distinct cellular processes, including vesicle transport, signal transduction and cytoskeletal regulation (see below). FYVE-FINGER PROTEINS AND MEMBRANE TRAFFICKING EEA1 in endocytic membrane docking/fusion Our most detailed knowledge about the function of the FYVEPtdIns(3)P interaction has originated from studies of the earlyendosomal autoantigen, EEA1 (Mu et al., 1995). EEA1 is a large coiled-coil protein that contains a C-terminal FYVE finger. It also contains two domains that bind to the active, GTP-bound form of the early-endosomal GTPase Rab5 (Simonsen et al.,
FYVE-finger proteins 4179 1998). One of these domains is adjacent to the FYVE finger, whereas the other one, which is a C2H2-type zinc finger, is found at the N terminus (Fig. 4A). The C terminus of EEA1, containing one Rab5-binding domain and the FYVE finger, is necessary and sufficient for the targeting of EEA1 to early endosomes, and the membrane binding requires both PI 3-kinase activity and Rab5:GTP (Patki et al., 1997; Simonsen et al., 1998). This has led to the idea that PtdIns(3)P and Rab5:GTP together form a specificity code that ensures the exclusive targeting of EEA1 to early endosomes (Simonsen et al., 1998). The relatively low affinities of the C-terminal Rab5-binding domain for Rab5:GTP and of the FYVE finger for PtdIns(3)P secure that, under physiological conditions, EEA1 is recruited only to membranes that contain both of these components. What is the function of EEA1 at the endosome membrane? Depletion of EEA1 inhibits homotypic endosome fusion in vitro (Mills et al., 1998; Simonsen et al., 1998), indicating that EEA1 plays a role in this process. Endosome fusion also requires Rab5:GTP (Gorvel et al., 1991) and PI 3-kinase acitivity (Jones and Clague, 1995; Li et al., 1995), and Rab5 is required on both membranes in order for the fusion to take place (Barbieri et al., 1998). EEA1 might therefore function as an effector of Rab5 and PtdIns(3)P in endosome fusion by tethering two Rab5-positive membranes (Fig. 4A). Such tethering is thought to facilitate the pairing of SNARE proteins (Rothman and Warren, 1994) present on the two opposing membranes, and SNARE pairing either drives membrane fusion or is closely linked to it (Pfeffer, 1999). The fact that excess EEA1 stimulates endosome fusion in vitro and, when SNARE function is impaired, leads to the occurrence of aggregated, unfused endosomes (Christoforidis et al., 1999a) supports this view. Furthermore, although EEA1 is found as a dimer in the cytosol (Callaghan et al., 1999), it appears to engage in oligomeric complexes during membrane fusion. These complexes contain the endosomal SNARE molecule syntaxin13 as well as its regulator NSF (McBride et al., 1999). EEA1 thus appears to be central to a machinery that drives the tethering and fusion of endosomes, and the role of the FYVE-PtdIns(3)P interaction could be to concentrate EEA1 at the right location. Hrs, a FYVE-finger protein involved in endosome function and ventral-folding morphogenesis Another mammalian FYVE-finger protein that has been implicated in membrane trafficking is Hrs, a hepatocyte growth-factor-regulated tyrosine-kinase substrate (Komada and Kitamura, 1995). Like EEA1, Hrs is localised to early endosomes in a manner that requires PI 3-kinase activity (Komada et al., 1997; Komada and Soriano, 1999), and mouse embryos that lack Hrs contain abnormally large early-endocytic structures. Little is known about these structures except that they contain transferrin receptors and seem to have a vacuolar appearance. Hrs might thus play a role in the formation of multivesicular endosomes, a process that requires PI 3-kinase activity (Fernandez-Borja et al., 1999). The Hrs knockout leads to a defect in ventral folding morphogenesis and is embryonic lethal at the 11-day stage. The defects in ventral folding could be a consequence of an impaired endosome function. However, Hrs might also play a role in signal transduction, given that it becomes tyrosine phosphorylated in an agonist-dependent manner (Komada and Kitamura, 1995; Komada et al., 1997) and forms a complex with a signaltransducing adapter molecule (STAM) that has been implicated in transcriptional regulation (Asao et al., 1997).
A two-hybrid screen for molecules interacting with the SNARE molecule SNAP-25 yielded Hrs-2, a molecule identical to or closely related to Hrs (Bean et al., 1997). Hrs-2 possesses a weak ATPase activity, but its exact biochemical function is not known. SNAP-25 participates in the docking of synaptic vesicles with the presynaptic plasma membrane (Rothman and Warren, 1994), and the association of Hrs-2 with this molecule thus suggests that Hrs-2 plays a role in regulated exocytosis. Indeed, recombinant Hrs-2 inhibits calcium-triggered noradrenaline release from permeabilized PC12 cells (Bean et al., 1997). However, no evidence for localisation of Hrs-2 to synaptic vesicles or to the presynaptic plasma membrane has been presented, and the possibility that the inhibitory effect of excess Hrs-2 on noradrenalin release reflects a nonphysiological sequestering of SNAP-25 cannot be excluded. Vac1p and Vps27p: putative homologues of EEA1 and Hrs in endocytic trafficking in yeast The only PI 3-kinase in S. cerevisiae, Vps34p, produces exclusively PtdIns(3)P. It was discovered in a screen for mutants in which vacuolar protein sorting is defective (vps mutants), and its inactivation is associated with improper sorting of a subset of hydrolases from the Golgi apparatus to the vacuole, the yeast equivalent of the lysosome (Schu et al., 1993). Vacuolar protein sorting appears to be the only pathway that is affected in vps34 mutants, indicating that PtdIns(3)P is exclusively needed in this pathway. Indeed, of five FYVEfinger proteins expressed in yeast, three of these, Vac1p, Vps27p and Fab1p, have already been implicated in membrane trafficking to the vacuole. (The function of the two remaining yeast FYVE-finger proteins, Pib1p and YGL023, is not known). Yeast vac1 mutants accumulate vesicles destined for the endosome, which suggests that Vac1p is involved in endocytic docking or fusion. The findings that Vac1p contains an N-terminal C2H2 zinc finger and binds to a yeast Rab5 homologue suggest that this protein functions similarly to EEA1. Interestingly, Vac1p contains a second, divergent FYVE finger N-terminal to the previously identified FYVE finger (see Fig.1, Vac1_B). However, this domains lacks most of the conserved basic residues of the FYVE finger, and its role in PtdIns(3)P binding is uncertain. Like EEA1, Vac1p also appears to be linked to the SNARE machinery of vesicle fusion: it binds directly to the SNARE-associated Sec1p-family protein, Vps45p (Peterson et al., 1999; Tall et al., 1999), and it is found in a NSF-dependent complex with Vps45p and the SNARE Pep12p, a possible homologue of mammalian syntaxin13 (Burd et al., 1997). However, the involvement of Vac1p and Vps45p in endocytic membrane fusion has not yet been studied in detail. Whereas Vac1p is thought to play a role in vesicle docking at the endosome, Vps27p appears to function later in the endocytic pathway. Its inactivation is associated with impaired sorting of certain hydrolases to the vacuole, a decreased number of intralumenal vesicles in endosomes and vacuoles, and the occurence of the so-called ‘class E compartment’, a cup-shaped multilamellar organelle that distinguishes the class E subset of vps mutants. The class E compartment has been proposed to represent an aberrant late-endocytic structure (Piper et al., 1995; Odorizzi et al., 1998), and Vps27p has thus been implicated at a late stage of the endocytic pathway. On the other hand, Vps27p shows significant sequence similarity
4180 H. Stenmark and R. Aasland
Fig. 3. A cartoon showing the domain organisation of the nine different groups of FYVE-finger proteins. Domains were identified by a combined use of Blast2 (Altschul et al., 1997); HMMER/Pfam (Bateman et al., 1999) and SMART (Schultz et al., 1998). The lengths of each protein are indicated at the right, and a key to the domain icons is given at the bottom. In the case of group 3, the KIAA0993 sequence is underlined, and the domain structure of the C. elegans homologue T23B5.2 is indicated in blue. The grey pentagons with question marks indicate putative WD repeats. Domains rich in proline (Pro), glutamine (Gln) and acidic residues (E) are indicated. References for the domains follow: FYVE (Stenmark et al., 1996); C2H2 and IQ (Mu et al., 1995); coiled-coil (Lupas, 1996); ankyrin repeat (Sedgwick and Smerdon, 1999); WD repeat (Smith et al., 1999); BTB/POZ (Albagli et al., 1995); VHS (Lohi and Lehto, 1998), DEP (Boutros et al., 1998); PH (Gibson et al., 1994), GEF (Aghazadeh et al., 1998).
to mammalian Hrs, which is associated with early endosomes. If Hrs and Vps27p do indeed have analogous functions, this could perhaps mean that these proteins are involved in the formation of early multivesicular endosomes. Fab1p, a PI 5-kinase implicated in endocytic membrane traffic Another FYVE finger protein that has been implicated in the formation of multivesicular endosomes in yeast is Fab1p, whose inactivation is associated with a reduced number of intralumenal vesicles (Odorizzi et al., 1998). However, the most striking phenotype of fab1 mutants is their expanded vacuole. This phenotype can in part be explained by a decreased formation of intralumenal vesicles, but it is also conceivable that Fab1p is involved in a membrane-recycling pathway from the vacuole to other organelles. Fab1p has a very interesting biochemical property: it is a PI 5-kinase that converts PtdIns(3)P into the newly discovered phospoinositide, PtdIns(3,5)P2 (Cooke et al., 1998; Gary et al., 1998). PtdIns(3,5)P2 thus seems to be required for the formation of multivesicular endosomes and/or membrane recycling from the vacuole, presumably by interacting with (so far unidentified) effector molecules that specifically recognise this lipid. The recent discovery of a putative mammalian orthologue of Fab1p, PIKfyve (Sbrissa et al., 1999; Shisheva et al., 1999), may shed light on the role of PtdIns(3,5)P2 in mammalian cells.
The functions of the yeast FYVE-finger proteins are likely to be associated with their ability to bind PtdIns(3)P, as illustrated by the fact that FYVE finger mutations impair the function of Vps27p as well as Vac1p (Piper et al., 1995; Burd et al., 1997). One surprising difference between Vac1p, Vps27p, Fab1p and their putative mammalian counterparts is that interactions between yeast FYVE domains and PtdIns(3)P appear not to be required for membrane binding (Piper et al., 1995; Burd et al., 1997; Burd and Emr, 1998). This suggests that additional domains contribute to membrane binding but does not exclude the role of FYVE/PtdIns(3)P interactions in precise subcellular targeting. SARA, A FYVE-FINGER PROTEIN IN SIGNAL TRANSDUCTION Even though the best-characterised role of FYVE-finger proteins is in membrane trafficking, several FYVE-finger proteins have other functions. An interesting example is SARA (Smad anchor for receptor activation), which is an important mediator of transforming growth factor β (TGFβ) signalling (Fig. 4B). The binding of TGFβ to its receptor causes a cytoplasmic serine/threonine kinase domain of the receptor to phosphorylate the signal mediators Smad2 and Smad3. The phosphorylated Smads then associate with Smad4, and the
FYVE-finger proteins 4181
A
B TGFβ
FYVE
P EEA1
GTP
Rab5
C2 H 2
P
P
PtdIns(3)P
SARA
GTP
Rab5
TGFβ receptor Smad2
FYVE P
P Smad2
PtdIns(3)P
Smad4
Nucleus
Fig. 4. FYVE-finger proteins in membrane trafficking and signal transduction. A Proposed tethering of two Rab5-positive organelles by EEA1. The organelles could either be one endocytic vesicle and one early endosome or be two early endosomes. The N-terminal C2H2 zinc finger binds to Rab5:GTP on one membrane, whereas the C-terminal Rab5 binding region binds to Rab5:GTP and the FYVE finger binds to PtdIns(3)P on the other membrane. B TGFβ-signalling via SARA. Binding of TGFβ to its receptor causes the phosphorylation of one of the receptor chains (for simplicity the receptor is visualised as one chain). This causes the receptor to bind to Smad2, which is kept in the right position by binding to SARA, which is membrane-localised via binding of its FYVE finger to PtdIns(3)P. Smad2 is then phosphorylated by the receptor kinase, and this causes it to associate with Smad4. The Smad2-Smad4 complex translocates to the nucleus, where it regulates transcription of target genes.
resulting complexes translocate to the nucleus, where they control the transcription of target genes. The role of SARA is to recruit Smad2 and Smad3 to intracellular membranes (presumably early endosomes) that contain the receptor (Tsukazaki et al., 1998). The FYVE finger of SARA is thus essential for the intracellular localisation of both SARA and Smad2. Many agonists lead to the rapid production of PtdIns(3,4,5)P3, which is involved in the recruitment and activation of a variety of effectors (Leevers et al., 1999; Rameh and Cantley, 1999). In contrast, PtdIns(3)P appears to be constitutively produced by the cell, and there is no evidence that TGFβ modulates its formation. This suggests that the role of this lipid in TGFβ signalling could be to ensure that signalling occurs at a specific intracellular location. A putative C. elegans homologue of SARA, D1022.7, is an AKAP (A-kinase anchoring protein) that mediates the membrane binding of a specific regulatory subunit of protein kinase A (PKA) (Angelo and Rubin, 1998). Mammalian SARA might thus also be responsible for the recruitment of certain PKA isoforms to membranes that contain PtdIns(3)P. Given the importance of AKAPs and PKA signalling in a variety of cellular processes (Colledge and Scott, 1999), it will be interesting to examine this possibility further.
FGD1, A FYVE-FINGER PROTEIN IMPLICATED IN CYTOSKELETAL REGULATION One of the first FYVE-finger proteins to be identified was Fgd1, the product of the faciogenital dysplasia (Aarskog syndrome) gene (Pasteris et al., 1994). Faciogenital dysplasia is an X-linked developmental disease primarily associated with skeletal, facial and genital anomalies. Overexpression of Fgd1 transforms fibroblasts (Whitehead et al., 1998), and many (but not all) of the functions of Fgd1 appear to be linked to its role as a GEF for Cdc42, a Rho-family GTPase that controls the submembraneous actin cytoskeleton. The FYVE domain of Fgd1 is flanked by two PH domains, which suggests that this protein binds to PtdIns(3)P as well as additional phosphoinositides. Three proteins closely related to Fgd1 (Fgd2, Fgd3 and Frabin) have been identified. All these proteins contain the FYVE and PH domains, although their FYVE domains differ slightly from that of Fgd1. Interestingly, the FYVE domains of Fgd1 and Fgd3 lack the tryptophane residue conserved in all other FYVE finger proteins (position 5 in Fig. 1), and one should thus determine whether these FYVE domains do bind PtdIns(3)P. Another interesting possibility is that they may bind to a closely related molecule, such as the newly discovered PtdIns 5-phosphate (PtdIns(5)P)
4182 H. Stenmark and R. Aasland (Tolias et al., 1998), whose headgroup resembles that of PtdIns(3)P. CONCLUSIONS FYVE-finger proteins are recruited to specific membranes by virtue of their ability to bind PtdIns(3)P. The affinity of a FYVE finger for PtdIns(3)P is relatively low, and the binding of nearby domains to additional membrane molecules, such as small GTPases, might therefore be essential for membrane recruitment. EEA1 is one example, but Rabip4, a protein that interacts with the GTP-bound form of Rab4, also contains a FYVE finger and might be recruited in a similar fashion (Mari et al., 1999). Likewise, the synaptic vesicle protein Rabphilin3A, implicated in regulated exocytosis, contains a Rab3Abinding domain adjacent to its FYVE-related zinc finger (Ostermeier and Brunger, 1999), which might bind to some (unidentified) lipid headgroup other than PtdIns(3)P. Even though structural and functional data indicate that FYVE fingers bind PtdIns(3)P, the possibility that some FYVE fingers may interact with additional ligands cannot be excluded. The headgroup of PtdIns(5)P is sufficiently similar to that of PtdIns(3)P that it might function as a ligand for certain FYVE fingers. Moreover, the FYVE finger of EEA1 is involved in its interaction not only with PtdIns(3)P, but also with the SNARE proteins, syntaxin-6 and syntaxin-13 (McBride et al., 1999; Simonsen et al., 1999). This is reminiscent of several PH domains, which have been implicated in interactions with proteins as well as with phosphoinositides (Rebecchi and Scarlata, 1998). However, whereas the FYVE finger is an autonomous PtdIns(3)P binding domain, its SNARE binding requires the participation of other domains as well, and PtdIns(3)P binding is likely to be the most prominent function of the FYVE finger. It is striking that the FYVE-finger proteins whose intracellular localisations have been studied all localise to early endosomes. Moreover, the PI 3-kinase that probably is responsible for the bulk of PtdIns(3)P in mammalian cells is an effector of the early-endosomal GTPase Rab5 (Christoforidis et al., 1999b). This suggests that the FYVEPtdIns(3)P interactions may serve to recruit proteins specifically to early endosomes. In order to verify this it will now be important to determine the subcellular localisation of PtdIns(3)P. Even though few FYVE-finger proteins have so far been studied in detail, it is evident that different FYVE finger proteins have highly distinct functions, and their only common structural domain is the FYVE finger itself. The interaction between PtdIns(3)P and FYVE fingers appears to be a way to recruit a limited array of proteins to perform various tasks at a specific cellular location. An additional possibility that needs further investigation is that the interaction of FYVE-finger proteins with PtdIns(3)P could affect their conformation and thus directly modulate their activity. Future work will need to clarify the relationship between the intracellular location and the functions of these proteins. This work was supported by a biotechnology network grant from the Research Council of Norway. H.S. was supported by the Norwegian Cancer Society and the Novo Nordisk Foundation. Rein Aasland was supported by the L. Meltzer Legacy.
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