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

Kinases that phosphorylate phosphatidylinositol (PtdIns) 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 (1–3). 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 (4, 5). 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 (6). 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) (7–9). The lipid composition of vesicular transport intermediates is also critical, and roles for phosphoinositides, phosphatidic acid, and lysobisphosphatidic acid have been documented (6, 10, 11). 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.

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 (1)(2)(3). 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 (4,5). 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 (6). 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) (7)(8)(9). The lipid composition of vesicular transport intermediates is also critical, and roles for phosphoinositides, phosphatidic acid, and lysobisphosphatidic acid have been documented (6,10,11). 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 Traffic
The importance of PtdIns-3-P in vesicular transport was first revealed during the study of Golgi to vacuole/lysosome transport in yeast (12). The yeast vacuole, an acidified organelle that contains active hydrolytic enzymes, is the functional analog of the mammalian lysosome (13). Newly synthesized hydrolases and cargo destined for degradation by these hydrolases are transported to the vacuole along several specialized trafficking pathways (14 -17). 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 (14,18). This sequence of transport steps requires the function of more than 40 gene products designated Vps, for vacuolar protein sorting (19 -22). One of these genes, VPS34, encodes a PtdIns 3-kinase (12,23). In vitro studies revealed that Vps34p specifically phosphorylates Ptd-Ins but not PtdIns-4-P or PtdIns-4,5-P 2 at the D-3 position of the inositol ring (12). 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 (12). These results suggest a role for PtdIns-3-P in the sorting of proteins to the yeast vacuole.
3Ј-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 (24 -26). A human homolog of the yeast Vps34 PtdIns 3-kinase has been cloned and found to be sensitive to nanomolar levels of wortmannin (27). 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 (28). Interestingly, the block in homotypic endosome fusion caused by wortmannin can be overcome by the overexpression of Rab5 in its active GTP-bound form (29), suggesting a link between Rab5 function and PI 3-kinase activity.

PtdIns-3-P Transits Golgi/Endosomal
Compartments to the Vacuole In yeast, Vps34p is recruited from the cytosol to the Golgi and/or endosome by a membrane-associated serine/threonine protein kinase, Vps15p (30 -32). 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 Ͼ10-fold (32). Inactivation of the Vps15p protein kinase results in severe decreases in cellular levels of PtdIns-3-P and the missorting of vacuolar hydrolases (30,32). 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 (33). 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 (30,31).
In yeast, dramatic decreases in the cellular levels of PtdIns-3-P occur within minutes after inactivation of Vps34p (32), 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 (40). Cellular levels of PtdIns-4-P and PtdIns-4,5-P 2 are not affected in hydrolase-deficient strains (40). Indeed, the vacuole/lysosome contains candidate lipases and phosphatases (41,42), which may function in the turn-* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This is the second article of five in "A Thematic Series on Kinases and Phosphatases That Regulate Lipid Signaling." This work was supported by National Institutes of Health Grant CA58689 (to S. D. E. and A. E. W.).
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) (43) or YPT7 (Rab GTPase) (44) causes severalfold increases in PtdIns-3-P, presumably by blocking delivery of vacuole-bound PtdIns-3-P that accumulates in a prevacuolar endosomal compartment (40).
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 (40,45). 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 (40,46). 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 Signaling
Several proteins have been postulated to function as downstream effectors of PtdIns 3-kinases (47,48). One of these, the mammalian EEA1 (early endosome antigen 1) protein, is required for homotypic endosome-endosome fusion in vitro (49). Critical to the involvement of EEA1 in this process is its ability to associate with endosomal membranes (49), an interaction that is inhibited by wortmannin (47). EEA1 thus represents a potential PtdIns-3-Pbinding 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 (50 -52). 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 (53,54). FYVE domains coordinate 2 Zn 2ϩ ions via 8 cysteine/histidine residues spaced in a specific manner (CX 2 CX 9 -39CX 1-3 (C/H)X 2-3 CX 2 CX 4 -48 CX 2 C) (54). This protein motif also contains a basic amino acid patch adjacent to the 3rd cysteine residue (50,54), which is critical for binding of acidic PtdIns-3-P (50). In addition to binding PtdIns-3-P in vitro, Aequorea victoria green fluorescent protein coupled to the EEA1-FYVE domain localizes to endosomal and vacuolar compartments in yeast (50). This localization is dependent on Vps34 PtdIns 3-kinase activity, demonstrating that the FYVE domain is sufficient to mediate membrane association in vivo (50). Conversely, deletion of the FYVE domain of EEA1 (54) or the treatment of cells with wortmannin (47) disrupts the endosomal association of this protein.
As mentioned above, overexpression of Rab5 rescues wortmannin-induced inhibition of homotypic endosome fusion (29), 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 (49). In fact, in addition to binding PtdIns-3-P, EEA1 acts as an effector of Rab5 as it directly interacts with Rab5-GTP (49). 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 (53,54). Like the FYVE domain of EEA1, the FYVE domains of these proteins also bind PtdIns-3-P (50, 51). 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 (47,55), functions as a multivalent regulatory protein that interacts with the yeast Rab5 GTPase (Vps21p), the endosomal t-SNARE, Pep12p, and the Sec1p homolog, Vps45p (56 -60). 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 (58). Vps27p, the likely yeast counterpart of mammalian Hrs (61), mediates the maturation of endosomes (e.g. receptor recycling, multivesicular body formation), a process required for endosome fusion with the vacuole (18,62). An intact FYVE domain is required for Vps27p function (63)

. Vacuolar hydrolases and the Fab1 PtdIns-3-P 5-kinase terminate signals mediated by PtdIns-3-P.
Vps15p-Vps34p complexes synthesize PtdIns-3-P at the late Golgi/endosome. PtdIns-3-P regulates forward transport to the vacuole (solid arrows) through two FYVE domain effector proteins, Vac1p and Vps27p. The bulk of PtdIns-3-P is turned over by hydrolases within the lumen of the vacuole. This is likely to entail the sorting of PtdIns-3-P into vesicular invaginations formed at the endosome or vacuole and the delivery of these vesicles, which contain PtdIns-3-P in their inner leaflet, to the vacuole. PtdIns-3-P-containing vesicles can then be degraded in a hydrolase-dependent manner within the vacuole lumen. Another pool of PtdIns-3-P is converted to PtdIns-3,5-P 2 in the cytoplasmic membrane leaflet by the Fab1p 5-kinase. PtdIns-3,5-P 2 may be required for the efflux of vacuolar membranes to prevacuolar compartment(s) (dashed arrows) or for the invagination and degradation of vacuolar membranes. role for Fab1p in vacuolar membrane efflux/degradation. Point mutations within the FYVE domain of Fab1p also result in enlarged vacuole phenotypes, 2 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-P 2 , a Signaling Lipid Required for Vacuole Membrane Homeostasis
PtdIns-3,5-P 2 is a newly identified phosphoinositide, discovered both in yeast and higher eukaryotic cells (66,67). This lipid is synthesized directly from a preexisting pool of PtdIns-3-P, indicating the existence of a PtdIns-3-P 5-kinase (66,67). Thus, yeast and mammalian cells presumably maintain at least two pathways for the turnover of PtdIns-3-P, one that requires the activity of vacuolar/lysosomal hydrolases and a second mediated by a 5-kinase, which utilizes PtdIns-3-P as a substrate (40,66,67).
Sequence comparisons of the catalytic domains of many known phosphoinositide kinases has allowed grouping of these kinases into separate classes, consistent with their substrate specificities (65). This analysis led to the identification of a new subgroup defined by Fab1p (65), a protein essential to the maintenance of normal vacuole morphology (64). 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 (65). Cells lacking Fab1p or expressing Fab1p mutants, which contain point mutations within the kinase domain, produce undetectable levels of PtdIns-3,5-P 2 , without dramatically affecting the levels of other phosphoinositides (65,68). In addition, purified full-length Fab1p phosphorylates PtdIns-3-P at the 5-position of the inositol ring (68). Fab1p also contains an N-terminal FYVE domain, which functions to bind PtdIns-3-P (see above) (50). Therefore, Fab1p not only functions as a downstream effector of PtdIns-3-P through its FYVE domain (50) but also converts PtdIns-3-P to PtdIns-3,5-P 2 , a distinct lipid second messenger (65,67).
What is the function of PtdIns-3,5-P 2 ? vps34 deletion mutants lack PtdIns-3-P, the immediate product of the Vps34 PtdIns 3-kinase and as a result deplete the cell of the Fab1p substrate and hence PtdIns-3,5-P 2 (12,65,68). Depletion of these lipids in vps34⌬ strains results in severe defects in vacuolar protein sorting, enlarged vacuole morphologies, and growth defects (23). The identification of Fab1p as a PtdIns-3-P 5-kinase has allowed for the dissection of defects resulting from the loss of PtdIns-3,5-P 2 . fab1 mutants that lack PtdIns-3,5-P 2 do not missort or secrete resident vacuolar enzymes (65), indicating that PtdIns-3-P directly regulates vacuolar protein transport (see Fig. 2) (12,32). This is consistent with the finding that PtdIns-3-P, but not PtdIns-3,5-P 2 , binds FYVE domain-containing proteins, which mediate anterograde traffic to the vacuole (50). Instead, the primary phenotype associated with loss of Fab1p function is the dramatic enlargement of the vacuole (64,65). This indicates that PtdIns-3,5-P 2 is involved in regulating vacuole membrane homeostasis and that loss of Vps34p PtdIns 3-kinase activity may cause enlargement of the vacuole as a secondary consequence of depleting the Fab1p substrate (see Fig. 2). Increases in vacuole surface area in fab1 mutants may be because of defects in the turnover or efflux of vacuolar membranes. Control of vacuole size by a degradative mechanism could occur through the invagination and hydrolase-mediated breakdown of endosomal/vacuolar membranes within the vacuole lumen (see Fig. 1). Indeed, although intravacuolar vesicles can be detected (40), fab1⌬ mutants lack these vesicles (65), suggesting a possible role for PtdIns-3,5-P 2 in their formation. Mutations within the class E subset of VPS genes also block this pathway, indicative of a functional connection between PtdIns-3,5-P 2 signaling and class E Vps proteins (71). Alternatively, vacuolar membranes could be redistributed to prevacuolar compartments via a retrograde trafficking pathway (see Fig. 1). Retrieval sequence-alkaline phosphatase (RS-ALP), a transmembrane vacuolar hydrolase that has been engineered to contain a Golgi retrieval signal (FXFXD) within the cytoplasmic domain of ALP, traffics to the vacuole where it is proteolytically matured and is then sorted back to a prevacuolar compartment (69). Thus, RS-ALP and other proteins (69) may be the cargo of a vacuolar recycling mechanism regulated by Fab1p PtdIns-3-P 5-kinase activity. Recycling of RS-ALP was found to be dependent upon Vac7p, an integral membrane protein identified in a screen for yeast mutants defective in vacuole inheritance (69,70). Vac7p may function in the same signaling pathway as Fab1p because both vac7 and fab1 mutants have dramatically enlarged vacuoles (65,70). Moreover, cells deleted for VAC7 do not produce detectable levels of PtdIns-3,5-P 2 (65). Therefore, Vac7p may act as an upstream activator of Fab1p, triggering vacuole membrane recycling through Fab1p-mediated production of PtdIns-3,5-P 2 (65). Downstream effectors of Fab1p are also likely to exist, and these proteins would be expected to interact with PtdIns-3,5-P 2 . Identification of candidate effectors of Fab1p should help clarify the mechanism by which PtdIns-3,5-P 2 signaling allows for the maintenance of normal vacuolar membrane homeostasis.
Recent studies have elucidated the downstream effectors and degradation mechanism of PtdIns-3-P as well as the role of PtdIns-3-P in the biosynthesis of PtdIns-3,5-P 2 . The components of the PtdIns-3-P synthesis machinery, the requirement for PtdIns-3-P in membrane transport, as well as the effectors of PtdIns-3-P appear to be conserved between yeast and mammalian cells. These studies have also revealed the role of phosphoinositides in compensatory trafficking pathways, which function to maintain vacuole size by modulating the protein and lipid composition of the organelle. PtdIns-3-P appears to function as an anterograde vacuolar transport signal whereas conversion to PtdIns-3,5-P 2 appears to regulate retrograde vacuolar membrane efflux/degradation. The importance of tightly regulating these competing pathways is underscored by the fact that an imbalance in either pathway results in organelle dysfunction.
Acknowledgments-We thank Tamara Darsow for the critical reading of the manuscript and Peter Parker and Stephen Dove for sharing unpublished results. 2 J. D. Gary and S. D. Emr, unpublished observations.