From the Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, California 92093-0668
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 (1-3). 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.
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 PtdIns but not PtdIns-4-P or
PtdIns-4,5-P2 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.
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 (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.
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
>10-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).
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 (34, 35). PtdIns-4,5-P2 turnover is
also mediated by Type II 5-phosphatases like synaptojanin and OCRL (36,
37). 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 (38) and
purified (39), respectively.
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-P2 are not affected in
hydrolase-deficient strains (40). Indeed, the vacuole/lysosome contains
candidate lipases and phosphatases (41, 42), 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) (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.
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-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 (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 Zn2+ ions via 8 cysteine/histidine residues spaced in a specific manner (CX2CX9-39CX1-3(C/H)X2-3CX2CX4-48CX2C) (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), 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 (64, 65), suggesting a 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.
PtdIns-3,5-P2 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-P2, 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-P2, a distinct lipid
second messenger (65, 67).
What is the function of PtdIns-3,5-P2? 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-P2 (12, 65, 68). Depletion of these
lipids in vps34
INTRODUCTION
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INTRODUCTION
Requirement for PtdIns-3-P in...
PtdIns-3-P Transits...
FYVE Domain-containing Proteins...
Conversion of PtdIns-3-P, a...
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Requirement for PtdIns-3-P in Vesicular Traffic
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INTRODUCTION
Requirement for PtdIns-3-P in...
PtdIns-3-P Transits...
FYVE Domain-containing Proteins...
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PtdIns-3-P Transits Golgi/Endosomal Compartments to the
Vacuole
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INTRODUCTION
Requirement for PtdIns-3-P in...
PtdIns-3-P Transits...
FYVE Domain-containing Proteins...
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Fig. 1.
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-P2 in the cytoplasmic membrane leaflet by the
Fab1p 5-kinase. PtdIns-3,5-P2 may be required for the
efflux of vacuolar membranes to prevacuolar compartment(s)
(dashed arrows) or for the invagination and
degradation of vacuolar membranes.
FYVE Domain-containing Proteins as Effectors of Vps34 PtdIns
3-Kinase Signaling
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INTRODUCTION
Requirement for PtdIns-3-P in...
PtdIns-3-P Transits...
FYVE Domain-containing Proteins...
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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 Homeostasis
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INTRODUCTION
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PtdIns-3-P Transits...
FYVE Domain-containing Proteins...
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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-P2. fab1 mutants that lack
PtdIns-3,5-P2 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-P2, 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-P2 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-P2 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-P2 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-P2 (65). Therefore, Vac7p may act as an upstream
activator of Fab1p, triggering vacuole membrane recycling through
Fab1p-mediated production of PtdIns-3,5-P2 (65). Downstream
effectors of Fab1p are also likely to exist, and these proteins would
be expected to interact with PtdIns-3,5-P2. Identification
of candidate effectors of Fab1p should help clarify the mechanism by
which PtdIns-3,5-P2 signaling allows for the maintenance of
normal vacuolar membrane homeostasis.
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Fig. 2.
Biosynthesis and function of
3-phosphoinositides in yeast. The sequential phosphorylation of
PtdIns by the Vps34 PtdIns 3-kinase and the Fab1p PtdIns-3-P
5-kinase yields PtdIns-3-P and PtdIns-3,5-P2,
respectively. PtdIns-3-P regulates anterograde Golgi-to-vacuole traffic
through FYVE domain-containing downstream effectors such as Vac1p and
Vps27p. PtdIns-3,5-P2 may be essential to signal the efflux
or turnover of vacuolar membranes.
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-P2. 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-P2 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.
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ACKNOWLEDGEMENTS |
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We thank Tamara Darsow for the critical reading of the manuscript and Peter Parker and Stephen Dove for sharing unpublished results.
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FOOTNOTES |
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* 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.).
Supported as an investigator of the Howard Hughes Medical
Institute. To whom correspondence should be addressed. Tel.:
619-534-6462; Fax: 619-534-6414; E-mail: semr{at}ucsd.edu.
2 J. D. Gary and S. D. Emr, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PtdIns, phosphatidylinositol; PI, phosphoinositide; SNARE, SNAP (soluble NSF (N-ethylmaleimide-sensitive factor)) receptor; CPY, carboxypeptidase Y; EEA1, early endosome antigen 1; RS-ALP, retrieval sequence-alkaline phosphatase.
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