Department of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA 92093-0668, USA
* Author for correspondence (e-mail: semr{at}ucsd.edu)
Accepted 2 August 2002
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Summary |
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Key words: Vesicular transport, Endosome-to-Golgi retrograde transport, PX domain, Phosphatidylinositol 3-phosphate, Saccharomyces cerevisiae
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Introduction |
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Studies from both yeast and mammalian systems have demonstrated that
phosphatidylinositol 3-phosphate (PtdIns3P) is specifically
recognized by a set of proteins containing the FYVE domain. These proteins act
as downstream effectors in the regulation of membrane trafficking
(Corvera, 2000;
Odorizzi et al., 2000
;
Gillooly et al., 2001
). A
novel phosphoinositide-binding motif, the Phox homology (PX) domain, has
recently been characterized, which also targets proteins to intracellular
membranes by interacting with specific phosphoinositides
(Sato et al., 2001
;
Simonsen and Stenmark, 2001
;
Wishart et al., 2001
;
Xu et al., 2001
;
Yu and Lemmon, 2001
). The PX
domain was first identified in two subunits of the phagocyte NADPH oxidase
complex (Ponting, 1996
) and
contains two conserved basic motifs. More than 100 PX-containing proteins have
now been identified by sequence homology. In contrast to mammalian PX domain
proteins, which are able to recognize various phosphoinositides, the PX domain
proteins of S. cerevisiae, which have been characterized by
protein-lipid overlay assays, specifically bind PtdIns3P
(Yu and Lemmon, 2001
).
A significant number of the PX domain proteins are localized to membranes
or vesicular structures required for vesicular transport between intracellular
organelles. In S. cerevisiae, vacuolar recruitment of Vam7p, a
homologue of the mammalian t-SNARE SNAP-25, has been shown to be modulated via
specific interaction between the Vam7p-PX domain and PtdIns3P
(Cheever et al., 2001).
Vps5p and Vp17p are PX domain proteins that function as subunits of a
multimeric complex including Vps26p, Vps29p and Vps35p. This complex, dubbed
the retromer, is proposed to form a membrane coat required for cargo retrieval
from endosomes to the late-Golgi
(Horazdovsky et al., 1997;
Kohrer and Emr, 1993
;
Seaman et al., 1997
;
Seaman et al., 1998
;
Nothwehr et al., 1999
;
Reddy and Seaman, 2001
). The
cargo molecules in this pathway include the carboxypeptidase Y (CPY) receptor
Vps10p (Marcusson et al.,
1994
; Cereghino et al.,
1995
; Cooper and Stevens,
1996
) and the late-Golgi endoprotease Kex2p
(Wilcox et al., 1992
;
Voos and Stevens, 1998
).
Analysis of mutant strains defective for retromer complex function revealed
that these cells are unable to recycle Vps10p back to the Golgi. As a
consequence, Vps10p is mislocalized to the vacuolar membrane and CPY is
secreted in its Golgi-modified p2 precursor form.
Interestingly, phenotypic analysis of vps30 mutant cells revealed
similar phenotypes to those observed in mutants of the retromer complex
(Seaman et al., 1997).
However, Vps30p could not be identified as part of the retromer complex
(Seaman et al., 1998
).
Recently, Kihara and co-workers demonstrated that Vps30p assembles together
with the protein kinase Vps15p, the PtdIns 3-kinase Vps34p and Vps38p to form
a complex that is required for maturation of CPY
(Kihara et al., 2001
). In
addition, they characterized a second Vps34p kinase complex that functions in
autophagy. These two distinct Vps34p kinase subcomplexes, named complex I and
II, differ from each other by only one subunit: Vps38p, is a subunit of
complex II, whereas Apg14p is unique for complex I. The VPS30 gene is
allelic to APG6 (Kametaka et al.,
1998
) and functions in the autophagy pathway when associated with
the complex I-specific subunit Apg14p, or in the CPY pathway together with the
unique complex II subunit Vps38p. Since synthesis of PtdIns3P in
yeast is mediated by the sole PtdIns 3-kinase Vps34p, it suggests that
distinct pools of PtdIns3P are synthesized for distinct cellular
functions.
Based on the observations described above, we investigated whether the PtdIns 3-kinase complex II might influence endosome-to-Golgi retrograde transport. In this study, we show that vps30 and vps38 mutant strains are impaired in recycling of cargo molecules from the endosome to the late-Golgi. In addition, PtdIns3P levels in vps30 and vps38 mutant cells are about threefold lower compared with wild-type cells. We further show that the retromer subunits Vps5p and Vps17p are redistributed to the cytoplasm in vps30 and vps38 mutants, indicating a role for PtdIns3P in retrograde transport. Taken together, we suggest that complex II, which consists of Vps15p, Vps34p, Vps30p and Vps38p, produces a specific pool of PtdIns3P that is needed for recruitment/assembly of the retromer complex, which in turn is required for endosome-to-Golgi retrograde transport.
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Materials and Methods |
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DNA manipulation and plasmids
Restriction enzymes and DNA-modifying enzymes were purchased from Roche
(Indianapolis, IN) and Life Technologies Gibco BRL (Gaithersburg, MD).
Otherwise standard molecular biology techniques were used
(Maniatis et al., 1982). Yeast
transformations were performed using the lithium acetate method
(Ito et al., 1983
) and genomic
DNA was isolated as described (Hoffman and
Winston, 1987
). The KEX2-HA gene was cloned by PCR
amplification of genomic DNA derived from strain PBY82, with primers
containing the 5' SpeI and 3' XhoI sites
approximately 500 base pairs upstream and 200 base pairs downstream of the
KEX2-HA open reading frame. The KEX2-HA ORF was then
inserted into the SpeI-XhoI poly-linker site of the pRS416
vector resulting in pPB1. The Vps5-GFP gene was cloned by PCR
amplification of genomic DNA derived from strain PBY86 with primers containing
the 5' SacII and 3' XhoI sites approximately 500
base pairs upstream and 200 base pairs downstream of the VPS5-GFP
open reading frame resulting in pPB2. The plasmids pPB3
(vps5Y322A,R360A-GFP; pRS416) and pPB4
(vps5Y322A,R360A; pRS416) were constructed by PCR-based
site-directed mutagenesis. pPB2 was linearized with BstXI and was
co-transformed with the mutagenized PCR product into strain BHY152
(
vps5). The recombined plasmid was rescued from the yeast
strain and amplified in E. coli. The mutation was subsequently
confirmed by DNA sequencing. The construction of Vam7-GFP (pTKS35) and GFP-CPS
(pGO45) have been described (Odorizzi et
al., 1998
; Sato et al.,
1998
).
Pulse-labeling and protein immunoprecipitation
Protein transport assays and immunoprecipitations were carried out as
described (Audhya et al.,
2000).
Subcellular fractionation and western blot analysis
Subcellular fractionation and immunoblot analyses were performed as
previously described (Gaynor et al.,
1994; Babst et al.,
1998
). Monoclonal antibody against the HA or the myc epitope
(Boehringer Mannheim Biochemicals) was used at a 1:1500 dilution.
Protein purification and protein-lipid overlay assay
A DNA fragment encoding amino acids 268-406 of Vps5p (which corresponds to
the PX domain) was amplified by PCR using primers that added 5'
NcoI and 3' XhoI sites. This fragment was inserted
into the pGEX-KG vector, and the resulting plasmid was expressed in E.
coli. The protein was purified with Glutathione Sepharose 4B beads,
eluted with 5 mM reduced glutathione, and used immediately after purification.
To construct a GST-Vps17PX domain fusion protein, a DNA fragment encoding
amino acids 95-238 of Vps17p was amplified by PCR using primers that added
5' XmaI and 3' XhoI sites. The fragment was
processed as described above. The construction of a GST-Vam7PX domain fusion
protein has been described (Cheever et al.,
2001). To construct the GST-Vps5PX domain fusion protein carrying
the point mutations Y322A and R360A, DNA from plasmid PB4 encoding amino acids
268-406 of Vps5p was amplified by PCR using primers that added 5'
NcoI and 3' XhoI sites. The fragment was then cloned
into pGEX-KG vector as described, resulting in
pGEX-Vps5PXY322A,R360A. The GST fusion proteins were then subjected
to protein-lipid overlay assays as described
(Cheever et al., 2001
;
Dowler et al., 2002
). The
lipid-strips were purchased from Echelon.
In vivo analysis of phosphoinositides
Analysis of phosphoinositide levels was carried out as described previously
(Audhya et al., 2000;
Foti et al., 2001
). Briefly,
cells were grown in synthetic medium with the appropriate amino acids. Five
OD600 units of cells from a log-phase culture were harvested,
washed and resuspended in inositol-free synthetic medium. Cells were then
shifted to the appropriate temperature for 10 minutes, followed by the
addition of 50 µCi of myo-[2-3H]inositol (Nycomed
Amersham), and labeled for 45 minutes. Next, the cells were lysed by
mechanical agitation with glass beads in 4.5% perchloric acid to generate
extracts. Further processing of extracts is described
(Stack et al., 1993
). Analysis
of 3H-labeled glycerol-phosphoinositols was performed by separation
using HPLC (column #4611-1505, Whatman, Clifton, NJ) on a Beckman System Gold
HPLC and quantified by liquid scintillation counting by a Packard online
radiomatic detector.
FM4-64-labeling of vacuoles and endosomes and fluorescence
microscopy
To examine vacuolar structures in vivo, FM4-64 (Molecular Probes, Eugene,
OR) labeling was carried out as previously described
(Vida and Emr, 1995). The
labeling was carried out at a concentration of 16 µM FM4-64 at 30°C for
15 minutes and the cells were chased for a period of 1 hour. To stain
endosomal structures, FM4-64 was diluted in YPD to a concentration of 3.2 nM
and the labeling procedure was performed as described
(Shin et al., 2001
). GFP-CPS
fluorescent images were collected on a fluorescent microscope equipped with a
FITC filter and acquired using a CCD camera (model4995; COHU), an integrator
box (model 440A; Colorado Video Inc.) and an LG-3 Frame Grabber. All other
fluorescent images were acquired using a Ziess Axiovert S1002TV inverted
fluorescent microscope and subsequently processed using a Delta Vision
deconvolution system (Applied Precision, Seattle, WA). The software used was
Adobe PhotoShop 6.0.
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Results |
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VPS30 and VPS38 are two previously characterized genes
which, when mutated, affect transport of CPY from the Golgi to the vacuole
(Luo and Chang, 1997;
Seaman et al., 1997
;
Kihara et al., 2001
). Since
CPY precursors were often degraded when analyzed by western blot techniques,
we monitored processing of CPY by pulse-chase immunoprecipitation. As shown in
Fig. 1A, CPY molecules were
mostly detected in the Golgi-modified p2 form, which is in agreement with
previously published data. In addition, the
vps30
vps38 double mutant strain exhibited a
similar sorting phenotype compared with each single mutant, suggesting that
Vps30p and Vps38p act along a common pathway.
|
To test whether vps30 and
vps38 mutant
cells exhibit defects in the transport of other vacuolar hydrolases that
traverse the CPY and ALP pathways, respectively, we examined the delivery of
carboxypeptidase S (CPS) and ALP. Whole cell lysates from
vps30 and
vps38 mutants were generated, and
maturation of CPS and ALP was analyzed using western blot techniques. CPS
exists in two glycoforms, which can be separated by SDS-PAGE (data not shown).
To exclude co-migration of different CPS-glycoforms, protein lysates were
treated with endoglycosidase H, resulting in deglycosylated CPS molecules. In
contrast to the CPY maturation defects, we observed normal ALP and CPS
processing in both
vps30 and
vps38 cells
(Fig. 1B). A minor amount of
the unprocessed Golgi-form of ALP (pALP) could be detected in
vps30
vps38 double-mutant cells.
We also analyzed the processing of CPS and ALP in vps30 and
vps38 mutants by pulse-chase immunoprecipitation and observed
a slight kinetic delay in CPS processing (data not shown) compared with the
processing under steady-state conditions
(Fig. 1B). ALP processing
remained unaffected when analyzed by pulse-chase immunoprecipitation (data not
shown). CPS is a biosynthetic cargo molecule that requires a functional
multivesicular body (MVB) sorting pathway for its delivery to the vacuolar
lumen (Odorizzi et al., 1998
).
To exclude a defect in the MVB pathway,
vps30 and
vps38 cells were transformed with a plasmid coding for CPS
protein fused to green fluorescent protein (GFP). In wild-type cells, GFP-CPS
is delivered to the lumen of the vacuole
(Fig. 1C). Normal delivery of
GFP-CPS to the vacuole was observed in
vps30 and
vps38 mutant cells, indicating a functional MVB pathway
(Fig. 1C). Together, these data
show a role for Vps30p and Vps38p in vacuolar trafficking of CPY, but not for
the vacuolar hydrolases CPS and ALP.
Vp10p and Kex2p are mislocalized to the vacuolar membrane in
vps30 and vps38 mutant cells
Aberrant sorting of CPY to the vacuole can be caused by either a defect in
Golgi-to-vacuole transport or deficient recycling of the CPY receptor Vps10p.
The function of Vps10p is to deliver CPY to endosomes, where the receptor
releases its cargo before cycling back to the trans-Golgi for further rounds
of sorting (Horazdovsky et al.,
1997; Nothwehr and Hindes,
1997
; Seaman et al.,
1997
; Seaman et al.,
1998
; Nothwehr et al.,
1999
; Reddy and Seaman,
2001
).
Since vps30 and
vps38 mutants showed a
vacuolar sorting defect specific for CPY, we wanted to investigate whether
Vps10p localization is altered in these mutant strains. To monitor the
cellular localization of Vps10p, a chromosomal copy of the VPS10
locus was tagged with GFP at the 3'-end. The resulting fusion protein
appeared to be functional, as no CPY sorting defects were observed (data not
shown). In wild-type cells, Vps10-GFP was localized to punctate structures
typical of the Golgi and/or endosomes (Fig.
2, upper panel). In
vps38 mutant cells, however,
Vps10-GFP was redistributed to the vacuolar membrane in a fashion also
observed for
vps30 mutant cells
(Fig. 2, middle and lower
panel). To visualize vacuolar membranes, cells were incubated with FM4-64, a
lipophilic fluorescent dye. FM4-64 incorporates into the plasma membrane, is
then transported into cells via the endocytic pathway and ultimately
accumulates at vacuolar membranes (Vida
and Emr, 1995
). In
vps30 and
vps38
strains, co-localization of Vps10-GFP with FM4-64-stained vacuoles was
observed, demonstrating that Vps10-GFP is mislocalized to the limiting
membrane of the vacuole (Fig.
2).
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To confirm the defect of Vps10p localization in vps30 and
vps38 cells, we investigated the subcellular distribution of
Vps10p by cell fractionation. For this purpose, Vps10p was tagged with
hemagglutinin (HA) at its C-terminus and introduced into wild-type,
vps30 and
vps38 cells. Total cell lysates were
separated by sequential centrifugation into low and high speed membrane
pellets (P13, P100, respectively) and the soluble/cytosolic (S100) fraction.
The fractions were then analyzed by immunoblotting using anti-HA-specific
antibodies. In wild-type cells, Vps10-HA was found almost exclusively in the
P100 fraction enriched for Golgi and endosomal markers
(Table 2). In
vps38 cells, however, redistribution of Vps10-HA to the P13
pool was observed. This pelletable pool is enriched for proteins residing in
the endoplasmic reticulum, plasma membrane and vacuoles (ALP was used as a P13
marker protein; see Table 2). Glucose-6-phosphate-dehydrogenase (G6PDH), a cytosolic protein, served as a
marker for the S100 fraction (Table
2). A similar shift of Vps10p from the P100 to the P13 pool was
previously reported for
vps30 mutant cells
(Seaman et al., 1997
).
Together, these data show that, in
vps30 and
vps38 mutants, a major fraction of Vps10p is redistributed to
the vacuolar membrane leading to the selective CPY missorting phenotype
observed in these mutant cells.
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Kex2p is an endoprotease responsible for processing of -factor and
M1 killer toxin precursors in the late-Golgi
(Gluschankof and Fuller,
1994
). Like Vps10p, Kex2p cycles between the Golgi and endosomes
(Wilcox et al., 1992
;
Voos and Stevens, 1998
). We
fused GFP to the cytosolic tail of Kex2p and the fusion protein allowed
processing of pro-
-factor indicating a functional fusion (data not
shown). We found that, in
vps30 and
vps38
mutant strains, Kex2-GFP was redistributed to the vacuolar membrane in a very
similar manner to that observed for Vps10-GFP in these strains (data not
shown). Previous studies revealed that, in mutant cells deficient for
endosome-to-Golgi retrograde transport, Kex2p is rapidly degraded in the
vacuole (Nothwehr and Hindes,
1997
). To investigate the stability of Kex2p in
vps30 and
vps38 strains, a single copy plasmid
carrying KEX2-HA gene was constructed and transformed into the mutant
cells. Kex2-HA protein stability was subsequently assessed by pulse-chase
immunoprecipitations using anti-HA specific antibodies. By following the
kinetics of Kex2-HA, we observed that Kex2-HA stability was significantly
decreased in
vps30 and
vps38 mutant cells
(Fig. 3, lanes 4,6), compared
with wild-type cells (Fig. 3,
lane 2). Kex2-HA degradation was also observed in
vps35 cells
deficient in retrograde transport (Fig.
3, lane 8). The cytosolic protein G6PDH remained stable in all
mutant cells, indicating that the instability of Kex2p was specific
(Fig. 3, lower panel). Further
studies revealed that Kex2-HA degradation in
vps30 and
vps38 mutant cells was dependent upon the PEP4 gene
product protease A, a vacuolar proteinase (data not shown). These results
demonstrate that Kex2-HA fails to be recycled to the late-Golgi in
vps30 and
vps38 mutants and is instead
degraded by vacuolar proteases. In summary, our data strongly suggest that
Vps30p and Vps38p are essential for efficient endosome-to-Golgi retrograde
transport.
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vps30 and
vps38 cells exhibit defects
in PtdIns3P synthesis
Previous biochemical studies revealed that the Vps15 protein kinase
activity is required for both the stable interaction between Vps15p and Vps34p
and the activation of the PtdIns 3-kinase activity of Vps34p
(Stack et al., 1993;
Stack et al., 1995
).
Similarly, Vps30p and Vps38p, which form a complex with Vps15p and Vps34p,
might regulate PtdIns3P synthesis. Thus, we addressed whether Vps30p
and Vps38p act as regulators of the Vps15p/Vps34p kinase complex.
To investigate this possibility, we analyzed intracellular
PtdIns3P levels in vps30 and
vps38
mutant strains. For this purpose, phosphoinositides were labeled in vivo with
[3H]myoinositol, extracted and analyzed using HPLC. We found that
in
vps30 and
vps38 cells levels of
PtdIns3P were significantly decreased (approximately threefold)
compared with wild-type cells (Fig.
4). In addition,
vps30
vps38
double-mutant cells showed no further decrease in PtdIns3P compared
with each single mutant, again suggesting that Vps30p and Vps38p function
together at a common step.
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Diminished PtdIns3P levels in vps30 and
vps38 cells specifically affect localization of the retromer
subunits Vps5p and Vps17p
Even though Vps30p and Vps38p are not part of the retromer complex, our
data clearly show recycling defects of Vps10p and Kex2p in
vps30 and
vps38 mutant cells, mimicking
phenotypes observed in retromer mutants. Interestingly, two of the retromer
subunits, Vps5p and Vps17p, harbor the PX domain, a PtdIns-binding motif
(Sato et al., 2001
;
Wishart et al., 2001
;
Yu and Lemmon, 2001
). Based on
the observation that
vps30 and
vps38 mutant
cells showed decreased PtdIns3P levels, we wanted to determine
whether membrane recruitment of Vps5p and Vps17p may be altered in these
mutants.
To monitor the in vivo localization of Vps5p and Vps17p, the proteins were
chromosomally tagged at their C-terminus with GFP. Both Vps5- and Vps17-GFP
were functional in that CPY transport was not impaired in these strains (data
not shown). Using fluorescence microscopy, the fusion proteins were detected
as punctate structures (Fig.
5A). To show that the punctate structures corresponded to
endosomes, cells harboring the GFP fusion proteins were incubated briefly with
FM4-64 dye. The reaction was stopped before the dye was delivered to the
vacuolar membrane, thereby allowing visualization of prevacuolar endosomes
(Shin et al., 2001). We
observed overlapping signals between Vps5/Vps17-GFP and FM4-64 and concluded
that Vps5p and Vps17p are recruited to prevacuolar endosomes (data not
shown).
|
We subsequently tested whether PtdIns3P levels would affect
recruitment of Vps5p and Vps17p to the endosomes. Both Vps5-and Vps17-GFP were
expressed in vps34 mutant cells. Neither Vps5-GFP nor
Vps17-GFP was associated with endosomal membranes, but were instead
distributed in the cytoplasm (Fig.
5A). Importantly, a similar distribution of Vps5- and Vps17-GFP
was observed in
vps38 cells
(Fig. 5A). This is in agreement
with the observation that PtdIns3P levels are reduced in these
cells.
We also investigated the localization of Vam7-GFP in vps38
mutant strains. Vam7p, the vacuolar t-SNARE has previously been reported to be
targeted to vacuolar membranes in a PtdIns3P-dependent manner
(Cheever et al., 2001
). In
wild-type cells, Vam7-GFP localized primarily to the vacuolar membrane
(Fig. 5B). Interestingly, we
found a very similar distribution of Vam7-GFP localization in
vps38 strains (Fig.
5B). Since Vps38p is the component unique for complex II, and is
necessary for retrograde transport, we reasoned that it might specify the site
of synthesis of the PtdIns3P pool used in this process. Therefore,
these observations suggest that the PtdIns3P pool synthesized by
complex II is specific for retrograde transport.
To confirm that redistribution of Vps5p and Vps17p to the cytosol was
primarily due to the loss of PtdIns3P synthesis, we expressed
Vps17-GFP in a temperature-conditional vps34tsf mutant
strain (Stack et al., 1995).
At the nonpermissive-temperature of 37°C, PtdIns3P synthesis is
blocked in this strain. At permissive temperature (26°C), we observed
endosomal localization of Vps17-GFP (Fig.
5C, upper panel). However, after shifting the cells to the
non-permissive temperature of 37°C for 60 minutes, Vps17-GFP predominantly
localized to the cytoplasm (Fig.
5C, lower panel). Western blot analysis using anti-GFP antibodies
confirmed that during the shift of vps34tsf cells to the
non-permissive temperature, the Vps17-GFP fusion protein was not degraded, but
remained as a stable fusion protein (data not shown). We performed identical
experiments using Vps5-GFP expressed in vps34tsf mutant
strains and found that upon temperature shift to 37°C, Vps5-GFP
redistributed to the cytosol as shown for Vps17-GFP (data not shown).
The results described above indicate that PtdIns3P is required for
recruitment of Vps5p and Vps17p, a prerequisite for efficient
endosome-to-Golgi retrograde transport of cargo molecules, such as Vps10p. To
test this, Vps10-GFP was expressed in temperature-conditional
vps34tsf mutant strains. At the permissive temperature
(26°C), we observed wild-type localization of Vps10-GFP
(Fig. 6). After shifting the
cells to the non-permissive temperature of 37°C for 60 minutes, Vps10-GFP
was localized to the limiting membrane of the vacuole
(Fig. 6), in a manner
previously observed in vps30 and
vps38 cells
(Fig. 2). These data are in
agreement with the idea that recruitment of Vps5p and Vps17p to endosomal
membranes is triggered by the synthesis of PtdIns3P in vivo.
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Vps5p and Vps17p bind PtdIns3P via their PX domain in vitro
and in vivo
Since PtdIns3P is required for efficient recruitment of Vps5p and
Vps17p to endosomes, we reasoned that these proteins may directly interact
with PtdIns3P through their PX domain. To test this, the PX domain of
Vps5p and Vps17p, respectively, was fused to glutathione S-transferase (GST).
The resulting fusion proteins were subsequently purified and used in a
protein-lipid overlay assay to determine their capability to bind to
phosphoinositides. As shown in Fig.
7A, the GST-PX domain fusion proteins of Vps5p and Vps17p
specifically bound PtdIns3P. No binding of GST to phospholipids was
detected (data not shown).
|
To compare the PtdIns3P-binding specificity of Vps5 and Vps17 PX
domain fusion proteins, various concentrations of PtdIns3P were
spotted onto nitrocellulose membranes. The membranes were then subjected to
protein-lipid overlay assays. The GST-Vam7 PX domain fusion protein was used
as a positive control for PtdIns3P-binding
(Cheever et al., 2001). The
Vps5 and Vps17 PX domain fusion proteins had approximately a threefold lower
affinity for PtdIns3P compared with the Vam7 PX domain fusion protein
(Fig. 7B). These findings are
in agreement with previous studies reporting that the PX domain of Vam7p binds
to PtdIns3P with higher affinity than the PX domain of Vps5p and
Vps17p, respectively (Yu and Lemmon,
2001
).
To confirm that PtdIns3P is required for endosomal localization in
vivo, we expressed Vps17-GFP in PtdIns-kinase mutants defective for the
synthesis of other known yeast phosphoinositides and monitored the
localization of Vps17-GFP by fluorescence microscopy. In strains deleted for
FAB1, the PtdIns3P 5-kinase
(Cooke et al., 1998;
Gary et al., 1998
), Vps17-GFP
was detected on puncate structures (Fig.
7C). Next, we made use of the stt4isf and
pik1tsf kinase mutants, both defective for
PtdIns(4)P synthesis (Flanagan et
al., 1993
; Yoshida et al.,
1994
; Audhya et al.,
2000
), and mss4tsf strains deficient for
PtdIns(4)P 5-kinase activity
(Desrivières et al.,
1998
; Homma et al.,
1998
; Stefan et al.,
2002
). Upon shift to the non-permissive temperature of 37°C,
Vps17-GFP localization did not change in these lipid kinase mutants
(Fig. 7C) compared with the
permissive temperature (26°C; data not shown). Together with the in vitro
lipid binding assays (Fig.
7A,B), the in vivo studies strongly suggest that Vps5p and Vps17p
specifically interact with PtdIns3P.
Amino acid sequence alignments of multiple PX domains have revealed highly
conserved residues and previous studies have shown that point mutations in the
conserved residues result in diminished binding to phosphoinositides and
redistribution of the corresponding PX domain proteins
(Cheever et al., 2001;
Xu et al., 2001
). To test
whether the PX domain of Vps5p was essential for PtdIns3P binding and
proper protein function, we substituted two highly conserved PX domain
residues in the Vps5-GFP fusion protein. Tyr322 and Arg360 were each changed
to alanine. Western blot analysis using anti-GFP antibodies showed that the
mutant protein was stably expressed (data not shown). Using fluorescence
microscopy, we observed that the Vps5-GFP mutant protein did not localize to
endosomes, but rather localized to the cytoplasm as observed in
vps38 cells (Fig.
8A). Importantly, we found a strong CPY processing defect for the
Vps5Y322A,R360APX mutant cells, a defect very similar to that of
vps5 mutant strains (Fig.
8B).
|
Next, we tested the ability of the Vps5Y322A,R360A PX domain to bind PtdIns3P. The mutated PX domain of Vps5p was fused to GST and the resulting fusion protein was subjected to the lipid-protein overlay assay as described above. Consistent with the observed loss of function in CPY processing in the Vps5Y322A,R360APX mutant strain, binding of the mutated Vps5 PX domain fusion protein to PtdIns3P was abolished (Fig. 8C, right panel). In summary, a functional PX domain is crucial for the proper recruitment of Vps5p to endosomes, which is a prerequisite for efficient CPY sorting.
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Discussion |
---|
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---|
Previous studies demonstrated that the yeast t-SNARE Vamp7p is localized to
vacuolar membranes in a manner that is dependent on its PX domain and on the
synthesis of PtdIns3P (Cheever et
al., 2001). Importantly, in
vps38 mutant cells,
Vam7-GFP still localized to vacuolar membranes. In addition, Vam7p function is
maintained in
vps38 mutants as transport of ALP and CPS to the
vacuole, which is dependent on Vam7p function, is not blocked. We therefore
propose that complex II is responsible for the synthesis of a specific pool of
PtdIns3P that is required for recruitment of the retromer complex to
endosomes, which directs endosome-to-Golgi retrograde transport.
How can the cell generate spatially distinct pools of PtdIns3P? In
S. cerevisiae, Vps34p is the sole PtdIns3P kinase,
nevertheless there are multiple downstream effectors of PtdIns3P that
act in different pathways within the cell. These effectors include proteins
containing a FYVE domain (5 gene products in yeast) or a PX domain (15 gene
products in yeast) (Odorizzi et al.,
2000; Sato et al.,
2001
). The synthesis of different PtdIns3P pools may be
achieved by recruiting the Vps15p-Vps34p kinase complex to specific
subcellular compartments via the formation of distinct protein complexes.
Indeed, two Vps34p kinase subcomplexes, complex I and II, have been
discovered, each of them functioning in different cellular processes
(Kihara et al., 2001). Vps30p
and Vps38p assemble together with the protein kinase Vps15p and the lipid
kinase Vps34p to form complex II, which is required for CPY maturation. In
contrast, complex I has been reported to function in autophagy and differs
from complex II only by one subunit; Vps38p is replaced by Apg14p. Vps38p and
Apg14p may individually regulate localization and/or activity of the
corresponding Vps34p kinase complex, thereby generating distinct
PtdIns3P pools, either for autophagy or for CPY sorting. Since the
Vps15p-Vps34p kinase complex is also required for Golgi-to-vacuole anterograde
transport, it seems likely that additional complexes exist in the cell. This
idea is supported by the fact that PtdIns3P is still detected in
vps30
vps38 double mutant strains. In
vps15 mutants cellular PtdIns3P levels are almost
completely absent (Stack et al.,
1993
) and no PtdIns3P can be detected in
vps34 cells (Schu et al.,
1993
). The Vps15p-Vps34p kinase complex may cycle between
membranes and the cytosol, forming distinct subcomplexes, when interacting
partners are available at the target membrane.
Our data are consistent with a model in which complex II and the retromer communicate with each other. Our preliminary findings indicate that Vps30p and Vps38p are able to associate in the cytosol prior to their recruitment to the membrane (P.B., unpublished). One could imagine that an increase of cargo molecules in the endosome might trigger recruitment of Vps30p-Vps38p to endosomal membranes, followed by binding to Vps15p-Vps34p to form the final complex II. The synthesis of a specific PtdIns3P pool by complex II that is localized to a subdomain of the endosome could then trigger assembly of the retromer and ultimately vesicle formation for retrograde transport.
Data obtained from yeast and mammalian cells show that PtdIns3P
synthesis occurs mainly at endosomal membranes. In vivo localization studies
using a PtdIns3P-specific GFP-FYVE domain fusion protein revealed
that PtdIns3P is enriched in prevacuolar compartments
(Burd and Emr, 1998). Vacuolar
membrane labeling was also observed, albeit to a lesser extent. These results
were confirmed by studies using electron microscopy techniques to localize
PtdIns3P (Gillooly et al.,
2000
). In agreement with these findings, many proteins containing
PtdIns3P-binding modules, are recruited to endosomal membranes
(Odorizzi et al., 2000
;
Teasdale et al., 2001
;
Stenmark et al., 2002
).
The prevacuolar endosome represents a complex sorting compartment where
biosynthetic and endocytic cargoes merge and are then sorted to the vacuole.
In addition, other proteins such as the CPY receptor Vps10p are recycled back
to the late-Golgi from this compartment. PtdIns3P appears to be a key
regulator in each of the endosomal sorting events. However, PtdIns3P
alone can hardly be sufficient to provide specificity in these different
pathways. Apparently, it is the combination of PtdIns3P-binding
domains acting together with protein-protein interaction domains that ensures
the specific location and function of each effector protein. Previous studies
revealed that the yeast t-SNARE Vam7p contains both the PX domain and an
-helical coiled-coil domain, which are required to stabilize the
interaction of Vam7p with the vacuolar membrane by binding PtdIns3P
and to the vacuolar t-SNARE Vam3p, respectively
(Sato et al., 1998
). Within
the retromer complex, Vps35p provides the cargo-selective protein-protein
interaction by directly binding to Vps10p
(Nothwehr et al., 1999
;
Nothwehr et al., 2000
). It is
possible that, together with the PtdIns3P-dependent recruitment of
Vps5p and Vps17p, specific recruitment and/or assembly of the retromer complex
at the prevacuolar endosome is assured. Thus, spatially restricted synthesis
of PtdIns3P could concentrate PtdIns3P effectors at the
membrane, thereby allowing the effector proteins to interact with specific
membrane-bound molecules via other protein-protein interaction domains.
Recently, SNX1 and SNX2, homologues of Vps5p, were found to associate with
human orthologues of Vps26p, Vps29p and Vps35p
(Haft et al., 1998). The
structural similarities to the yeast retromer strongly suggest that the
mammalian complex performs a related function in protein trafficking. In
summary, the present data suggest that the restricted localization of distinct
Vps15p-Vps34p complexes results in PtdIns3P synthesis at specific
organelles, such that membrane microdomains enriched in PtdIns3P are
generated. Further studies, including in vitro reconstitution assays will be
required to elucidate the mechanisms involved in regulating the dynamic
organization of these lipid microdomains and to understand, how these domains
recruit and activate the appropriate set of effector molecules.
![]() |
Acknowledgments |
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