* Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229; Department of Physiology, University of
Iowa, Iowa City, Iowa 52242; and § Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242
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Abstract |
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A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid-containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.
Key words: endosome; SNARE; TGN; vacuole; VPS ![]() |
Introduction |
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PROTEINS traveling to the lysosome of mammalian
cells and the analogous vacuole in yeast cells reach
their destination by following one of many pathways (Griffiths et al., 1988; Ludwig et al., 1991
; Rabouille et
al., 1993
; Hunziker and Geuze, 1996
; Liou et al., 1997
; Bryant and Stevens, 1998
). With the exception of resident lysosomal/vacuolar proteins, which tend to be degradative enzymes, most of these proteins are ultimately degraded and
as such the lysosome and vacuole are viewed as the terminal compartments of the endosomal system (DeDuve,
1963
; Kornfeld and Mellman, 1989
). The endosomal system
is clearly a very dynamic system with many proteins cycling
between various compartments within it as part of their
normal cellular itinerary (Kornfeld and Mellman, 1989
; Nothwehr and Stevens, 1994
). To date, recycling of proteins is thought to occur from compartments proximal to
the lysosome/vacuole, and the possibility that proteins can
recycle out of these hydrolytic compartments has been
largely overlooked because of the difficulty of measuring
such movement. Recent studies, however, indicate that lysosomes are far more dynamic than was previously appreciated, and that they are in close communication with endocytic compartments perhaps making retrograde traffic
from the lysosome more likely (Hunziker and Geuze,
1996
; Storrie and Desjardins, 1996
). These studies indicate
that transport to the lysosome involves the fusion of late
endosomes with pre-existing lysosomes (van Deurs et al., 1995
; Futter et al., 1996
; Storrie and Desjardins, 1996
;
Bright et al., 1997
; Mullock et al., 1998
), or possibly involves a distinct class of transport vesicles (Berg et al.,
1995
). Either scenario invokes the need to reacquire at
least the specific transport proteins, such as putative SNAP
receptor (SNARE)1 proteins, that may mediate such a process.
Studies on the trafficking of lysosomal membrane proteins in the presence of cycloheximide, wortmanin, or the
vacuolating toxin, VacA, indicate that proteins such as
Lamp-1 continually cycle between lysosomes and late
endosomes (Lippincott-Schwartz and Fambrough, 1987;
Akasaki et al., 1993
, 1995
; Reaves et al., 1996
). Using a different model system, Brachet et al. (1997)
showed protein
traffic out of the lysosome-like MHC class II compartment is possible for MHC class II a/b chain complexes when
degradation of the invariant chain is blocked. Other experiments indicate that soluble lysosomal constituents may
also participate in retrograde transport (Jahrous et al.,
1994
). A mechanism for how proteins could be retrieved
from the lysosome is provided by the observation that AP-2/clathrin coats can assemble specifically on the surface of
lysosomes thus providing a specific mechanism for the
budding of transport intermediates (i.e., vesicles) from the
lysosome (Traub et al., 1996
). While these above studies
provide support for the occurrence of retrograde transport
from the lysosome, they are complicated by the use of pharmacological experimental manipulations that may compromise the distinction and function of late endosomes
and lysosomes. Furthermore, it has not been possible to
find a particular protein that clearly follows such a pathway under normal conditions.
Vacuolar biogenesis in Saccharomyces cerevisiae has
very strong parallels with lysosomal biogenesis in mammalian cells (Stack et al., 1995; Bryant and Stevens, 1998
).
The delivery of vacuolar hydrolases such as carboxypeptidase Y (CPY) relies on the cycling of the CPY receptor,
Vps10p, between the TGN and the prevacuolar/endosomal compartment (PVC) in a manner analogous to the delivery of modified hydrolases by the mannose-6-phosphate
receptor (Stack et al., 1995
). In this pathway the fusion of
Golgi-derived vesicles that contain Vps10p with the PVC
is mediated by the Sec1p-like protein, Vps45p, the target
(t)-SNARE protein Pep12p, the Rab protein, Vps21p/
Ypt51, and the vesicle (v)-SNARE protein Vti1p (Cowles et al., 1994
; Horazdovsky et al., 1994
; Piper et al., 1994
;
Singer-Kruger et al., 1994
; Becherer et al., 1996
; Fischer
von Mollard et al., 1997
). Efflux of traffic from the PVC is
controlled by class E Vps proteins such as Vps27p and
Vps4p and these proteins may function by creating the
necessary transport intermediates that ultimately fuse with
the vacuole (Piper et al., 1995
; Babst et al., 1997
). Fusion
to the vacuole itself is controlled in part by the vacuolar t-SNARE Vam3p, although it is unclear whether final delivery of CPY from the PVC requires a vesicular carrier or
the fusion of a larger endosomal compartment with the
vacuole as has been proposed for the delivery to lysosomes (Futter et al., 1996
; Storrie and Desjardins, 1996
;
Bright et al., 1997
). A second intracellular route to the vacuole from the TGN is taken by the membrane proteins alkaline phosphatase (ALP) and Vam3p (Cowles et al.,
1997
; Piper et al., 1997
). These proteins reach the vacuole
by a transport pathway that is independent of Vps45p,
Pep12p, and Vps27p function implying that they do not
enter into Golgi-derived vesicles that fuse with the PVC
(Piper et al., 1997
). Transport of ALP to the vacuole does
require Vam3p and the adaptor complex AP-3 as well as
the dynamin homologue Vps1p indicating that ALP may
be specifically sorted into a separate class of transport vesicles that rely on the vacuolar t-SNARE Vam3p for fusion
to the vacuole (Nothwehr et al., 1995
; Cowles et al., 1997
;
Stepp et al., 1997
). This pathway appears to parallel a similar pathway in mammalian cells where a subset of lysosomal proteins such as LIMP-II and tyrosinase may be
sorted by AP-3 in the TGN for ultimate delivery to the lysosome (Honing et al., 1998
).
Previous studies have shown that the cytosolic tail of
ALP contains sorting information sufficient for direction
into the alternative pathway (Cowles et al., 1997; Piper et al.,
1997
). Our laboratory has previously reported that the
motif FXFXD identified within the cytosolic tail of the
resident TGN protein dipeptidyl aminopeptidase (DPAP)
A mediates retrieval from the PVC back to the TGN
(Nothwehr et al., 1993
; Bryant and Stevens, 1997
). Transplantation of this motif into the cytosolic tail of ALP results in a protein, RS-ALP (retention sequence-ALP),
which achieves steady state localization to the TGN through
continual retrieval from a post-Golgi compartment. In RS-ALP, the FXFXD retrieval motif is adjacent to a sorting domain for the alternative pathway. In this present study we
report that like the wild-type ALP protein, RS-ALP follows
the alternative route to the vacuole in a manner that was independent of Vps45p and Vps27p function. Furthermore,
this protein was efficiently retrieved from the vacuole to the
TGN via the PVC. We have identified vac7 mutants as being defective in this retrograde trafficking pathway out of
the vacuole and also found that the v-SNARE protein Vti1p uses this pathway to cycle out of the vacuole as part of its normal cellular itinerary.
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Materials and Methods |
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Materials
Enzymes used in DNA manipulations were purchased from New England Biolabs Inc. (Beverly, MA), Boehringer Mannheim Corp. (Indianapolis, IN), Bethesda Research Laboratories (Gaithersburg, MD), or United States Biochemical Corp. (Cleveland, OH). Secondary antibodies used for indirect immunofluorescence (all cross-species absorbed) were purchased from Jackson Immunoresearch Laboratories Inc. (West Grove, PA). mAbs specific for ALP (1D3-A10) are available from Molecular Probes, Inc. (Eugene, OR). Fixed Staphylococcus aureus cells (Ig sorb) were obtained from The Enzyme Center (Malden, MA). [35S]Express label was from New England Nuclear (Boston, MA). Oxalyticase was from Enzogenetics (Corvallis, OR). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Strains, Media, and Microbiological Techniques
Yeast strains used in this study are listed in Table I. Strains were constructed by standard genetic techniques and grown in rich media (1% yeast
extract, 1% peptone, 2% dextrose; YEPD) or standard minimal medium
with appropriate supplements (Sherman et al., 1986). Strain NBY88 was
derived from RPY2 (Piper et al., 1995
) by transforming with pSN111
(pho8
-X construct) linearized with SalI (Nothwehr et al., 1995
). Ura+
transformants were plated onto media containing 5-FOA to select for Ura
loopouts and pho8
-X colonies were identified through immunoblot analysis. NBY72, NBY86, NBY73, and NBY89 were similarly derived from
SF838-9D (Rothman et al., 1989
), RHY6210 (Gomes de Mesquita et al.,
1996
), and RPY3 (Piper et al., 1995
) and LWY2809, respectively. LWY2809
is a sister spore of LWY2806 (Bonangelino et al., 1997
). (NBY85 was
derived from NBY89 by transforming cells with EcoRI-linearized pLO2010
(Nothwehr et al., 1995
). Ura+ transformants were plated onto media containing 5-FOA and pep4
-X colonies were identified using the APNE plate
assay (Wolf and Fink, 1975
). NBY100 was made by using pSN111 to delete
PHO8 in RPY1017. RPY1017 was made by transforming SEY6210 to His+
using a PCR product encompassing the HIS3 gene flanked by 49-100 and
1,351-1,402 relative to the start codon. This HIS3 disruption resulted in the
deletion of the region corresponding to amino acids 33-450 of the APM3
gene was confirmed by PCR analysis of the resulting APM3 locus.
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Plasmid Construction
Plasmids used in this study are listed in Table II. DNA manipulations and
DNA-mediated transformation of E. coli strains CJ236 and XL-1 blues
were performed by routine procedures (Sambrook et al., 1989). pSN123, a
CEN-URA3 plasmid encoding (F/A)RS-ALP was constructed by oligonucleotide directed mutagenesis of pSN97 according to the method of
Kunkel et al. (1987)
. The resulting plasmid encodes a version of RS-ALP
in which the two phenylalanine residues contained within the Golgi localization motif (RRESFQFNDI) transplanted from Ste13p into the cytosolic
tail of ALP (Nothwehr et al., 1993
) have been mutated to alanine residues.
pNB7, a CEN-LEU2 plasmid encoding RS-ALP, was constructed by subcloning the 4-kb BamHI fragment from pSN97 into the BamHI site of
pRS315 (Sikorski and Hieter, 1989
). pNB8, a CEN-LEU2 plasmid encoding (F/A)A-ALP, was constructed by subcloning the SacI-EcoRV fragment
encompassing the STE13-PHO8 gene fusion encoding (F/A)A-ALP from
pSN100 into the SacI-SmaI sites of pRS315 (Sikorski and Hieter, 1989
).
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Radiolabeling and Immunoprecipitation
[35S]Methionine labeling and immunoprecipitation of ALP fusion proteins, Vps10p-related proteins, and CPY were performed as previously
described (Piper et al., 1994; Nothwehr et al., 1995
; Cooper and Stevens,
1996
). In brief, yeast cultures were grown overnight in selective synthetic
media without methionine to OD600 = 1. Cells were harvested and resuspended in fresh media to the same OD600. Cells were pulse labeled for 10 min with 100 µCi 35S-Express label per 0.5 OD600, followed by the addition
of unlabeled methionine and cysteine both to 50 µg/ml. At specified times
samples were removed and treated by the addition of sodium azide to 10 mM
at 4°C. Vps10p, CPY, ALP, and related proteins were quantitatively immunoprecipitated from protein extracts of these cells using the relevant
specific polyclonal antibodies as previously described (Piper et al., 1994
;
Nothwehr et al., 1995
; Cooper and Stevens, 1996
). Half-times of processing of ALP- and Vps10p-related proteins were determined as previously
described (Nothwehr et al., 1993
; Cooper and Stevens, 1996
) using an
AMBIS Radioanalytic Imaging System (Ambis, San Diego, CA) and linear regression analysis plotting percentage total protein processed as a
function of time.
Immunofluorescence Microscopy
The preparation of fixed yeast cell spheroplasts, their attachment to microscope slides, and co-staining of ALP fusion proteins using the mouse
anti-ALP mAb 1D3-A10 (Molecular Probes Inc.) and Vph1p using affinity-purified polyclonal antibodies was carried out as previously described
(Cooper and Stevens, 1996; Piper et al., 1997
). Before use, the mouse anti-ALP mAb 1D3-A10 (Molecular Probes Inc.) was adsorbed against pho8
yeast cells to increase the ALP-specific signal. pho8
yeast cells were
fixed, converted to spheroplasts, and then permeabilized using 1% SDS
for 1 min. These were resuspended in 1D3-A10, diluted in PBS containing
5 mg/ml BSA, and then incubated for 1 h at 25°C. Spheroplasts were removed by centrifugation and the resultant supernatant was used to stain
fixed cells attached to microscope slides. Essentially, fixed spheroplasts attached to slides were incubated with the following solutions, followed by
extensive washing with PBS containing 5 mg/ml BSA after each step (all
antibody incubations were performed at 25°C for 1 h with the exception of
those involving the mouse anti-ALP mAb 1D3-A10 cultured supernatant;
these were performed at 4°C for 12-16 h): (a) PBS-BSA containing a 1:3
dilution of adsorbed 1D3-A10 cultured supernatant; (b) 1:500 dilution of
a biotin-conjugated donkey anti-mouse IgG (H + L) and a 1:20 dilution of
affinity-purified rabbit anti-Vph1p polyclonal antibody; and (c) 1:500
dilution of FITC-conjugated streptavidin and 1:2,000 dilution of Texas
red-conjugated goat anti-rabbit IgG (H + L). Co-staining of ALP with
Vti1p was performed using 1D3-A10 cultured supernatant at a dilution of
1:3 in conjunction with affinity-purified antibodies specific for Vti1p as
previously described (Fischer von Mollard et al., 1997
). In brief, the incubations were as follows: (a) PBS-BSA containing a 1:50 dilution of the affinity-purified polyclonal antibody, (b) followed by a 1:500 dilution of a biotin-conjugated donkey anti-rabbit IgG (H + L) and a 1:3 dilution of
1D3-A10, and (c) 1:500 dilution of FITC-conjugated streptavidin and
1:2,000 dilution of Texas red-conjugated goat anti-mouse IgG (H + L). Similarly, co-staining of RS-ALP and Vps10p was performed as previously described (Cooper and Stevens, 1996
) using a 1:200 dilution of affinity-purified polyclonal antibodies that recognize Vps10p in conjunction with a 1:3
dilution of 1D3-A10. Staining for Pep12p was performed using affinity-purified rabbit anti-Pep12p antibodies (Fischer von Mollard et al., 1997
)
in conjunction with biotin/streptavidin amplification as described above.
Images were captured on 35 mm Tmax
400 film with a 100× oil immersion lens on an Axioplan fluorescence microscope (Carl Zeiss,
Oberkochen, Germany). Film negatives were digitized using a Polaroid
SprintScan 35. Images were adjusted with standard settings using PhotoshopTM (Adobe Systems Inc., Mountain View, CA).
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Results |
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Ablation of the Retrieval Motif of the Model Recycling Golgi Membrane Protein, RS-ALP, Causes Mislocalization to the Vacuole
Our laboratory has previously described the protein A-ALP,
which was constructed by fusing the cytosolic domain of
the TGN protein DPAP A to the transmembrane and lumenal domains of the vacuolar protein ALP (Nothwehr et
al., 1993). A-ALP is found in the TGN of wild-type cells,
and achieves this localization through a combination of
static retention mechanisms and retrieval from the post-Golgi PVC (Nothwehr et al., 1993
; Bryant and Stevens, 1997
). Fig. 1 shows a schematic representation of A-ALP
and the other hybrid proteins used in this study, as well as
their cellular localization in wild-type cells. In (F/A)A-ALP, the two phenylalanine residues contained within the
FXFXD motif responsible for the retrieval of DPAP A
and A-ALP from the PVC back to the Golgi have been
mutated to alanine residues (Nothwehr et al., 1993
). Because of this mutation, (F/A)A-ALP cannot be retrieved
from the PVC to the TGN, and consequently travels on to
the vacuolar membrane (Nothwehr et al., 1993
; Bryant
and Stevens, 1997
). RS-ALP was constructed by transplanting a 10-residue stretch from DPAP A, encompassing
the FXFXD motif, into the cytosolic domain of ALP (Nothwehr et al., 1993
). This transplantation inserted the
retrieval motif (RRESFQFNDI) in place of residues
11-17 (TRLVPGS) from the 33-residue tail of ALP, and
the resulting protein localizes to the TGN of wild-type
cells (Nothwehr et al., 1993
). Using indirect immunofluorescence and subcellular fractionation, previous studies have shown that RS-ALP, like A-ALP, colocalizes with
the TGN marker protein Kex2p (Nothwehr et al., 1993
;
Bryant and Stevens, 1997
). Like A-ALP, RS-ALP achieves
its localization by continuous retrieval from a post-Golgi
compartment but does not use the static retention mechanism(s) used by DPAP A and A-ALP (Bryant and
Stevens, 1997
).
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Fig. 1 B shows double-labeling immunofluorescence for
the 100-kD subunit of the vacuolar ATPase (V-ATPase),
Vph1p, and the various ALP-based proteins depicted in
Fig. 1 A. Whereas ALP was found on the vacuolar membrane of wild-type cells (Fig. 1 B, a), both A-ALP and RS-ALP localized to punctate structures (Fig. 1 B, b and d)
characteristic of markers of the TGN (Franzusoff et al.,
1991; Redding et al., 1991
; Nothwehr et al., 1993
). This localization requires the presence of the two phenylalanine
residues within the FXFXD motif, as demonstrated by the
co-localization of both (F/A)A-ALP and (F/A)RS-ALP
with Vph1p on the vacuolar membrane (Fig. 1 B, c, h, e,
and j). Like (F/A)A-ALP (Nothwehr et al., 1993
), (F/
A)RS-ALP was constructed by mutation of the two phenylalanine residues within the FXFXD motif to alanine
residues. The differential localization of RS-ALP and (F/
A)RS-ALP (Fig. 1 B, d and e) demonstrates that it is the
FXFXD motif that allows RS-ALP to achieve its TGN localization and in its absence, the protein behaves like ALP
and is delivered to the vacuole.
Neither RS-ALP nor (F/A)RS-ALP Depend on VPS45 for Their Localization
At the TGN, recycling Golgi membrane proteins and vacuolar proteins that follow the VPS-dependent (or CPY)
pathway to the vacuole enter transport vesicles whose fusion with the PVC is controlled by the Sec1p-like protein
Vps45p (Cowles et al., 1994; Piper et al., 1994
, 1997
; Bryant et al., 1998
). In vps45 mutant cells vacuolar proteins
such as the 100-kD subunit of the V-ATPase, Vph1p, become trapped in these Golgi-derived transport vesicles, which are unable to fuse with the PVC (Bryant et al.,
1998
). This mislocalization can be observed using indirect
immunofluorescence microscopy where proteins caught in
these vesicles display a diffuse staining pattern (Piper et al.,
1994
, 1997
; Bryant et al., 1998
). Golgi membrane proteins,
such as the CPY receptor Vps10p, that continually cycle
between the TGN and the PVC, also enter into Vps45p-controlled vesicles and consequently Vps10p localizes to diffuse vesicular structures in vps45 mutant cells (Bryant
et al., 1998
). In contrast to this, proteins that follow the alternative, or ALP, pathway from the TGN to the vacuole
do not depend on Vps45p to reach their final destination
and are localized to the vacuolar membrane in vps45 mutant cells (Piper et al., 1997
). This is demonstrated in Fig. 2
A, which shows that ALP was delivered to the vacuolar membrane in vps45
cells (Fig. 2 A, a; Piper et al., 1997
).
In these same cells, in contrast to the vacuolar staining pattern observed in wild-type cells, Vph1p displayed a disperse, clearly non-vacuolar staining pattern consistent
with its entrapment inside the vesicles that accumulated in
vps45
cells (compare Fig. 1 B, f with Fig. 2 A, d). To determine whether the vacuolar proteins (F/A)A-ALP or (F/
A)RS-ALP entered into Vps45p-controlled vesicles, we
immunolocalized these proteins in vps45
cells. Like
Vph1p, (F/A)A-ALP (a version of A-ALP that cannot recycle from the PVC back to the Golgi and consequently
travels to the vacuole via the CPY pathway) accumulated
in transport vesicles in vps45
cells. This is in contrast to
its localization in wild-type cells where it was found on the
vacuolar membrane (compare Fig. 1 B, c with Fig. 2 A, b).
Fig. 2 A also shows that like ALP, whose trafficking to the
vacuole is not dependent on VPS45, (F/A)RS-ALP was localized on the vacuolar membrane while Vph1p accumulated in transport vesicles in vps45
cells (compare Fig. 2
A, c and f). These data demonstrate that while both (F/
A)A-ALP and (F/A)RS-ALP transit to the vacuole in
wild-type cells, they do so by different routes. (F/A)A-ALP enters the Golgi-derived transport vesicles controlled by Vps45p, whereas (F/A)RS-ALP bypasses these
trafficking intermediates as it travels to the vacuole.
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The observation that (F/A)RS-ALP, like ALP, does not
enter Vps45p-controlled transport vesicles demonstrated
that sorting information necessary to enter the alternative
pathway to the vacuole was fully functional within this
protein. We reasoned that the unmutated RS-ALP protein
would have both sorting information to enter the alternative pathway to the vacuole, and a functional retrieval signal. In both wild-type and vps45 mutant cells, RS-ALP displayed a punctate distribution (Fig. 1 B, d; data not shown) consistent with its previous colocalization with the TGN
protein Kex2p (Nothwehr et al., 1993). However, it was
difficult to quantitatively assess the degree to which RS-ALP had a vesicular pattern in vps45 mutant cells compared with its punctate distribution in wild-type cells (Fig.
1 B, d). Thus, to determine quantitatively whether RS-ALP did bypass Vps45p-controlled vesicles, we performed
a series of pulse-chase immunoprecipitation experiments.
Upon delivery to the vacuole, proteins containing the lumenal domain of ALP are processed in a PEP4-dependent
manner to lower molecular mass forms (Ammerer et al.,
1986; Klionsky and Emr, 1989
; Nothwehr et al., 1993
). For
(F/A)A-ALP, this processing occurred with a half-time of
~60 min in wild-type cells (Fig. 2 B; Nothwehr et al.,
1993
). In vps45
cells, this processing was blocked since
(F/A)A-ALP was trapped inside transport vesicles and unable to gain access to a proteolytically active compartment (Fig. 2 B). As expected from the observation that ALP
does not require VPS45 to reach the vacuolar membrane,
the kinetics of processing of ALP were similar in wild-type
and vps45
cells (Fig. 2 B; Piper et al., 1997
). Consistent
with the vacuolar localization of (F/A)RS-ALP in vps45
cells (Fig. 2 A, c), this protein was proteolytically processed
with similar kinetics in wild-type and vps45
cells (Fig. 2
B). RS-ALP was also processed with similar kinetics in
wild-type and vps45
cells (Fig. 2 B). These data indicate
that RS-ALP does not use VPS45-controlled vesicles as part of its normal cellular itinerary, and like (F/A)RS-ALP,
follows the VPS45-independent route out of the TGN.
The VPS45-independent route taken by ALP to the vacuole requires the adaptor protein complex AP-3 (Cowles
et al., 1997; Stepp et al., 1997
). Therefore we tested
whether transport of (F/A)RS-ALP and RS-ALP to the
vacuole was AP-3 dependent. Pulse-chase immunoprecipitation experiments revealed that the processing of ALP
was significantly impaired in cells defective for the AP-3
complex (apm3
), as previously reported (Cowles et al.,
1997
; Stepp et al., 1997
). The same set of experiments (Fig.
3) also revealed that the vacuolar delivery of (F/A)A-ALP, which transits to the vacuole through the CPY pathway, is unaffected by the apm3
mutation. As observed
for ALP, the proteolytic processing of (F/A)RS-ALP was
significantly slowed in apm3
cells (Fig. 3). The vacuolar
delivery of RS-ALP was similarly impaired in apm3
cells
(data not shown). These data indicate that (F/A)RS-ALP
and RS-ALP transport to the vacuole is AP-3 dependent.
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The data presented thus far fit with a model evoking the existence of an as yet undescribed retrograde trafficking pathway out of the vacuole (Fig. 2 C, broken arrow). RS-ALP and (F/A)RS-ALP would travel from the TGN to the vacuole along the alternative pathway taken by ALP, and after recognition of its FXFXD motif, RS-ALP (but not (F/A)RS-ALP) would be retrieved to the TGN via the PVC by the retrograde pathway.
RS-ALP Reaches the Prevacuolar/Endosomal Compartment via the Vacuole
Proteins such as ALP and Vam3p that follow the alternative pathway to the vacuole (Cowles et al., 1997; Darsow
et al., 1997
; Piper et al., 1997
) do not transit through the
PVC defined by mutations in the class E VPS gene, VPS27
(Fig. 2 C). vps27 mutants accumulate an exaggerated form
of the PVC because of a block of traffic out of this compartment both on to the vacuole and back to the TGN
(Piper et al., 1995
). This class E compartment contains both endocytosed proteins and recycling late-Golgi membrane proteins as well as vacuolar proteins that follow the
VPS-dependent, or CPY, pathway to the vacuole (Piper
et al., 1995
). Vacuolar proteins such as Vph1p and recycling Golgi membrane proteins such as Vps10p, enter the
class E compartment in Golgi-derived, Vps45p-controlled transport vesicles (Bryant et al., 1998
) while proteins that
follow the alternative pathway to the vacuole bypass this
compartment (Piper et al., 1997
).
We examined the behavior of RS-ALP and (F/A)RS-ALP in vps27 mutant cells to gain further insight into the
trafficking routes of these proteins. Fig. 4 A shows that
while Vph1p accumulated in the perivacuolar class E compartment (Fig. 4 A, f-j), ALP was localized to the vacuolar
membrane of vps27 cells (Fig. 4 A, a; Piper et al., 1997
).
Entry into the class E compartment from the TGN is controlled by Vps45p (Bryant et al., 1998
), and as expected,
proteins that get packaged into Vps45p vesicles become trapped in the class E compartment of vps27
cells. This is
demonstrated in Fig. 4 A where b and c show that A-ALP
and (F/A)A-ALP colocalized with Vph1p (g and h) in the
class E compartment. By contrast, (F/A)RS-ALP did not
require VPS45 to reach the vacuole (Fig. 2 A, c) and further evidence that this protein followed the alternative pathway to the vacuole is shown in Fig. 4 A (e). (F/A)RS-ALP did not accumulate in the class E compartment of
vps27
cells but instead localized to the vacuole. Interestingly, RS-ALP does localize to the class E compartment of
vps27 mutant cells (Fig. 4 A, d; Bryant and Stevens, 1997
).
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The observation that RS-ALP localizes to the class E
compartment is not inconsistent with the observation that
it does not travel via the CPY pathway in Vps45p-controlled vesicles. To test the model that RS-ALP visits the
vacuole before its arrival at the PVC, we used a regimen in
which the PVC of vps27 mutant cells can be made proteolytically inactive while the vacuole remains proteolytically active. This manipulation uses a temperature-sensitive allele of VPS27 and the phenomenon of phenotypic
lag (Zubenko et al., 1982). vps27-ts cells harboring a plasmid in which the PEP4 open reading frame was placed under the control of the inducible GAL1 promoter were
grown in media containing galactose as their sole carbon
source at the permissive temperature of 22°C. We have reported previously that these cells are Pep+ and that when
cells are exposed to glucose to shut off production of
Pep4p, active vacuolar proteases are flushed from prevacuolar/endosomal compartments (Piper et al., 1997
). Shifting these cells to the restrictive temperature of 37°C induces the formation of a proteolytically inactive PVC
(Piper et al., 1997
). Under this strategy, the vacuole itself
remains proteolytically active because of the phenomenon of phenotypic lag, where the autocatalytic activation cycle
of protease B results in this and other proteases remaining
active for many cell divisions after elimination of Pep4p
synthesis (Zubenko et al., 1982
; Jones, 1991
).
Cells were grown at 22°C in media containing 2% galactose for 24 h, to induce Pep4p production. Expression of
PEP4 in these cells was turned off when the cultures were
diluted in media containing 2% glucose and incubated at
22°C for an additional 24 h to ensure that Pep4p was
flushed out of the biosynthetic pathway to the vacuole.
Cells were then either shifted to 37°C for 10 min, or left at
22°C, and subjected to radiolabel pulse/chase analysis to
follow the fate of both newly synthesized ALP and newly
synthesized Vps10p-10*. Vps10p-
10* is a mutant form
of the CPY receptor, Vps10p, which lacks the information
allowing the protein to cycle between the PVC and the
TGN and is instead mislocalized to the vacuole (Cooper
and Stevens, 1996
). Consequently, Vps10p-
10* follows
the VPS-dependent pathway to the vacuole and has been shown previously to depend on both VPS27 and VPS45 for
its trafficking to the vacuole (Cooper and Stevens, 1996
;
Piper et al., 1997
). Fig. 4 B shows that at 22°C both ALP and
Vps10p-
10* were proteolytically processed indicating that
both had reached the vacuole. These data indicate that the
vacuole in these cells is fully capable of processing both
ALP and Vps10p-
10* despite the fact that there had been
no Pep4p biosynthesis for several generations.
At 37°C, the VPS-dependent pathway was blocked as
demonstrated by the severe inhibition of Vps10p-10*
processing. Since Vps10p-
10* localizes to the PVC of
vps27 mutant cells (Piper et al., 1997
), the observation that
no processing occurred indicates that the PVC within
these vps27-ts cells at 37°C is proteolytically inactive. In
contrast, ALP within the same cells was processed with
the same kinetics as observed in wild-type cells, demonstrating that the vacuole was proteolytically active. Fig. 4
B also shows that the processing of (F/A)A-ALP, which
is delivered to the vacuole in vps27-ts cells at 22°C (Piper
et al., 1997
), with 55% of the protein processed after 60 min.
The processing of (F/A)A-ALP was blocked at 37°C,
with no detectable processing after 40 min, consistent with the protein being trapped within the proteolytically
inactive PVC that accumulates within these cells. Recall
that like Vps10p-
10* and (F/A)A-ALP, RS-ALP becomes trapped in the PVC of vps27
cells (Fig. 4 A, d) and
vps27-ts cells at 37°C (Bryant and Stevens, 1997
). However, unlike Vps10p-
10* and (F/A)A-ALP, the processing of RS-ALP was not blocked in vps27-ts cells at 37°C
whose PVC had been made proteolytically inactive (40%
of RS-ALP has been processed after 40 min at 37°C compared with 35% of the protein after 60 min at 22°C). These
data strongly suggest that RS-ALP visits the proteolytically active vacuole before becoming trapped in the PVC
of vps27 mutant cells.
RS-ALP and A-ALP Follow Different Intracellular Routes to the PVC
We have previously reported that the entry of proteins
such as Vph1p, that follow the CPY pathway to the vacuole, into the class E compartment of vps27 cells requires
the function of VPS45 (Bryant et al., 1998). We found that
for Vph1p trafficking, VPS45 is epistatic to VPS27 since
mutations in VPS45 prevent the accumulation of Vph1p in
the PVC of vps27 mutant cells. Similarly, VPS45 is epistatic to VPS27 for Vps10p trafficking, since the recycling receptor becomes trapped inside Golgi-derived transport
vesicles and cannot gain entry into the PVC of cells carrying mutations in both VPS45 and VPS27 (Bryant et al.,
1998
). It is clear that these vps45 vps27 double mutant cells
do accumulate a PVC since the endocytosed protein
Ste3p, whose trafficking does not depend upon VPS45, accumulates there (Bryant et al., 1998
).
While both A-ALP and RS-ALP use continuous retrieval from the PVC to achieve steady-state localization
to the TGN (Bryant and Stevens, 1997), it appears that the
two proteins are delivered to the PVC via different trafficking pathways. If RS-ALP reaches the PVC via retrograde transport from the vacuole (Fig. 2 C), we predict
that while VPS45 function is required for the entry of
A-ALP and Vph1p into the PVC (Bryant et al., 1998
), RS-ALP will not require Vps45p to enter the same compartment. Fig. 5 shows that both A-ALP and (F/A)A-ALP
were prevented from reaching the PVC in vps27
vps45
double mutants. Neither A-ALP nor (F/A)A-ALP displayed characteristic staining of localization to the class E
compartment in these cells, but instead displayed a disperse staining pattern, as seen for Vph1p within the same
cells, consistent with their entrapment inside transport vesicles (Fig. 5, b, c, g, and h). These data are consistent with
the finding that A-ALP and (F/A)A-ALP use the CPY
pathway to transit between the TGN and the PVC (Figs. 2
and 3). Like A-ALP and (F/A)A-ALP, RS-ALP accumulated in the PVC of vps27
cells (Fig. 4 A, b-d) but its trafficking to this compartment did not require VPS45, RS-ALP localized to the PVC of vps27
vps45
double mutants
while Vph1p accumulated within VPS45 controlled vesicles
within the same cells (compare Fig. 5, d and i).
|
Like ALP, which does not require the function of
VPS45 or VPS27 to reach the vacuole (Piper et al., 1997),
(F/A)RS-ALP was found on the vacuolar membrane of
vps27
vps45
double mutants (Fig. 5, e). This is consistent with the data presented in Figs. 2 and 3, which show
that (F/A)RS-ALP followed the alternative pathway to
the vacuole. This, taken with the observation that RS-ALP
localized to the PVC of vps27
vps45
double mutants
supports a model in which RS-ALP traffics to the vacuole
along the alternative (ALP) pathway before traveling to
the PVC and finally back to the TGN as part of its normal
cellular itinerary. The unique properties of RS-ALP have
thus provided the basis for an assay for the retrograde trafficking step from the vacuole to the prevacuolar/endosomal compartment in yeast.
RS-ALP Cannot Be Retrieved from the Vacuole of vac7 Mutant Cells
The Class III vac mutant vac7 is defective in vacuolar inheritance likely resulting from an inability to perform a
scission step necessary to segregate the budding vacuole
from the mother into the daughter cell (Bonangelino et al.,
1997). We reasoned that the same machinery on the vacuolar membrane might be involved in a trafficking step out
of the vacuole, and investigated whether vac7 mutant cells
were defective in the transport of RS-ALP out of the vacuole. RS-ALP and A-ALP colocalize with Kex2p to the TGN in wild-type cells (Nothwehr et al., 1993
; Bryant and
Stevens, 1997
) showing the punctate staining pattern in indirect immunofluorescence that is characteristic of localization to this compartment (Fig. 1 B, b and d; Nothwehr
et al., 1993
). In vac7-1 cells, RS-ALP was found on the
vacuolar membrane, colocalizing with the V-ATPase (Fig.
6 B, a and b) while A-ALP still displayed a punctate staining pattern (Fig. 6 C, a and b). Fig. 6 D also shows double
labeling of RS-ALP and the TGN protein Vps10p. These
two proteins both localize to the TGN in wild-type cells
(Nothwehr et al., 1993
; Cooper and Stevens, 1996
), but in
vac7-1 cells, RS-ALP was mislocalized to the vacuolar
membrane while Vps10p maintained its punctate, non-vacuolar staining pattern (Fig. 6 D, a and b). Fig. 6 demonstrates that even though vac7-1 cells possessed a morphologically recognizable TGN (as defined by A-ALP and
Vps10p), RS-ALP was not found in this organelle as it is in
wild-type cells but instead accumulated on the vacuolar
membrane. These data suggest that VAC7 is required for a
membrane transport pathway out of the vacuole, which
RS-ALP uses to achieve its localization to the TGN.
|
The v-SNARE Vti1p Is Mislocalized in Cells That Block Retrograde Traffic Out of the Vacuole
The SNARE hypothesis proposes that interactions between specific v-SNARE molecules on transport vesicles
and cognate t-SNAREs on target membranes are involved
in controlling the fidelity of membrane fusion (Sollner et al.,
1993; Ferro-Novick and Jahn, 1994
; Rothman, 1994
). In
this model, vesicle fusion results in delivery of v-SNAREs
to the target membrane and it is likely that these proteins
are recycled back to the compartment from where their
vesicles bud so that they can be involved in subsequent rounds of targeting. Vti1p is a v-SNARE that has been
shown to interact with the t-SNARE Pep12p to control entry of proteins into the PVC (Fischer von Mollard et al.,
1997
). Vti1p has also been shown to interact with the
t-SNARE Sed5p to control protein transport between the
ER and the cis-Golgi (Fischer von Mollard et al., 1997
; Lupashin et al., 1997
). More recently, Vti1p has been shown
to interact with Vam3p (Holthius et al., 1998) and to be involved in the alternative pathway taken by ALP and
Vam3p to the vacuole (Fischer von Mollard, G., and T.H.
Stevens, in preparation). Since Vti1p presumably travels
to the vacuole in its role of directing ALP containing trafficking intermediates there, it is likely to be recycled back
to the TGN so that it can be involved in further rounds of
protein transport. Thus, we identified Vti1p as a candidate
for an endogenous cargo protein of the retrograde pathway out of the vacuole. In wild-type cells, Vti1p displayed
a punctate immunofluorescence staining pattern (Fig. 7 A,
a), but in vac7-1 cells the protein was found on the vacuolar membrane colocalizing with ALP (Fig. 7 A, d and e).
|
The endosomal t-SNARE protein Pep12p (Becherer et al.,
1996) was also immunolocalized in vac7-1 cells to determine whether this protein would maintain a non-vacuolar
localization. Consistent with subcellular fractionation localization of Pep12p (Becherer et al., 1996
), Pep12p displayed a punctate, non-vacuolar staining pattern in wild-type cells (Fig. 7 B, a) (similar to that seen for Vti1p in
wild-type cells [Fischer von Mollard et al., 1997
; Fischer
von Mollard, G., unpublished data]). Wild-type cells contained 30-50 of these punctate structures per cell, not seen
in cells that do not produce Pep12p (Fig. 7 B, b). Pep12p
was observed to maintain a punctate, non-vacuolar distribution in vac7-1 cells (Fig. 7 B, c) and was not mislocalized
to the vacuole, arguing that Pep12p does not use the retrograde pathway controlled by VAC7 to maintain its localization.
Vti1p has been shown to be involved in both the VPS-dependent, or CPY pathway, and the alternative, or ALP
pathway to the vacuole (Fischer von Mollard et al., 1997;
Fischer von Mollard, G., and T.H. Stevens, manuscript in
preparation). Since vac7 mutant cells accumulate Vti1p on
the vacuolar membrane, vac7 mutant cells could be defective for transport along both the ALP and CPY pathways. In fact, it has been reported that vac7 mutant cells accumulate Golgi-modified forms of CPY intracellularly (Gomes
de Mesquita et al., 1996
). Fig. 7 C shows pulse-chase immunoprecipitation experiments, which revealed that vac7
mutant cells were also defective in processing of Vps10p-
10*, a membrane protein that follows the VPS-dependent pathway to the vacuole (Cooper and Stevens, 1996
;
Piper et al., 1997
; Fig. 4 B). In wild-type cells, Vps10p-
10* underwent PEP4-dependent processing with a half-time of ~15 min (Fig. 7 C; Cooper and Stevens, 1996
), but
in vac7-1 cells, little processing was observed even after
chase times of 60 min. Fig. 7 C also shows that the processing of ALP was significantly slower in vac7 mutant cells
than in wild-type cells, with unprocessed forms of ALP
still present after 60 min of chase. These data suggest that
the vacuolar delivery of ALP is slower in vac7 mutant cells than in wild-type cells, but it is worth noting that processed forms of ALP are obvious after longer chase times, consistent with the immunolocalization of ALP to the vacuole in
vac7 mutant cells (Fig. 6 A, a; Fig. 7 A, d; and B, d). This
defect in both the CPY and the alternative pathway to the
vacuole in vac7-1 cells may reflect mislocalization of proteins such as Vti1p in these cells, since Vti1p uses retrograde transport out of the vacuole as part of its normal cellular itinerary (Fig. 7 A).
![]() |
Discussion |
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In this study we report that a hybrid protein (RS-ALP) resident to the TGN of the yeast Saccharomyces cerevisiae achieves its localization by transiting through the vacuole (Fig. 8). Directed by sorting information contained within its cytosolic tail, RS-ALP follows the alternative pathway, as taken by ALP, to the vacuole thus bypassing Golgi- derived transport vesicles that carry proteins from the TGN to the PVC. By virtue of the retrieval motif (FXFXD) that it also carries in its cytosolic tail, RS-ALP then uses a retrograde membrane trafficking step out of the vacuole to reach the PVC from where it is delivered back to the TGN. This work has also revealed that VAC7 function is required for membrane traffic out of the vacuole, and that the v-SNARE Vti1p uses this retrograde trafficking pathway as part of its normal cellular itinerary. This study represents the first report of retrograde membrane traffic out of the vacuole in the yeast S. cerevisiae.
|
Identification of a Retrograde Membrane Trafficking Step Out of the Vacuole
The yeast vacuole and its mammalian counterpart the lysosome have typically been thought of as terminal destinations for proteins with regard to membrane trafficking
(Kornfeld and Mellman, 1989; Bryant and Stevens, 1998
).
Recently however, evidence that this might not be the case
has been building from work carried out on mammalian
cells (see Introduction for details), suggesting that the lysosome acts as a donor membrane in protein trafficking and is therefore a more dynamic organelle than has been
widely appreciated. Although the possible existence of a
retrograde pathway out of the yeast vacuole has been suggested (Wilcox et al., 1992
), there has been no experimental test of this idea. In this study, we have taken advantage
of the unique properties of a hybrid protein, RS-ALP
(Nothwehr et al., 1993
) to demonstrate the existence of a
retrograde membrane trafficking step out of the vacuole in
the yeast S. cerevisiae. RS-ALP carries a motif (FXFXD)
in its cytosolic tail that is sufficient to specify retrieval of
the protein from the PVC to the TGN, affording steady
state localization to the TGN (Bryant and Stevens, 1997
).
RS-ALP was constructed in such a way that information
sufficient to direct ALP away from the CPY pathway and
into the alternative pathway taken by ALP to the vacuole
remains present (Nothwehr et al., 1993
; Piper et al., 1997
).
The transport of ALP, RS-ALP, and (F/A)RS-ALP to
the vacuole has been found to be Vps45p independent but
requires the adaptor protein complex AP-3. Although
ALP, RS-ALP, and (F/A)RS-ALP all travel to the vacuole
along the alternative pathway, they are not all proteolytically processed with the same kinetics. Vowels and Payne
(1998) recently identified the amino acid residues LV as
being the most important signal within the ALP cytosolic
domain for targeting into the alternative pathway. The residues LV are missing in (F/A)RS-ALP, and although it is
clear that this protein uses the alternative pathway (as
does a version of ALP lacking residues 2-21 and therefore
the LV signal [Piper et al., 1997
]), its slower transport rate
to the vacuole likely results from its compromised targeting information. Like (F/A)RS-ALP, RS-ALP also lacks
the LV motif, and therefore enters the alternative pathway at a rate slower than ALP, but likely at the same rate as
(F/A)RS-ALP. However, RS-ALP is processed in wild-type cells with even slower kinetics than (F/A)RS-ALP.
This difference can be explained if the signal-dependent
retrieval from the vacuole occurs rapidly enough to prevent RS-ALP from becoming fully processed during one
round of transit through the vacuole.
The AP-3-dependent, alternative pathway to the vacuole is unaffected by mutations in either VPS45 or VPS27,
which control transit through the PVC (Cowles et al.,
1994, 1997
; Piper et al., 1994
, 1995
, 1997
; Bryant et al.,
1998
). We have demonstrated that the trafficking of RS-ALP occurs independently of Vps45p and that it reaches
the PVC by first traveling through the proteolytically active vacuole. The observation that RS-ALP localizes to the
PVC of vps27 mutant cells (Bryant and Stevens, 1997
),
whereas a mutant version of the same protein (F/A)RS-ALP is found on the vacuolar membrane, was crucial to
the formation of our model in which RS-ALP reaches the
PVC from the vacuole via a retrograde membrane trafficking step (Fig. 8).
Although both RS-ALP and the hybrid protein A-ALP
achieve TGN localization through continual retrieval from
the PVC (Bryant and Stevens, 1997), the route taken by
the two proteins to reach the PVC differs. A-ALP consists
of the cytosolic domain of the TGN protein DPAP A
fused to the transmembrane and lumenal domains of ALP
(Nothwehr et al., 1993
). A-ALP and its derivative (F/A)A-ALP leave the TGN by entering the CPY pathway out of
the Golgi (Fig. 8, pathway 1). Fusion of the vesicles that
these proteins enter with the PVC is controlled by the
Sec1p-like protein Vps45p. After recognition of its
FXFXD motif, A-ALP is recycled from the PVC back to
the TGN (Fig. 8, pathway 2) and thus achieves steady state localization to the TGN (Nothwehr et al., 1993
; Bryant
and Stevens, 1997
). Since it does not carry an FXFXD motif, (F/A)A-ALP cannot be retrieved from the PVC to the
TGN, but instead travels on from the PVC to the vacuole
(Bryant and Stevens, 1997
; Fig. 8, pathway 3). In contrast
to this, RS-ALP and (F/A)RS-ALP do not enter Vps45p controlled vesicles and instead follow the alternative pathway to the vacuole (Fig. 8, pathway 4). After delivery to
the vacuolar membrane, the FXFXD motif of RS-ALP directs its retrograde transport from the vacuole to the PVC
(Fig. 8, pathway 5) from where it follows the same pathway as A-ALP to return back to the TGN (Fig. 8, pathway
2). As a result of ablation of the FXFXD motif, (F/A)RS-ALP is not retrieved from the vacuole to the PVC and is
therefore localized to the vacuolar membrane of both
wild-type and vps27 mutant cells.
This model can be used to explain why RS-ALP undergoes processing by vacuolar proteases in wild-type cells,
whereas the CPY receptor, Vps10p, which leaves the TGN
and enters the PVC with similar kinetics to RS-ALP, is not
exposed to vacuolar proteases (Bryant and Stevens, 1997).
RS-ALP becomes processed as it transits through the vacuole en route to the PVC, whereas Vps10p enters the PVC
in Vps45p controlled vesicles (Bryant et al., 1998
). These data also suggest that the levels of active vacuolar proteases contained within the PVC are not as high as those
found in the vacuole.
Machinery and Physiological Relevance of Retrograde Vacuolar Transport
We have demonstrated that mutations in the class III VAC
gene VAC7 (Gomes de Mesquita et al., 1996; Bonangelino
et al., 1997
) cause mislocalization of RS-ALP to the vacuolar membrane. This implies that Vac7p, a vacuolar protein required for vacuolar inheritance and normal vacuolar morphology (Bonangelino et al., 1997
), is required for
the transport of RS-ALP from the vacuole to the PVC.
The identification of the v-SNARE Vti1p (Fischer von
Mollard et al., 1997) as a cargo molecule of the retrograde
membrane trafficking pathway out of the vacuole is important since it provides some insight into the physiological
relevance of this pathway. Vti1p is required for the CPY
pathway, where it interacts with the t-SNARE Pep12p to
control the entry of proteins into the PVC (Fischer von
Mollard et al., 1997
). In addition, Vti1p is required for the
trafficking of ALP to the vacuole as demonstrated by the observation that cells carrying a conditional allele of VTI1
accumulate unprocessed ALP under restrictive conditions
(Fischer von Mollard, G., and T.H. Stevens, manuscript in
preparation). Like RS-ALP, Vti1p accumulates on the vacuolar membrane of vac7 mutant cells. In its role of directing trafficking intermediates to the vacuole along the ALP
pathway at least a portion of Vti1p must be transported to
the vacuolar membrane. Our observation that Vti1p follows the retrograde trafficking step defined by mutations in
vac7 allows us to propose a mechanism for recycling of this
v-SNARE to allow it to be involved in further rounds of
targeting. Although vac7 mutant cells do not secrete CPY
as might be expected from their mislocalization of Vti1p,
they do accumulate Golgi-modified, or p2, precursor forms
of CPY intracellularly consistent with a defect in TGN to
PVC trafficking (Gomes de Mesquita et al., 1996
; our unpublished data). In addition, vac7 mutant cells are defective in the processing of ALP as well as in that of Vps10p-
10*, a membrane protein marker of the CPY pathway
(Cooper and Stevens, 1996
; Piper et al., 1997
).
While vac7 mutant cells mislocalize Vti1p, overexpression of VTI1 is not sufficient to suppress these processing
phenotypes (Bryant, N.J., and T.H. Stevens, unpublished
data). This suggests that mislocalization of Vti1p alone is
not responsible for the trafficking defects displayed by
vac7 mutants. Such an observation is not surprising since
there are likely to be additional proteins that travel to the
vacuole along with Vti1p by the alternative pathway and it
is easy to imagine that these molecules will also be recycled using retrograde transport out of the vacuole. VTI1 is
unusual among genes involved in vacuolar protein sorting
in that it is an essential gene (Fischer von Mollard et al.,
1997). The essential nature of VTI1 arises from the involvement of Vti1p in ER to cis-Golgi trafficking though
interactions with the t-SNARE Sed5p (Fischer von Mollard et al., 1997
; Lupashin et al., 1997
). Although vac7 mutant cells mislocalize Vti1p to the vacuole, these cells are
still viable (although it is worth noting that vac7
cells do
display a slow growth phenotype; Bonangelino et al., 1997
). It may be that newly synthesized Vti1p in these cells
is sufficient to allow enough membrane traffic to occur
through the cis-Golgi to sustain life.
Our studies with RS-ALP and its derivative (F/A)RS-ALP indicate that the delivery of RS-ALP from the vacuole to the PVC depends on the FXFXD motif. There is no
such motif within the cytosolic sequence of Vti1p (Fischer
von Mollard et al., 1997), which suggests that there are at
least two ways of entering this pathway, or perhaps overlapping signals. A similar phenomenon is seen with the signal-dependent alternative pathway taken by ALP to the
vacuole (Bryant and Stevens, 1998
). The t-SNARE
Vam3p also follows this pathway and yet there are no obvious similarities between the sequences of ALP and
Vam3p (Wada et al., 1997
). To date, very little is known
regarding the trafficking of SNARE proteins and it is
likely that as more information regarding the intracellular pathways followed by these proteins is uncovered, signals
that control the trafficking of Vam3p, Vti1p, and SNARE
proteins in general will be identified.
![]() |
Footnotes |
---|
Received for publication 11 May 1998 and in revised form 24 June 1998.
Address all correspondence to T.H. Stevens, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229. Tel.: (541) 346-5884. Fax: (541) 346-4854. E-mail: stevens{at}molbio.uoregon.eduWe thank S. Nothwehr for the construction of pSN123 and C. Bonangelino for strain LWY2809. We are grateful to L. Graham for her generous supply of affinity-purified antibodies against Vph1p and discussions regarding this work. S. Gerrard is thanked for her help with photography, and we are grateful to members of the Stevens lab for discussion of this work. We would also like to thank G. Fischer von Mollard for sharing her unpublished data and supplying us with affinity-purified antibodies against Vti1p.
This work was supported by National Institutes of Health grants GM 32448 to T.H. Stevens and GM 50403 to L.S. Weisman, and an American Cancer Society grant CSM 87938 to R.C. Piper.
![]() |
Abbreviations used in this paper |
---|
ALP, alkaline phosphatase; CPY, carboxypeptidase Y; DIC, differential interference contrast; DPAP, dipeptidyl aminopeptidase; PVC, prevacuolar/endosomal compartment; SNARE, SNAP receptor; V-ATPase, vacuolar ATPase; VAC, vacuolar inheritance; VPS, vacuolar protein sorting.
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References |
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