Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
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Abstract |
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The transport of newly synthesized proteins
through the vacuolar protein sorting pathway in the
budding yeast Saccharomyces cerevisiae requires two
distinct target SNAP receptor (t-SNARE) proteins,
Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport
of proteins from the Golgi to the vacuole through a well
defined route, the carboxypeptidase Y (CPY) pathway.
Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently
described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in
sorting of vacuolar proteins, overexpression of VAM3
can suppress the protein sorting defects of pep12 cells.
Based on this observation, we developed a genetic
screen to identify domains in Vam3p (e.g., localization and/or specific protein-protein interaction domains)
that allow it to efficiently substitute for Pep12p. Using
this screen, we identified mutations in a 7-amino acid
sequence in Vam3p that lead to missorting of Vam3p
from the ALP pathway into the CPY pathway where it
can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required
for AP-3 adaptor-dependent transport in both yeast
and mammalian systems. Furthermore, disruption of
AP-3 function also results in the ability of wild-type
Vam3p to compensate for pep12 mutants, suggesting
that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein-specific sorting signal
in a t-SNARE protein, and suggest that AP-3-dependent sorting of Vam3p acts to restrict its interaction
with compartment-specific accessory proteins, thereby
regulating its function. Regulated transport of cargoes
such as Vam3p through the AP-3-dependent pathway
may play an important role in maintaining the unique
composition, function, and morphology of the vacuole.
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Introduction |
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IN eukaryotic cells, accurate transport of proteins between intracellular compartments is essential to maintain the biochemical identity of each organelle. Proteins trafficking through the secretory pathway use a vesicular transport mechanism in which proteins are actively concentrated into budding vesicles and then delivered in a vectorial manner to subsequent compartments. Efficient sorting of proteins in this system depends upon both selective packaging of proteins into the appropriate vesicles and recognition and fusion of cargo-containing transport vesicles with the correct target organelle.
Formation of many of the vesicle populations that transport cargo through the endocytic and lysosomal pathways
requires both the coat protein clathrin and distinct heterotetrameric adaptor protein complexes (Robinson, 1994).
While clathrin acts to deform membranes into vesicles,
adaptor proteins provide both a binding site for clathrin
on the membrane, and act to select cargo for inclusion into
vesicles by recognizing sorting signals contained within the
cargo proteins themselves (Marks et al., 1997
; Robinson, 1997
). Three distinct adaptor protein complexes, AP-1,
AP-2, and AP-3, have been identified in both mammalian
cells and in yeast and are thought to direct transport of
cargo proteins into the endocytic/lysosomal pathways
(Phan et al., 1994
; Stepp et al., 1995
; Dell'Angelica et al.,
1997
; Simpson et al., 1997
) by recognition of two main
classes of sorting signals; tyrosine- (Chen et al., 1990
) and
di-leucine-based (Letourneur and Klausner, 1992
; Marks et al., 1997
) sorting motifs. In mammalian cells, AP-1 and
AP-2 are associated with budding clathrin-coated vesicles
at the TGN and the plasma membrane, respectively, although assigning sorting function to these proteins in yeast
has been difficult as deletions of these genes do not result
in detectable protein sorting phenotypes (Robinson, 1994
).
AP-3 has been assigned a sorting function in the process of
pigment deposition in Drosophila (Simpson et al., 1997
)
and cargo selective transport of proteins to the vacuole in
yeast (Cowles et al., 1997a
; Stepp et al., 1997
).
Once vesicles have budded from a donor membrane,
specific cognate interactions between SNAP receptor
(SNARE)1 proteins, which are found on both vesicle (v)-
SNARE and target (t)-SNARE membranes, are thought
to provide the targeting specificity required for a transport
vesicle to dock and fuse with the appropriate acceptor organelle (Bennett et al., 1993; Bennett and Scheller, 1993
;
Sollner et al., 1993
). At each intracellular compartment, SNAREs act with members of several other protein families, including compartment-specific Sec1p and Rab proteins and the general factors N-ethylmaleimide-sensitive
factor (NSF) (Sec18p) and soluble NSF attachment protein (SNAP) (Sec17p), which together are thought to regulate the formation and/or activity of SNARE complexes
(for review see Novick and Brennwald, 1993
; Rothman,
1994
). In accordance with their targeting function,
t-SNAREs are associated for the most part with individual
compartments and become, in effect, markers for that
compartment. Thus, t-SNAREs represent a large family of
related proteins, each of which must be sorted to a distinct
cellular location to maintain accurate intracellular protein
trafficking.
In yeast, protein transport to the vacuole has proven to
be a powerful model system for the study of protein sorting, as many of the required components (i.e., adaptor
proteins, SNAREs, Sec1, and Rab proteins) are evolutionarily conserved (Bennett and Scheller, 1993; Stack and
Emr, 1993
). The characterization of a large collection of
vacuolar protein sorting (VPS) genes has revealed two distinct routes by which proteins traffic from the TGN to the
vacuole. These routes can be distinguished in temperature-sensitive mutants of transport components that display differential effects on the transport of two vacuolar
hydrolases, carboxypeptidase Y (CPY) and alkaline phosphatase (ALP). Many vacuolar resident proteins, such as
CPY, are delivered to the vacuole through a well defined route that requires the function of the VPS genes, one of
which encodes the endosomal t-SNARE, Pep12p (Becherer
et al., 1996
). In contrast, the vacuolar integral membrane
protein ALP is transported to the vacuole in a manner that
is independent of PEP12 (Cowles et al., 1997b
), and is
therefore presumed to bypass this endosomal compartment. Instead, ALP transport to the vacuole follows an AP-3 adaptor protein-dependent pathway (Cowles et al.,
1997a
; Stepp et al., 1997
). Vam3p, the vacuolar t-SNARE,
is required for delivery of both CPY and ALP to the vacuole, indicating that these distinct pathways ultimately converge at this docking site (Darsow et al., 1997
). Interestingly, localization studies indicate that Vam3p may also be
transported to the vacuole via the ALP pathway, suggesting that it too may bypass the endosomal compartment defined by Pep12p (Cowles et al., 1997b
; Piper et al., 1997
).
Although Vam3p and Pep12p perform analogous biochemical t-SNARE functions, these genes clearly have distinct sites of action (Becherer et al., 1996; Burd et al., 1997
;
Darsow et al., 1997
). Surprisingly, however, overexpression of VAM3 can compensate for the loss of PEP12 function (Darsow et al., 1997
; Gotte and Gallwitz, 1997
), suggesting that under some conditions, Vam3p can substitute for Pep12p. We designed a random genetic screen to define the sequence determinants in Vam3p that allow it to
replace Pep12p. This screen specifically identified a small
region within the Vam3p sequence (NEQSPLL), which is
similar to a region of the ALP cytosolic tail that contains a
di-leucine sequence required for proper sorting (Cowles
et al., 1997b
; Vowels and Payne, 1998
). Furthermore, many
of the mutations within the presumptive Vam3p sorting
determinant caused mislocalization of Vam3p. Together, these results suggest that the Vam3p t-SNARE is likely to
be transported to the vacuole through the AP-3/ALP
pathway via recognition of an acidic di-leucine sorting signal. Thus, trafficking of Vam3p to the vacuole through the
AP-3 pathway appears to play an important role in restricting its site of function and regulating its association with other components of the transport machinery.
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Materials and Methods |
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Strains and Media
Saccharomyces cerevisiae strains used for these studies are listed in Table
I. Yeast strains were grown in standard yeast extract-peptone-dextrose
(YPD) or synthetic medium (YNB) supplemented with essential amino
acids. Standard bacterial medium, containing 100 µg/ml ampicillin for
plasmid selection, was used to propagate Escherichia coli. Transformation
of S. cerevisiae was done by the lithium acetate method (Ito et al., 1983).
E. coli transformations were done by the method of Hanahan (1983)
.
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Plasmid Construction and Nucleic Acid Manipulations
Restriction and modification enzymes were purchased from Boehringer
Mannheim (Indianapolis, IN), New England Biolabs (Beverly, MA), or
U.S. Biochemical Corporation (Cleveland, OH). Alleles of vam3 were
constructed in pVAM3.414 and were subcloned into pRS416 by excising
the NotI-XhoI fragment and ligating into NotI-XhoI-digested pRS416
(Sikorski and Hieter, 1989). For construction of NH2-terminal green fluorescent protein (GFP) fusion constructs, a 900-bp fragment containing the
VAM3 open reading frame (ORF) was amplified from either wild-type or
mutant VAM3 template using primers that induced an in-frame BglII site
at the start codon and an EcoRI site after the stop codon. BglII-EcoRI-
digested PCR product was then ligated into BamHI-EcoRI-digested
pGOGFP fusion vector (Cowles et al., 1997a
). Plasmids pVAM3.414 and
pVAM3.424 were described previously (Darsow et al., 1997
).
Alleles of VAM3 were constructed by PCR based mutagenesis (Muhlrad et al., 1992). Primers complimentary to chromosomal sequences immediately adjacent to the start and stop codons of VAM3 were used to
amplify a 900-bp fragment (containing the entirety of the VAM3 ORF)
under limiting dATP, dTTP, or dGTP conditions (20 µM) in separate reactions. The resulting PCR products were precipitated in 95% ethanol,
combined, and then gel purified. A gapped plasmid was generated by digesting pVAM3.414 with BsmI and isolating the vector by gel purification.
The mutagenized PCR product and gapped plasmid were co-transformed
into CBY31 cells and transformants in which homologous recombination
resulted in integration of mutagenized PCR product were selected by
amino acid prototrophy. Transformants were replica plated onto YPD
containing 50 µg/ml geneticin (Gibco Laboratories, Grand Island, NY)
and grown at 30°C for 2 d. Presumptive mutant colonies were picked, retested, and then plasmid linkage of the G418 resistance phenotype was
confirmed by retransformation of isolated plasmids into CBY31 cells.
Site-directed mutagenesis of Q156 was performed by amplifying the VAM3 locus by PCR using mutagenic primers in a gene SOE (splicing by overlap extension) reaction. Complimentary primers containing the appropriate mutation were used in conjunction with primers complimentary to sequences ~300 bp both 5' and 3' of the VAM3 ORF for the initial amplification. A secondary amplification using the initial PCR products as template was performed using only the outside primers to amplify the full-length vam3 mutant. The resultant PCR product was cloned into TOPO TA vector (Invitrogen Corp., Carlsbad, CA). The EcoRI fragment of the TA clones was excised and ligated into EcoRI-digested pRS414 vector for yeast expression. The mutation introduced a unique SacI restriction site and positive insert-containing clones were confirmed to be point mutants by digestion with SacI.
Plasmids isolated from the G418 resistance screen were purified from E. coli using miniprep spin columns (QIAGEN Inc., Valencia, CA). Resultant plasmids were denatured, hybridized to sequencing primers, and then subjected to dideoxy chain termination sequence analysis using the Sequenase enzymes and protocol (U.S. Biochemical Corporation).
Metabolic Labeling and Immunoprecipitation
To analyze the transport of vacuolar proteins, yeast cells were grown at
26°C in synthetic medium supplemented with amino acids to an OD600 of
0.5-1.0. Cells were harvested and converted to spheroplasts as described
previously (Paravicini et al., 1992). Spheroplasts were resuspended at a
concentration of 3 OD600/ml in synthetic medium containing amino acids
and supplemented with 100 µg/ml
2-macroglobulin and 1 mg/ml BSA to
stabilize secreted proteins. Cultures were pre-incubated at the appropriate
experimental temperature for 5 min, and then labeled with 60 µCi
[35S]cysteine/methionine per ml of cell suspension. After labeling, cultures
were chased with the addition of methionine, cysteine, yeast extract, and
glucose to a final concentration of 5 mM, 1 mM, 0.4%, and 0.2%, respectively. After appropriate chase periods, samples were harvested and precipitated by addition of TCA (10% final concentration). For analysis of
CPY in whole cells, metabolic labelings were done in a similar manner except BSA and
2-macroglobulin were excluded from the labeling medium. Whole cells lysates were generated by glass bead disruption in urea
buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 1% SDS, and 6 M urea). In
both spheroplast and whole cell experiments, immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by autoradiography. Antibodies to CPY and Pep12p have been previously described (Klionsky
and Emr, 1989
; Becherer et al., 1996
). mAbs to Vph1p and ALP were purchased from Molecular Probes (Eugene, OR).
Subcellular Fractionation and Gradient Analysis
For intracellular localization of proteins, cells were converted to spheroplasts and lysed in Hepes KOAc lysis buffer containing protease inhibitors
to the following final concentrations: 20 µg/ml PMSF; 5 µg/ml antipain; 1 µg/ml aprotinin; 0.5 µg/ml leupeptin; 0.7 µg/ml pepstatin; and 10 µg/ml
2-macroglobulin (Gaynor et al., 1994
). After a 5-min clearing spin at 300 g,
the spheroplast lysate was sequentially centrifuged at 13,000 g (15 min)
and 100,000 g (60 min) to generate both high and low speed pellet and
supernatant fractions. Resulting samples were TCA precipitated and analyzed by immunoblotting and ECL detection as described previously
(Babst et al., 1997
). Gradient Accudenz (Accurate Chemical and Scientific Corporation, Westbury, NY) solutions were prepared (wt/vol) in 10 mM
Hepes KOAc, pH 7.6, with protease inhibitors. The gradient was generated using the following Accudenz concentration steps from bottom to
top: 0.5 ml 60%; 1 ml 50%; 1 ml 43%; 1 ml 37%; 1 ml 31%; 1 ml 27%; 1 ml
23%; 1 ml 20%; 1 ml 17%; 1 ml 13%; 1 ml 7%. Gradient analysis was performed on 15 OD600 equivalents of cleared spheroplast lysate in a volume
of 1.5 ml loaded on top of the gradient. The gradient was subjected to centrifugation at 4°C in a Beckman SW41 rotor at 170,000 g for 20 h. 12 fractions were harvested manually from the top of the gradient, proteins were
TCA precipitated and analyzed by immunoblotting. Quantitation of proteins on gels was done by densitometry using NIH Image.
Fluorescence Microscopy
To examine vacuolar and endocytic structures in live yeast cells, FM4-64
(Molecular Probes) labeling was done as previously described (Vida and
Emr, 1995) except that the labeling was done at a concentration of 16 µM
FM4-64 at 26°C for 1 h and the cells were chased for a period of 1.5 h. Visualization of FM4-64 and GFP was done either by confocal microscopy or fluorescence microscopy using rhodamine and fluorescein filters, respectively.
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Results |
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Identification of VAM3 Mutants Capable of Substituting for PEP12
Vam3p and Pep12p are two homologous yeast t-SNARE
proteins involved in protein transport to the vacuole in
yeast. Although Vam3p and Pep12p have clearly distinguishable functions (i.e., Pep12p acts at the endosome and
Vam3p acts at the vacuole) (Fig. 1 A), when overexpressed, these proteins are able to partially substitute for
one another (Darsow et al., 1997; Gotte and Gallwitz, 1997
). This suggests that the specificity of Vam3p and
Pep12p functions may be dependent on interactions with
other compartment-specific transport components (Sec1
homologues, Rab proteins). Based on this observation, we
reasoned that mutations in Vam3p that allow it to substitute for Pep12p might uncover domains required for localization and/or specific protein-protein interactions of
Vam3p with Pep12p-specific transport components.
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Several vps mutants, including pep12, are hypersensitive to geneticin (G418), an aminoglycoside antibiotic related to gentamicin (Fig. 1 B). The mechanism of this hypersensitivity is unknown but for pep12
mutants it
appears to correlate with extent of vacuolar protein sorting defects. For example, overexpression of VAM3 from a
multi-copy vector in pep12
cells restored viability to cells
grown on media containing 50 µg/ml G418, while VAM3
expressed at single copy (which does not improve CPY
sorting when expressed in pep12
cells) did not rescue the
growth defects of pep12
cells on media containing 50 µg/ml
G418 (Fig. 1 B). Therefore, selection by growth on G418-containing media presented an easily scoreable phenotype
that could be used to select for vam3 mutants capable of
suppressing the growth defects of pep12
cells on media
containing G418.
The entire VAM3 ORF was randomly mutagenized by
error-prone PCR-mediated mutagenesis and then co-transformed with a gapped, single-copy plasmid into
pep12 mutant cells (Muhlrad et al., 1992
). Transformants
were replica plated onto media containing 50 µg/ml G418,
and viable colonies were selected. Approximately 10,000 colonies were screened, and >100 vam3 mutant clones
that had acquired the ability to suppress the growth defects of pep12
cells at 50 µg/ml G418 were recovered. We
selected ~50 representative clones that grew well at 50 µg/ml
G418 and rescued plasmids from these strains. 30 mutants
that showed plasmid linkage for the G418 resistance phenotype were selected for further analysis. As a secondary
screen, we tested the growth of these mutants at elevated
concentrations of G418. This criteria allowed us to separate the mutants into two general classes: (1) strong mutants that conferred resistance to 100 µg/ml G418, and (2)
weaker mutants that conferred resistance to only 50 µg/ml
G418.
Sequence analysis revealed that each of the 30 mutants
recovered in this screen contained at least one mutation
within a 7-amino acid region of Vam3p, corresponding to
amino acids 154-160 (Fig. 2). This sequence of Vam3p lies
approximately in the middle of the protein, between the
two coiled-coil domains, and it is distanced well away from
the transmembrane domain in a region that does not exhibit high sequence similarity to Pep12p. However, the sequence closely resembles a region in the cytosolic tail of
ALP that has been shown to be necessary for transport of
ALP through the AP-3-dependent pathway to the vacuole
(Cowles et al., 1997b; Vowels and Payne, 1998
). These
characteristics, together with the presence of a leucine pair
at positions 159-160, suggested that this region may represent a di-leucine-type sorting signal in Vam3p.
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Somewhat surprisingly, the mutations in the 30 plasmids
represented only seven separate single mutations in the
following five residues: asparagine 154, glutamate 155, serine 157, leucine 159, and leucine 160 (Fig. 2). No other
amino acid substitutions in Vam3p were found outside of
this region. The recovery of multiple identical mutants in
each residue indicates that the screen was near saturation
and that the most relevant residues in Vam3p that confer
suppression of the pep12 mutation had likely been mutated. The mutants that conferred the highest level of
G418 resistance (100 µg/ml) were glutamic acid 155 (at
4
relative to the di-leucine pair) to lysine (vam3E155K), and
leucine 160 to proline (vam3L160P). However, when these
same residues were mutated to either glycine (vam3E155G)
or glutamate (vam3L160Q), respectively, the cells grew only
at the lower concentration of G418 (50 µg/ml). Independently isolated clones containing mutations in asparagine
154 at the
5 position from the di-leucine (vam3N154K),
serine 157 at the
2 position (vam3S157G), and leucine 159 (vam3L159S) also resulted in maximum G418 resistance at
50 µg/ml G418 (Fig. 2).
The ALP cytoplasmic tail sequence and the Vam3p sequence both contain a conserved glutamine at the 3 position (Fig. 2). Since glutamine mutants were not recovered
in the G418 selection, we wanted to determine whether it
was required for the function of the Vam3p domain. Using
site-directed mutagenesis, we changed the glutamine 156 to
leucine (vam3Q156L), transformed the mutant into pep12
cells and assayed for G418 resistance. The vam3Q156L mutant did not confer G418 resistance to pep12
cells. However, when the vam3Q156L point mutation was transformed
into vam3
cells, it complemented both the morphological
defects and the CPY sorting defects of the vam3
cells
(data not shown), indicating that a full-length, functional
protein was still produced. Therefore, the glutamine at the
3 position of the Vam3p motif is not required for normal Vam3p function.
Vam3p Mutants Partially Substitute for Pep12p Function in Vacuolar Protein Sorting
To confirm that the G418 resistance phenotype used to
isolate vam3 mutants correlated with suppression of CPY
defects of pep12 cells harboring these mutant forms of
VAM3 we examined CPY sorting by pulse-chase/immunoprecipitation analysis. As expected, in pep12
cells CPY
was recovered almost exclusively as the Golgi-modified p2
form, consistent with severe defects in Golgi-to-endosome
transport. Similarly, pep12
cells expressing VAM3 from a
single-copy plasmid did not show any significant improvement in CPY sorting. However, in all cases, pep12
cells
expressing vam3 mutants from a single-copy plasmid that
conferred G418 resistance also exhibited improved CPY
sorting. Moreover, efficiency of CPY sorting in pep12
cells harboring the various vam3 mutants correlated with
degree of G418 resistance as mutants that exhibited resistance to high concentrations of G418 sorted and matured
more CPY than mutants resistant to only lower (50 µg/ml)
G418 concentrations. For example, pep12
cells expressing the vam3N154I mutant, which is resistant to G418 concentrations up to 50 µg/ml, converted ~50% of p2 CPY to
the mature form, whereas pep12
cells expressing the
vam3L160P mutant, which is resistant to 100 µg/ml G418,
matured ~80% of CPY (Fig. 3 A).
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As Vam3p requires AP-3 for its normal transport and
localization to the vacuole, disruption of the AP-3 pathway may mimic the effects of the Vam3p sorting signal
mutations. To examine this possibility, we analyzed CPY
sorting in both pep12 cells and apm3
pep12
double
mutant cells, each expressing an additional copy of wild-type VAM3 to mimic the conditions of the mutant screen. In contrast to pep12
cells containing wild-type VAM3, in
which CPY is blocked exclusively in the p2 form, ~80% of
CPY was converted to the mature vacuolar form in
apm3
pep12
double mutant cells (Fig. 3 A). This was remarkably similar to results obtained for Vam3p mutants
such as vam3L160P expressed in pep12
cells, and suggests
that these mutations allow CPY maturation because they
prevent normal recognition of Vam3p by the AP-3 adaptor complex and result in missorting of Vam3p to the pre-vacuolar endosome where it can partially substitute for
Pep12p.
VAM3 Mutants Still Maintain Vam3p Function
We were interested in determining whether the vam3 mutants were capable of normal Vam3p t-SNARE function
at the vacuole. Since the screen was performed in a strain
containing a wild-type VAM3 gene, it was possible that
mutations allowing for suppression of pep12 sorting defects would also result in loss of the ability of these proteins to function as Vam3p. Therefore, we examined CPY
sorting by pulse-chase analysis in vam3
cells transformed with the mutants recovered from the screen. As expected,
vam3
cells accumulated only p2 precursor CPY, indicating that transport of CPY to the vacuole was blocked.
However, vam3 mutants that resulted in both strong
(vam3L106P) and weak (vam3N154I) suppression of pep12
mutant phenotypes completely complemented the CPY
sorting defects of the vam3
strain (Fig. 3 B), indicating that although these mutants can substitute for the Pep12p
t-SNARE, they also retain Vam3p t-SNARE function.
These results suggest that the mutant Vam3 proteins must
at least partially localize to the vacuole, the normal site of
Vam3p function.
Mutant Vam3 Proteins Are Missorted into the CPY Pathway
The ability of the vam3 mutants to suppress pep12 vacuolar protein sorting defects suggests that mutant Vam3
proteins are at least partially localized to the pre-vacuolar
endosome, the site of Pep12p function. To examine the localization of the Vam3 mutant proteins, we fractionated
cells and examined the distribution of the Vam3 mutant
proteins relative to wild-type Vam3p by differential centrifugation. Vam3p fractionates exclusively in a low speed (P13) pellet fraction by differential centrifugation, which is consistent with its vacuolar localization (Darsow et al.,
1997
). However, the mutant forms of Vam3p fractionated
differently than the wild-type protein, with a significant
portion of the proteins localized to a high speed P100 pellet, (data not shown) which indicated that at least a portion of the mutant proteins localized to a non-vacuolar
fraction. Although the P13 fraction is enriched in vacuoles,
other compartments also fractionate in the P13. For example, the endosomal t-SNARE Pep12p typically exhibits a
40% P13/60% P100 fractionation pattern, even though this
protein is not localized to the vacuole (Becherer et al.,
1996
). To further resolve vacuoles away from other P13
material, vam3
cells harboring either single-copy wild-type VAM3 or the vam3L160P mutant were analyzed by
equilibrium density gradient fractionation. Cleared spheroplast lysates were applied to the top of Accudenz step gradients, which were then centrifuged to equilibrium. Fractions were collected from the top of the gradient and
analyzed for the presence of Vam3p, Vph1p, and the endosomal t-SNARE, Pep12p. In both gradients, Vph1p
fractionated primarily in the low density fractions (1-4) of
the gradient, as is typical for vacuolar membrane proteins
(Darsow et al., 1997
) (Fig. 4 A). In contrast, Pep12p fractionated exclusively in more dense regions of the gradient (fractions 5-7), indicating that distinct separation of vacuoles and endosomes was achieved in these gradients (Fig. 4
B). As expected, wild-type Vam3p primarily co-fractionated with Vph1p in the first four fractions of the gradient.
However, while a small percentage of Vam3pL160P also
fractionated in the top (vacuolar) region of the gradient, the majority of the mutant Vam3 protein (~80%) was recovered in more dense fractions (5-7), which also contained Pep12p (Fig. 4 B). Thus, while some Vam3pL160P is
localized to the vacuole, most of the protein appears to reside in a non-vacuolar compartment that co-fractionates
with Pep12p-containing endosomes.
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The cell fractionation data suggested that a significant
portion of the mutant Vam3 proteins were mislocalized to
compartments other than the vacuole. To visualize the
compartments in which the mislocalized proteins were residing, we constructed GFP fusions with wild-type and mutant Vam3 proteins. We transformed the plasmid constructs into vam3 cells and examined the cells by
fluorescence microscopy. Both the wild-type and mutant
Vam3 fusion proteins complemented the protein sorting
(data not shown) and morphology defects associated with
vam3
cells (Fig. 5 A) and thus functioned as the native
proteins. Consistent with the previous localization studies,
wild-type GFP-Vam3 fusion protein localized almost exclusively to vacuolar membranes (Fig. 5 A). While GFP-Vam3L160P still accumulated in vacuolar membranes, a
large portion of the protein was also seen in tubular and
punctate structures throughout the cell (Fig. 5 A). GFP fusion proteins of the other Vam3p mutants also behaved in
a similar manner (data not shown), indicating that these
mutant proteins accumulate in non-vacuolar structures.
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Class E vps mutants contain large aberrant endosomal
structures (class E compartments), which accumulate both
biosynthetic cargoes (e.g., CPY) as well as endocytic cargoes (e.g., the lipophilic dye FM4-64) (Rieder et al., 1996).
However, proteins that travel through the AP-3-dependent pathway (e.g., ALP, Vam3p) do not accumulate in
the class E compartment, as this pathway bypasses the endosome (Babst et al., 1997
; Piper et al., 1997
). Unlike wild-type Vam3p, if the mutant Vam3 proteins travel through
the CPY pathway to the vacuole, they should accumulate
in the class E compartment. We transformed class E mutant vps24
cells with plasmids encoding either GFP-Vam3 or GFP-Vam3L160P mutant fusion protein, labeled
the cells with FM4-64, and examined the cells for both
markers by fluorescence microscopy. Wild-type GFP-Vam3 fusion protein accumulated primarily on vacuolar
membranes and was excluded from the class E compartment (Fig. 5 B). In contrast, a large percentage of the cells
expressing GFP-Vam3pL160P displayed brightly fluorescent
signal in a perivacuolar compartment, in addition to a
lower intensity fluorescent signal on the vacuolar membrane (Fig. 5 B). FM4-64 counterstaining showed that this
perivacuolar compartment was coincident with the class E
compartment. GFP fusion proteins made with the other
Vam3p mutants also accumulated in the E compartment
in a similar manner (data not shown), indicating that mislocalization through the CPY pathway occurred in all of the mutants. Together, these localization data provide
compelling evidence that the mutant proteins do not traffic to the vacuole through the AP-3-dependent pathway
but instead transit the CPY pathway and accumulate in
pre-vacuolar compartments.
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Discussion |
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We have described a novel genetic screen for mutations in
the vacuolar t-SNARE Vam3p that allow it to functionally
replace the endosomal t-SNARE, Pep12p. SNARE proteins would appear to be one of the most important cargoes to be selectively sorted, as they represent a core component of the vesicle docking/fusion machinery. The
correct sorting of many other cargo proteins depends on
proper localization of these t-SNARE molecules. Here we
provide the first identification of a defined sorting signal in
a t-SNARE protein. Previous studies have indicated that
both transmembrane and cytosolic domains of t-SNAREs
contain information that influences their localization
(Banfield et al., 1994; Rayner and Pelham, 1997
). However, these studies did not define primary sequence sorting
signals, nor did they identify specific transport components that may be involved in the sorting of these proteins
(e.g., sorting receptors). This study provides significant, in
vivo evidence that Vam3p, like ALP, is sorted by recognition of an acidic di-leucine motif through an AP-3-dependent pathway. Furthermore, disruption of the di-leucine
sorting signal in Vam3p results in mislocalization of
Vam3p to the pre-vacuolar endosome where it functionally substitutes for the endosomal t-SNARE Pep12p.
These results suggest that the trafficking route of a SNARE
acts to restrict its site of function and that localization plays
an important role in determining the functional specificity
of a given SNARE protein. Therefore, trafficking of Vam3p
through the AP-3/ALP pathway plays a vital role in maintaining the unique composition and function of the vacuole.
Furthermore, these observations provide insight into the
requirement for the AP-3-dependent alternative Golgi-to-vacuole transport pathway.
Vam3p Contains a Putative Di-leucine-type Sorting Signal That Directs It into the ALP Pathway
The only known cargo of the AP-3-dependent sorting
pathway to the vacuole in yeast is ALP (Cowles et al.,
1997a; Stepp et al., 1997
). However, several lines of evidence suggest that Vam3p also traffics through this route.
First, Vam3p localizes to the vacuole in a manner independent of the function of Pep12p (Cowles et al., 1997b
) and
the class E Vps proteins Vps4p and Vps27p, and is specifically excluded from the class E compartment (Babst et al.,
1997
; Piper et al., 1997
), suggesting that it does not transit
through the pre-vacuolar endosome in the CPY pathway
en route to the vacuole. Furthermore, in an AP-3 mutant,
Vam3p is mislocalized to a non-vacuolar compartment in
the same manner as ALP (Cowles et al., 1997a
). A di-leucine sorting signal in the cytosolic tail of ALP has been
shown to be necessary for AP-3-dependent sorting of
ALP to the vacuole (Cowles et al., 1997b
; Vowels and Payne, 1998
). While Vam3p contains multiple putative di-leucine sequences within its cytoplasmic domain, we have
found through a random mutagenesis screen that disruption of only one of the potential di-leucine signals in
Vam3p alters its trafficking route and allows it to function
at a non-vacuolar compartment. Furthermore, deletion of
AP-3 complex components results in similar phenotypes as disruption of the di-leucine sequence of Vam3p. Together, these data strongly suggest that Vam3p traffics
through the AP-3-dependent ALP pathway via recognition of a di-leucine motif. It was recently shown that di-leucine sorting signals in the mammalian proteins LIMP
II, a lysosomal membrane protein, and tyrosinase, a melanosomal membrane protein that functions in melanin synthesis, bind to AP-3-enriched fractions in an in vitro surface plasmon resonance (SPR) assay, which is suggestive
of direct interactions between di-leucine motifs and AP-3
(Honing et al., 1998
). Our work provides strong in vivo evidence that di-leucine motifs are required for directing
cargo into the AP-3-mediated transport pathway in yeast.
Vam3p Mutants Reveal a Role for Additional Residues in the Di-leucine Motif
Random mutagenesis of Vam3p identified a small 7-amino
acid region in the middle of the cytoplasmic domain of
Vam3p that includes a leucine pair as well as several
amino acids just to the NH2-terminal side of the di-leucine
sequence. Because di-leucine sorting signals have been implicated in endocytosis from the plasma membrane (Letourneur and Klausner, 1992; Pond et al., 1995
) and also
have been shown to be capable of direct binding to both
AP-1 and AP-2 in vitro (Heilker et al., 1996
; Dietrich et al.,
1997
; Rapoport et al., 1998
), it is likely that only a subset
of di-leucine motifs interact with AP-3. Mutations in the
glutamate at position
4 relative to the leucine pair in
Vam3p resulted in particularly strong phenotypes, comparable to mutations in either of the leucine residues. These results are consistent with in vitro studies that have shown
that mutation of the acidic residues in the di-leucine sorting signals in LIMP II and tyrosinase abrogate binding to
AP-3-enriched fractions (Honing et al., 1998
). However,
acidic residues in addition to the di-leucine pair do not
seem to be sufficient to direct AP-3 binding, since many
mammalian proteins containing similar sequences do not
seem to be sorted through AP-3-dependent pathways
(Honing et al., 1998
). Our analysis has identified several
additional residues that also contribute to the function of
the di-leucine motif. In addition to the acidic residue at
4
and the leucine pair, the Vam3p motif requires an upstream polar residue at
5 and a hydrophilic amino acid at
the
3 position for optimal sorting activity. In fact, the
sorting determinant in ALP contains conserved residues at
the same positions to each of the additional residues that
were mutated in Vam3p (Fig. 6).
|
Similar sequence characteristics can also be found in
many of the other candidate AP-3 cargoes that have been
identified in mammalian systems. Melanosomal resident
enzymes have been implicated as cargo for AP-3-directed
sorting, as mutant mice in both AP-3 components and melanosomal proteins result in similar aberrant coat color phenotypes, suggesting that these pigmentation defects may
be due to improper sorting of proteins to melanosomes
(Odorizzi et al., 1998). Di-leucine motifs in many melanosomal proteins also contain a conserved hydrophilic amino
acid at the
3 position (Fig. 6). In addition, mutation of
residues at the same positions in proteins such as invariant
chain, which do not bind to AP-3, have been shown to
have no effect on the transport of these proteins (Motta et
al., 1995
; Pond et al., 1995
; Honing et al., 1998
). Both the
Vam3p di-leucine sequence and the di-leucine sequences in many of the other potential AP-3 cargoes contain a conserved proline at the
1 position. However, we did not recover mutations in this residue and it is not conserved in
the ALP sequence, suggesting that this proline may not be
required for the function di-leucine sorting signals, at least
in yeast. Together, these data point to a conserved sorting
motif for most AP-3-dependent cargoes and a common
mechanism for the recognition and packaging of these cargoes. Further mutational analysis of conserved amino acids in other vacuolar/lysosomal and melanosomal protein
sequences will confirm the importance of these residues in
AP-3-directed sorting. Sequence search algorithms derived from such analysis may also be useful in identifying
new potential cargo proteins in the AP-3 pathway and may
help to further define the pathway's biological significance.
Disruption of the Vam3p Di-leucine Motif Causes Mislocalization of Vam3p into the CPY Pathway
The CPY pathway seems to be the "default" route to the
vacuole, as no sorting signals required for transport of
membrane proteins into this pathway have been defined
and proteins that normally transit through this pathway
can be diverted into other pathways by the addition of
positive sorting signals (Cowles et al., 1997b). Furthermore,
overexpression of either ALP or Vam3p results in their
overflow into the CPY pathway, consistent with saturation of the signal recognition/packaging machinery in the AP-3
pathway (Cowles et al., 1997b
; Darsow et al., 1997
). We
have shown that disruption of the di-leucine sorting signal
in Vam3p results in the mislocalization of the protein into
the CPY pathway. It has been previously shown that
Vam3p is missorted in AP-3 mutants and co-fractionates
with late Golgi and endosomal markers (Cowles et al.,
1997a
). Consistent with these observations, we found that Vam3p sorting signal mutants accumulate in a Pep12p-containing intermediate compartment, most likely a pre-vacuolar endosome. The following observations support
this model: (a) the Vam3 mutant proteins are capable of
suppressing the defects of pep12
cells, which argues that
the proteins are reaching a compartment where Pep12p
normally operates, (b) the mutant proteins co-fractionate with Pep12p and are in a distinct location from vacuolar
markers in fractionation experiments and finally, and (c)
the accumulation of the Vam3 mutant proteins in a class E
mutant endosome implies that the proteins are not being
specifically retained in the Golgi complex but instead are
being transported forward at least until the class E block is
initiated, which is thought to be at the point of exit from
the endosomal compartment (Babst et al., 1997
). Together, these data suggest that a large pool of the Vam3
mutant proteins reside at steady state in a pre-vacuolar endosome.
The localization data also indicate that only a minor
fraction of the mutant proteins arrive at the vacuole
(~20%), and thus, that transport of Vam3p to the vacuole
through the CPY pathway is inefficient. It is therefore possible that mechanisms may exist which retain the majority
of Vam3 mutant protein in a stable, pre-vacuolar compartment. In wild-type cells, Pep12p is also retained within the
pre-vacuolar endosomal compartment, possibly by a similar mechanism. Perhaps within their highly conserved primary sequence, Pep12p and Vam3p contain retention motifs that maintain their localization in the endosome, either
by preventing forward transport or through recycling. Alternately, interactions with compartment-specific SNARE
accessory proteins that function at the pre-vacuolar endosome, such as Vps45p, Vps21p, or Vac1p (Cowles et al.,
1994; Horazdovsky et al., 1994
; Burd et al., 1997
), could act
to retain both Pep12p and the mutant Vam3 proteins in
the endosome. This would be consistent with our previous
findings that Vam3p-specific suppression of pep12
requires expression of endosomal transport components
(Darsow et al., 1997
).
AP-3-mediated Localization Restricts the Function of the Vam3p t-SNARE Protein
It has previously been shown that overexpression of
VAM3 can partially suppress the vacuolar protein sorting
defects of a pep12 null mutant and that this activity may be
dependent on mislocalization of Vam3p to the Pep12p
compartment (Darsow et al., 1997; Gotte and Gallwitz,
1997
). Our results presented here confirm that mislocalization of Vam3p allows it to efficiently substitute for
Pep12p. While we can not rule out that Vam3 mutant proteins have increased affinity for Pep12p-specific components (e.g., Vps45p), the data are most consistent with a
model in which these mutations result in quantitative missorting of Vam3p to the pre-vacuolar endosome where it
can efficiently substitute for Pep12p.
The finding that in the absence of a functional di-leucine
motif, Vam3p both localizes and functions at an earlier
step in the CPY pathway has several interesting implications for the function of t-SNARE proteins. Both Vam3p
and Pep12p act in what appear to be classical SNARE-
mediated transport steps. They both require specific accessory proteins such as Rab and Sec1 homologues, as well as
the general cytosolic fusion machinery, including Sec18p (NSF), for their activity (Burd et al., 1997; Darsow et al.,
1997
; Sato et al., 1998
). By simply driving Vam3p into a
different biosynthetic transport pathway, as may be accomplished either by Vam3p overexpression, disruption of
the Vam3p sorting signal, or by disruption of the sorting
machinery itself, Vam3p is able to function at a different compartment in the Golgi-to-vacuole transport pathway.
These results suggest that a major distinguishing characteristic between Pep12p and Vam3p is their biosynthetic
transport route and localization. Current models for
SNARE function postulate that cognate interactions between t- and v-SNAREs define the primary specificity of
each transport step. However, our observations are most
consistent with a model in which SNARE proteins are
much more promiscuous in their interactions with accessory proteins and, when localized improperly, are able to
partially function at alternate sites. For example, although
Vam3p normally acts in conjunction with the vacuolar
Sec1 homologue, Vps33p, the suppression of pep12
by
VAM3 overexpression also requires Vps45p, the endosomal Sec1 homologue (Darsow et al., 1997
). In this case,
Vam3p appears to use the docking and fusion machinery
of the endosome rather than recruiting these accessory
proteins from the vacuole. Clearly then, t-SNARE proteins do not define the sole component of specificity of vesicular transport, and other compartment specific proteins must also be present and act with the t-SNARE to accomplish this goal. It remains to be determined which proteins,
or combination of proteins are ultimately responsible for
transport specificity, but it seems likely that a set of complex interactions at each transport step may be required
for accurate vesicular transport.
![]() |
Footnotes |
---|
Received for publication 20 May 1998 and in revised form 15 July 1998.
Address all correspondence to Scott D. Emr, Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0668. Tel.: (619) 534-6462. Fax: (619) 534-6414. E-mail: semr{at}ucsd.eduWe thank both past and present members of the Emr lab, especially E. Gaynor, M. Babst, and B. Wendland for helpful comments and for critical reading of this manuscript. We would also like to thank G. Odorizzi for strains used in this study and D. Katzmann for assistance with the confocal microscopy.
This work was supported by grants GM32703 and CA58689 from the National Institutes of Health to S.D. Emr. S.D. Emr is an Investigator of the Howard Hughes Medical Institute.
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Abbreviations used in this paper |
---|
ALP, alkaline phosphatase; CPY, carboxypeptidase Y; GFP, green fluorescent protein; NSF, N-ethylmaleimide-sensitive factor; ORF, open reading frame; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; VPS, vacuolar protein sorting.
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