From the Department of Cell Biology and the
§ Howard Hughes Medical Institute, Yale University School of
Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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Intracellular membrane fusion events in eukaryotic cells are thought to be mediated by protein-protein interactions between soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins. We have identified and analyzed a new yeast syntaxin homolog, Tlg2p. Tlg2p is unique among known syntaxin family proteins in possessing a sizeable hydrophilic domain of 63 amino acids that is C-terminal to the membrane spanning region and nonessential for Tlg2p function. Tlg2p resides on the endosome and late Golgi by co-localization with an endocytic intermediate and co-fractionation with markers for both endosomes and late Golgi. Cells depleted for Tlg2p missort a portion of carboxypeptidase Y and are defective in endocytosis. In addition, we report that Tlg2p forms a SEC18-dependent SNARE complex with Snc2p, a vesicle SNARE known to function in Golgi to plasma membrane trafficking. Based on these findings we propose that Tlg2p is a t-SNARE that functions in transport from the endosome to the late Golgi and on the endocytic pathway.
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INTRODUCTION |
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The intracellular flux of lipids and proteins that constitutes vesicular trafficking in eukaryotic cells is a highly regulated process. The correct sorting of these components defines cellular compartmentalization and is thus vital for cellular function (1). Our understanding of the control of intracellular membrane fusion has been highlighted in recent years by the formulation of the SNARE1 hypothesis. Integral membrane proteins residing on different membrane-bound compartments (e.g. synaptic vesicles and plasma membrane) called t- and v-SNAREs are able to interact, and this interaction and its function are conserved from yeast to man (2-5). The complex formed between SNAREs is thought to span the lipid bilayers of both interacting compartments and to play a pivotal role in the docking and fusion processes. In agreement with this suggestion, a number of toxins that abrogate synaptic transmission do so by site-specific proteolysis of SNARE proteins (6-8), and antibodies to SNARE complex proteins inhibit in vitro membrane fusion assays (9). Complex formation between t-SNARE proteins on the target membrane and v-SNARE proteins on the vesicle membrane is thought to be modulated by a cycle of multiprotein complex assembly and disassembly that involves a number of accessory proteins (2). NSF, a multimeric ATPase, interacts with SNAREs via soluble NSF attachment proteins in an ATP-dependent manner. Hydrolysis of ATP by NSF induces conformational changes in SNAREs that lead to the disassembly of the SNARE complex. Recent data have indicated that this cycle may function at a point prior to docking, in rearranging intramembrane, inactive SNARE complexes (10). A basic implication of the SNARE hypothesis is that the fate of any patch of membrane, be it a transport vesicle or cellular compartment, is determined at least in part by the set of v- and/or t-SNAREs that are present on its surface (3). As such, an interesting question arises as to the precise fashion by which SNARE content correlates with membrane flux. It was originally suggested that t-SNAREs and v-SNAREs interact in a specific and exclusive fashion (11, 12) and that this specificity underlies the fidelity of vesicular transport. It has since been demonstrated, however, that a single v-SNARE can function at two different transport steps, with different t-SNAREs specific for each transport step (13). These findings have been accommodated within the SNARE model by suggesting that specificity arises out of additional, uncharacterized interactions that modulate the t-SNARE/v-SNARE coupling.
In the endocytic vacuolar system of Saccharomyces
cerevisiae, the endosome (also known as the prevacuolar
compartment) is a point of convergence for membrane traffic originating
in the Golgi complex and in the plasma membrane (14, 15). It has been
shown that Pep12p, a yeast protein that is homologous to the
prototypical mammalian t-SNARE syntaxin, acts as a t-SNARE required for
docking and/or fusion of Golgi-derived vesicles with the
endosome/prevacuolar compartment (16, 17). It has also been shown that
delivery of pheromone receptors to the prevacuolar compartment requires
the action of the NSF ATPase, Sec18p (18). In this paper we describe a
new syntaxin homologue, Tlg2p, which fractionates in a bimodal fashion
with protein markers for the endosome and late Golgi compartments in
density gradients and is required for efficient endocytosis. Unlike
Pep12p, tlg2 cells do not accumulate the p2 form of the
vacuolar protease carboxypeptidase Y (CPY) but missort a fraction of p2
CPY to the cell surface. We suggest that Tlg2p is a t-SNARE that is
required in trafficking from the prevacuolar compartment to the late
Golgi or alternatively for endocytosis-related membrane fusion
events.
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EXPERIMENTAL PROCEDURES |
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Materials-- Enzymes used in DNA manipulations were purchased from New England Biolabs (Beverly, MA) or Boehringer Mannheim Biochemicals. ECL reagents, 125I-protein A, horseradish peroxidase-labeled streptavidin, and 35S translabel were from Amersham Pharmacia Biotech. Nitrcellulose type HA85 paper was obtained from Schleicher & Schuell. Antiserum to yeast carboxypeptidase Y was a gift from Peter Novick. Zymolyase 100T (Kirin Brewery) was purchased from Seikagaku (Tokyo, Japan). Antisera to Pep12p and to Kar2p were kindly donated to us by Scott Emr and Mark Rose, respectively. Anti-Ste3p antiserum was a gift from G. Sprague. Lucifer Yellow-CH was from Fluka (Buchs, Switzerland). Protein A-Sepharose was from Amersham Pharmacia Biotech and Sigma Immunochemicals. Pronase and Biotinyl-anti-HA antibodies were from Boehringer Mannheim. Other chemicals were from Sigma, standard sources, or as indicated.
Strains, Media, and Microbiological Techniques--
The strains
used in this study are listed in Table I.
Strains were constructed by standard genetic techniques and grown in rich YEPD medium (1% yeast extract, 1% peptone, 2% dextrose), galactose-based rich YEPG medium (1% yeast extract, 1% peptone, 4%
galactose), or standard minimal medium (SD) with appropriate supplements as described by Sherman et al. (47). YEPD was
supplemented with 1 M NaCl for high salt selection assays.
Strain HAY31 was constructed by transforming SFNY 562 with the
tlg2::URA3 cassette. Disruption of the
TLG2 gene was confirmed by PCR analysis and immunoblotting.
To perform the blots, cell extracts (0.5 A600 cell equivalents) were resolved by SDS-PAGE (10%) and transferred onto
nitrocellulose by semi-dry immunoblotting. The immunoblot was probed
with anti-Tlg2p antibody at 1:1000 dilution, and the immune complexes
were detected with 125I-protein A (Amersham Pharmacia
Biotech).
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Plasmid Construction-- Genomic DNA isolated from SFNY 562 yeast was used as template in a PCR reaction to amplify an XbaI-SalI fragment carrying the TLG2 gene. The PCR product was digested, gel purified, and inserted into pRS305 and pRS315 or pRS316 (19) at the XbaI-SalI sites to form plasmids pHAB1, pHAB2, and pHAB36, respectively. Plasmid pHAB6 was constructed by first amplifying an in vitro, PCR-based recombination product that was obtained using the Splicing by Overlap Extension technique described in Ref. 20 (gene SOEing). Using two nested gene SOEing reactions, the TLG2 promoter region was fused to the GFP open reading frame, and the product of this reaction was in turn fused in-frame with the TLG2 coding sequence and 3' untranslated region. The flanking regions of this construct are identical with those of the XbaI-SacI fragment mentioned above, and the linear PCR product was inserted into pRS305 as before. Plasmid pHAB15 was constructed by cloning an Eco47III-BclI PCR amplified URA3 gene into the Eco47III and BclI sites of the TLG2 gene, in pHAB36. Plasmid pHAB16 was constructed by cloning a PCR product encoding amino acids 1-250 of Tlg2p into pET14b (Novagen). Plasmid pEG111 is an integrating vector containing the HA-SNC2 gene cloned between the GAL1 promoter and the ADH terminator. Triple HA tagging of the SNC2 gene at the C terminus was according to the method of Schneider et al. (21). The HA-SNC2 gene was PCR amplified using a PstI primer 49 base pairs upstream of the 5' end of the coding sequence and a HindIII primer 1351 base pairs downstream of the 3' end of the coding sequence. The PCR product was subcloned into pNRB529, a pRS305-based yeast expression vector containing GAL1 promoter and ADH terminator sequences. Site-directed mutagenesis to delete the C-terminal hydrophilic domain of Tlg2p was according to Kunkel et al. (22), using the Muta-Gene kit (Bio-Rad). All constructs were verified by DNA sequencing according to the Sanger dideoxy nucleotide method.
Recombinant Tlg2p Purification and Antibody
Production--
Plasmid pHAB16 was transformed into BL21 cells
(Stratagene), isopropyl-1-thio--D-galactopyranoside
induction of His6-tagged recombinant Tlg2p was confirmed by
Coomassie staining of acrylamide gels. Recombinant His6
Tlg2p (5 mg) was purified to homogeneity from 1 liter of
logarithmically growing bacterial cells using nickel-chelate
chromatography, following the pET system manual (Novagen). Antisera
were raised in New Zealand White rabbits by Cocalico, Inc. (Reamstown,
PA).
CPY Maturation Studies-- CPY maturation was followed essentially as described previously (23). Briefly, 4 OD units of cells from a culture grown to A600 of 0.5 were collected, treated for 10 min at room temperature with 100 mM Tris, pH 9.4, 10 mM dithiothreitol, washed, and resuspended in spheroplasting medium (SD plus nutrients, 1.2 M sorbitol, 1 mg/ml bovine serum albumin, 200 µg/ml zymolyase 100T). The cells were incubated with gentle agitation at 25 °C for 30 min. Spheroplasts were collected, washed with SD medium supplemented with 1.2 M sorbitol and auxotrophic requirements, and incubated in the same medium at 25 °C for 10 min. Labeling was initiated by adding 200 µCi 35S PRO-MIXTM ([35S]methionine and [35S]cysteine at 14.4 mCi/ml; Amersham Pharmacia Biotech). After a 5-min labeling period, chase was initiated by adding cysteine and methionine to 1 and 5 mM respectively, plus 0.2% yeast extract and 4% glucose (time 0).
Samples of 1 A600 cell equivalent in 100 µl were taken at 0, 10, 30, and 90 min after initiation of chase, brought to 5 mM NaN3, and cooled in ice. Samples were spun to separate intracellular and extracellular fractions. The spheroplast pellets were brought to 100 µl with SD supplemented with 1.2 M sorbitol and 5 mM NaN3, and all fractions were immediately precipitated with 10% trichloroacetic acid, 0.015% deoxycholate. The precipitates were washed twice with acetone, resuspended in SDS boiling buffer (50 mM Tris, pH 7.5, 1% SDS, 1 mM EDTA) and boiled for 5 min. The lysates were diluted with dilution buffer (50 mM Tris, pH 7.5, 2% Triton X-100, 0.1 mM EDTA, 150 mM NaCl) and immunoprecipitated with anti-CPY antibody. The immunoprecipitates were resolved by SDS-PAGE and 35S-labeled CPY was visualized and quantified using a Molecular Dynamics PhosphorImagerTM and ImageQuantTM software. The colony-lift immunoblotting technique (24) was used to assay gross CPY release from cells.Lucifer Yellow Uptake and Ste3p Endocytosis Assays-- Lucifer Yellow uptake was assayed as described previously (25). Briefly, 1 ml of logarithmically growing yeast cells cells (5 × 106 cells/ml in YEPD) were collected and resuspended in 900 µl of YEPD. 100 µl of Lucifer Yellow stock solution was added to a final concentration of 4 mg/ml, and the cells were incubated for 1 h at 25 °C with vigorous shaking. The cells were then washed three times in ice-cold buffer (50 mM succinate-NaOH, pH 5.0, 20 mM NaN3) and mounted in 0.8% low melt agarose. The cells were observed using a Zeiss axiophot microscope equipped with fluorescence and Nomarski optics. Photographs were taken using Kodak T-Max 400 film (Eastman Kodak Co.). Ste3p endocytosis and Pronase sensitivity assays were done as described by Davis et al. (26).
GFP Fluorescence Imaging--
For viewing GFP-tagged proteins,
logarithmically growing cells were fixed by suspension in 20 °C
methanol followed by
20 °C acetone and finally resuspended in TE
(10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Cells were
mounted in 0.8% low melting point agar and viewed using a Zeiss
Axiophot microscope equipped with a 100× Apochromat objective. For
fluorescence imaging of living cells, cells were mounted in YEPD medium
containing 0.8% low melting point agarose and viewed using a Zeiss
Axiovert 10 microscope coupled to a Bio-Rad MRC600 confocal laser
apparatus and equipped with a 63× Apochromat objective.
SNARE Complex Immunoprecipitations-- Cell were grown to early log phase in YEPD or YEPG medium, pelleted, and resuspended in medium pre-warmed to 25 or 37 °C. After a 10-min incubation, the cells were chilled to 4 °C by dilution into 10 volumes of ice-cold Tris buffer (10 mM, pH 7.4) and collected by centrifugation. The pellets were resuspended in HKDNE buffer (50 mM Hepes, pH 7.4, 150 mM KCl, 1 mM dithiothreitol, 0.5% Nonidet P-40, 1 mM EDTA) supplemented with 1 mM phenylmethylsulfonyl fluoride and then homogenized by vortexing for 3 min with acid washed glass beads. The homogenates were diluted to 1 mg/ml protein and then cleared by centrifugation for 15 min at 16,000 × g. Affinity purified anti-Tlg2p antibody was added to the supernatant and incubated for 6 h at 4 °C. The immunoprecipitates were collected on protein G-Sepharose beads, washed five times with HKDNE buffer, electrophoresed on an SDS 12% polyacrylamide gel, and transferred to nitrocellulose. The protein blot was probed with an anti-Snc1p antisera that cross-reacts with Snc2p (27) and detected by ECL. HA-Snc2p, which comigrates on the gel with antibody light chains, was detected with biotinylated 12CA5 (anti-HA) antibody and streptavidin-horseradish peroxidase.
FM4-64 Uptake-- FM4-64 uptake was essentially as described (28). Briefly, cells were grown to an A600 of 0.5 and 10 OD units were collected and resuspended in 5 ml of ice-cold YEPD containing 0.6 µM FM4-64 (Molecular Probes). After a 30-min incubation on ice with shaking, the cells were collected again and resuspended in 5 ml of ice-cold YEPD. The cells were then incubated at 15 °C for an additional 30 min for visualization of uptake intermediates or at 30 °C for vacuolar staining. Cells showing punctate staining after incubation at 15 °C were further incubated for 20 min at 30 °C to produce vacuolar staining patterns. Cells were mounted in 0.8% low melting agarose and visualized as above.
Yeast Cell Fractionation-- 600 A600 units of logarithmically growing yeast culture in YEPD were spun in the presence of 10 mM NaN3 and resuspended in 100 mM Tris, pH 9.4, 10 mM dithiothreitol, and 10 mM NaN3. The cells were incubated at room temperature for 10 min, collected, and resuspended in 12 ml of spheroplasting buffer (SPB buffer: 1.5 M sorbitol, 50 mM Tris-Cl, pH 7.2, 2 mM MgCl, 10 mM NaN3, and 0.2 mg/ml Zymolyase 100T) and incubated for 60 min at 30 °C with gentle shaking. Spheroplasts were cooled on ice (all subsequent handling was done at 4 °C unless stated otherwise) and centrifuged for 5 min at 700 × g through a cushion of SPB buffer without zymolyase and containing 1.9 M sorbitol.
The pellet was resuspended in 12 ml of lysis buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 0.2 M sorbitol, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml each of leupeptin, chymostatin, aprotinin, pepstatin A, and antipain), and the cells were lysed with a Dounce homogenizer (20 strokes). Lysates were cleared by centrifugation at 700 × g for 5 min. Low speed supernatant and low speed pellet were generated by a 15-min centrifugation at 13,000 × g. The low speed supernatant fraction was centrifuged for 1 h at 100,000 × g to generate high speed supernatant and high speed pellet fractions. Equivalent volumes of each fraction were used for SDS-PAGE and immunoblot analysis. 1 ml of high speed pellet fraction (120 A600 unit equivalents) in lysis buffer was loaded on a sucrose step gradient and fractionated as described (16). Tlg2p and Pep12p were detected by standard immunoblot technique. Kex2p activity was assayed according to Fuller et al. (29) using t-butoxycarbonyl-Gln-Arg-Arg 4-methylcoumarin-7-amide as substrate and following the evolution of 7-amino-4-methylcoumarin by fluorescence (385 nM excitation wavelength, 465 nM emission wavelength). One unit of Kex2p activity was defined as 1 pmol of 7-amino-4-methylcoumarin released per minute.Protease Protection Assays-- Spheroplast preparation was as described above. Spheroplasts were lysed by Dounce homogenizing in 0.3 M mannitol, 0.1 M KCl, 50 mM Tris, pH 7.5, 1 mM EGTA, and clarified extracts were centrifuged at 100,000 × g for 1 h. Protease protection assays (16) were conducted on the 100,000 × g pellet fraction. Membrane pellets (80 µg of protein) were resuspended in lysis buffer and incubated with combinations of 0.29 mg/ml proteinase K and/or 0.4% Triton X-100 for 5 min, and then the reactions were quenched with 10% trichloroacetic acid. Samples (15 µg of protein) were resolved by SDS-PAGE and analyzed by immunoblotting for Kar2p and Tlg2p.
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RESULTS |
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The TLG2 Gene Encodes a Syntaxin-like Protein--
We scanned the
yeast data base for proteins showing similarity to the conserved
juxta-membrane coiled-coil domain of Sed5p, a known syntaxin-like yeast
t-SNARE that is localized to the Golgi and required for membrane
traffic between the endoplasmic reticulum and Golgi complex (30). An
open reading frame (YOL018c), located on chromosome XV, was found to
encode a novel syntaxin-like protein. Unlike other members of the
syntaxin family, the predicted transmembrane domain encoded by this
sequence is not at the C terminus of the open reading frame. Rather, it
is followed by a hydrophilic stretch of 63 amino acids that shows no
obvious homology to known proteins or motifs. Pep12p and Vam3p are the
highest scoring yeast proteins that are found when searching the data
base with the BLAST program using the new open reading frame as query
(P scores of 7.9e19 and 1.3e
11,
respectively). During preparation of this manuscript, YOL018c was
designated Tlg2p in the yeast data base, and we have chosen to follow
this nomenclature. The similarity between Pep12p, Vam3p, and Tlg2p is
illustrated in Fig. 1. It is evident that
these three proteins share features beyond those within the conserved
membrane-proximal coiled-coil domain that defines the syntaxin family
of proteins (31). For example, all three proteins have transmembrane
domains of 18 amino acids and show a high degree of sequence identity within the transmembrane domain region. Although the transmembrane domain is constrained to hydrophobic amino acids, its length, amino
acid composition, and sequence have been shown to be important in
localization and function of t-SNAREs (32).
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Tlg2p Is a Nonessential Protein That Is Required for Growth on High
Salt and for the Efficient Sorting of CPY--
To disrupt
TLG2, SFNY562 diploid yeast were transformed with the
XbaI-SalI TLG2 disruption cassette. Dissection of
tetrads from these cells gave four viable spores, and the absence of
Tlg2p from the Ura+ haploid segregants was confirmed by
Western blotting. The high degree of similarity between Tlg2p and
t-SNAREs of the vacuolar endocytic pathway led us to ask if
tlg2 yeast show phenotypes characteristic of
abnormalities in those cellular functions. Yeast defective in vacuolar
endocytic trafficking often show sensitivity to osmotic stress (33) and
defects in the sorting of vacuolar proteases (23, 33). In 40 tetrads
scored, the Ura+ phenotype marking the tlg2
disruption segregated 2:2 and co-segregated with a growth defect at
ambient temperatures (20-25 °C) on rich medium containing 1 M NaCl (Fig. 2a).
Colony filter assays for CPY missorting showed that these same
segregants also released CPY to the extracellular medium. In addition,
light microscopic analysis revealed that tlg2
yeast had
abnormally fragmented vacuoles. Detection of extracellular CPY by the
colony filter assay could be the consequence of cell lysis, secretion
of the late Golgi (p2) form, or some other mechanism. The mobility of
CPY in SDS gels can be used as a gauge of intracellular location within
its biosynthetic route in the cell. Upon translocation to the lumen of
the endoplasmic reticulum, CPY is glycosylated by the addition of four
N-linked core oligosaccharides, and this precursor form of
the protein, called p1 CPY, migrates as 67 kDa in SDS-PAGE. As CPY
transits through the later compartments of the Golgi complex, it
acquires additional mannose residues to give rise to p2 CPY, which
migrates at 69 kDa. Upon delivery to the vacuole, the 90-amino acid
propeptide on p2 CPY is proteolysed, giving rise to the 61-kDa mature
form of the protein (mCPY) (34). We performed pulse-chase analysis of
CPY maturation to understand the origin of the extracellular CPY in
tlg2
cells. Spheroplasts were formed, pulsed for 5 min with 35S-PRO-MIXTM, and chased for various times as
described under "Experimental Procedures." We found that CPY
maturation in tlg2
cells is not blocked. Although
tlg2
cells were slightly delayed in the appearance of the
p2 form at very early time points (not shown), they processed p2 CPY to
the mature vacuolar form within the same time frame as that of wild
type cells (Fig. 2c). However, approximately 20% of the
total CPY synthesized was secreted from these cells as the
Golgi-modified p2 form.
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tlg2 Yeast Are Deficient in Lucifer Yellow Uptake and Show a
Delay in Ste3p Turnover--
Because a number of vacuolar protein
sorting mutants that missort CPY are also deficient in endocytosis, we
examined tlg2
mutant cells for defects in aspects of
endocytosis. Yeast cells constitutively take up soluble material from
the medium and transport it through the endocytic vacuolar system. This
process can be studied using Lucifer Yellow, a fluorescent dye that is
an established marker for fluid phase endocytosis (25). Wild type cells
incubated with Lucifer Yellow at 25 °C for 1 h show a distinct
vacuolar staining. In tlg2
cells, however, only a small
amount of dye appears to have been internalized into vacuolar
structures, as defined by Nomarski optics (Fig.
3). This result suggests that Tlg2p is
necessary for efficient fluid phase endocytosis.
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Tlg2p Is a Type II Membrane Protein That Fractionates in a Bimodal Fashion with Endosomal and Late Golgi Marker Proteins-- In differential centrifugation experiments, 70% of the total cellular Tlg2p is found in the 100,000 × g pellet, whereas 15% is found in the 12,000 × g pellet, and 10% is found in the 100,000 × g supernatant (Table II). The association of Tlg2p with the 100,000 × g pellet was efficiently disrupted by 2% Triton X-100, but not by 1.5 M NaCl, 2 M urea, or 0.3 M Na2CO3, pH 11.4 (not shown). These results imply that Tlg2p is tightly associated with membranes, presumably through the putative transmembrane domain. The N-terminal domain of Tlg2p is sensitive to addition of proteinase K both in the absence and presence of 0.4% Triton X-100, whereas the lumenal marker protein, Kar2p, is protected from proteolysis in the absence but not in the presence of detergent (Fig. 5). Thus, Tlg2p appears to be an integral membrane protein oriented such that its N-terminal domain is facing the cytoplasm.
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GFP-Tlg2p Co-localizes with Endocytic Intermediates Observed during
FM4-64 Uptake--
In order to directly observe Tlg2p localization in
living cells, the open reading frame of GFP was fused to the
TLG2 promoter as an N-terminal fusion to the Tlg2p reading
frame, yielding a GFP fusion gene transcribed from the endogenous
TLG2 promoter ("Experimental Procedures"). GFP-Tlg2p is
expressed at normal levels when compared with wild type Tlg2p and
functionally complements the high salt growth defect and CPY missorting
of tlg2 (not shown). The GFP fusion protein shows
punctate cytoplasmic staining in wild type cells, with typically 2-5
bright dots/cell. In type E vacuolar mutants, the normally punctate
staining that is observed with markers of the endosome/prevacuolar
compartment is found as a single, abnormally large structure. We
reasoned that if this staining was endosomal or late Golgi, then a
class E type vacuolar mutant would show a consolidation of these
punctate structures to one perivacuolar compartment (15, 23). Indeed,
vps27 tlg2
cells containing the GFP-Tlg2p fusion show the
expected pattern, with an avarage of 1.29 dots/cell (n = 66 cells, SD = 0.65) as compared with an average of 3.21 dots/cell in wild type (n = 64 cells, SD = 0.88).
The single dot in vps27 cells is much larger than that in
wild type and has a perivacuolar localization.
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Tlg2p Interacts with the v-SNARE, Snc2p, in a
SEC18-dependent Fashion--
Although sequence homology
suggests that Tlg2p is a t-SNARE, we sought functional evidence for
this by assaying its ability to interact with known v-SNAREs. Golgi to
plasma membrane trafficking requires one of the two functionally
redundant v-SNAREs, Snc1p and Snc2p (37). The Snc proteins are known to
interact with the plasma membrane localized t-SNAREs Sso1p, Sso2p, and
Sec9p (38, 39). We sought to determine whether the Snc proteins also
interact with Tlg2p by assaying the ability of anti-Tlg2p antibodies to
co-precipitate Sncp. A small amount of Sncp co-precipitated with Tlg2p
in wild type cells (Fig. 8a).
Sncp from a tlg2 strain does not precipitate with
anti-Tlg2 antibodies (Fig. 8b). In sec18-1 cells,
the amount of Sncp co-precipitated with Tlg2p dramatically increased
after shifting the cells to the restrictive temperature, 37 °C, for
10 min. This phenomenon, typical for SNARE complexes in yeast, is
thought to reflect a defect in the ability of the mutant Sec18p to
disassemble SNARE complexes (38,
44).2
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DISCUSSION |
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Although the SNARE machinery of the early and late secretory pathway in yeast has been under intense study in the last few years, less is known about the corresponding SNARE complexes and auxiliary proteins that control the vacuolar endocytic system.
Recently, SNARE complexes required for vacuole-vacuole fusion and for
late Golgi to endosome transport have been described, and a requirement
for NSF (Sec18p) activity in early endosome to late endosome transport
in yeast was reported (17, 18, 40). We now report that a third
syntaxin-like protein, Tlg2p, is important for normal functioning of
the vacuolar endocytic system in yeast. Tlg2p shows a stronger
resemblance to Vam3p and Pep12p than to other characterized yeast
syntaxin-like proteins: Sso1p, Sso2p, Sed5p, Ufe1p, and Sft1p (31).
Pep12p and Vam3p receive the most significant BLAST probability scores
when Tlg2p is used as query against the yeast data base (P
values of 7.9e19 and 1.3e
11, respectively).
Perhaps significantly, one of the regions in which the similarity of
Tlg2p with Pep12p is higher is the hydrophobic transmembrane domain,
which has been shown to be important for localization of previously
characterized t-SNAREs, including Pep12p (32). These three proteins
thus appear to comprise a subfamily of yeast syntaxin-like t-SNAREs
involved in controlling membrane flux and protein trafficking through
the vacuolar/endocytic system.
Tlg2p contains an unusual hydrophilic C-terminal domain that is absent from all other known syntaxin family proteins. The presence of this domain does not alter the overall topology of the rest of the protein. Tlg2p thus appears to be a type II integral membrane protein, as are all the syntaxin family proteins. In addition, the C-terminal hydrophilic region is not required for complementation of two phenotypes that we have associated with deletion of the TLG2 gene: failure to grow on high salt medium and partial secretion of p2 CPY. This result implicates the syntaxin-like cytoplasmic and transmembrane regions as both necessary and sufficient for these functions.
Yeast carrying vacuolar biogenesis mutations known as "class E" mutants accumulate a membrane-bound compartment, the class E compartment, generally thought to be an exaggerated late endosome or prevacuolar compartment (23). The class E compartment accumulates several types of proteins: resident endosomal proteins, as well as proteins that cycle between the late Golgi and the endosome, proteins that transit between the plasma membrane and the endosome, and proteins en route to the vacuole (15). Consistent with the idea that Tlg2p functions in the vacuolar endocytic pathway, the pattern of GFP-Tlg2p fluorescence is shifted in the class E vps27 mutants to a single, perivacuolar compartment. The fact that Tlg2p co-fractionates in a bimodal fashion with both Pep12 and Kex2p in density gradient centrifugation experiments, together with the class E compartment localization in vps27 cells, suggests that Tlg2p may be cycling between the late Golgi and the endosome. As such, it may also function in transport from the endosome to a late Golgi compartment.
Several lines of evidence link Tlg2p to a membrane fusion step or steps
that pertain to endocytosis. Although pep12 yeast, vam3
yeast, and tlg2
yeast all show
aberrations in CPY processing (16, 41), tlg2
cells are
unique in that intracellular CPY processing is not significantly
impaired. Although these cells secrete 15-20% of the total CPY
produced, the maturation of intracellular protein to the mCPY form is
not blocked, and intracellular p2 CPY does not accumulate. Thus,
although these cells have a sorting defect, they are not blocked in
membrane fusion events that are required in transport from the late
Golgi to the vacuole via the endosome/prevacuolar compartment. In
contrast, the uptake of the fluid phase endocytosis marker Lucifer
Yellow is highly retarded in these cells, and the half-life of Ste3p is
doubled in tlg2
cells relative to that of wild type. This
increase is not the result of inefficient proteolysis, because
intracellular Ste3p did not accumulate in tlg2
cells
(Fig. 4b). In addition, a portion of Tlg2p appears to be
localized to endosomes; Tlg2p partially co-fractionates with Pep12p in
density gradients, and the Tlg2p-GFP fusion protein co-localizes with
FM4-64 internalization intermediates. FM4-64 is a styryl dye that has
previously been shown to be internalized via endocytic intermediates en
route to the vacuole in yeast. The fact that this marker passes through
a compartment that also contains Tlg2p is highly suggestive of an
endocytosis-related role for Tlg2p. These data are also consistent with
a portion of Tlg2p co-fractionating with Pep12p, an endosomal
marker.
We have demonstrated that Sncp co-precipitates with Tlg2p in a SEC18-dependent fashion. This result suggests that Snc may form a functional SNARE complex with Tlg2 as it recycles from the plasma membrane back to the late Golgi, presumably via the endosome. Because there is no other evidence that Sncp functions in endocytosis, we carried out a mixing experiment to prove that the Sncp-Tlg2p interaction occurs in vivo. The data support a model whereby Sncp is involved in SNARE complexes with distinct t-SNAREs, Ssop and Tlg2p, as it cycles between the Golgi and the plasma membrane. A similar model has previously been proposed for Sec22p, a v-SNARE that cycles between the endoplasmic reticulum and cis-Golgi. Sec22p interacts both with the cis-Golgi t-SNARE Sed5p for anterograde transport and with the endoplasmic reticulum t-SNARE Ufe1p for retrograde transport (42-44). Evidence is accumulating that the mammalian homologs of Sncp, the synaptobrevins (VAMPs) and cellubrevin, also interact with multiple t-SNAREs in a similar fashion. These proteins were originally postulated to interact with syntaxin 1 during fusion of vesicles with the plasma membrane (45). However, more recently there have been reports that they interact with Golgi/endosome localized t-SNAREs including syntaxin 6 (46). A clearer understanding of the specific membrane fusion events in which Tlg2p functions (endocytosis versus endosome to Golgi traffic) and knowledge of the protein-protein interactions required for these functions will be necessary to test this hypothesis.
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ACKNOWLEDGEMENTS |
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We thank Scott Emr, Mark Rose, and George Sprague for generously providing antisera. We also thank Chavela Carr, Ruth Collins, N. Barry Elkind, and Rob Piper for fruitful discussions and Laurent Caron for help with confocal microscopy.
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FOOTNOTES |
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* This work was supported by Program Project Grant CA46128 from the National Cancer Institute (to S. F. N. and P. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a fellowship from the Human Frontier Science Program.
To whom correspondence should be addressed: Dept. of Cell
Biology, Boyer Center for Molecular Medicine, Howard Hughes Medical Inst., 295 Congress Ave., Rm. 254B, New Haven, CT 06510. Tel.: 203-737-5207; Fax: 203-737-5746; E-mail:
susan_ferronovick{at}quickmail.yale.edu.
1 The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; t-SNARE, target SNARE; v-SNARE, vesicle SNARE; NSF, N-ethylmaleimide-sensitive factor; CPY, carboxypeptidase Y; GFP, green fluorescent protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
2 E. Grote and P. Novick, unpublished data.
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REFERENCES |
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