From the Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Medical School, Houston, Texas 77030
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ABSTRACT |
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Molecular mechanisms of vesicle transport between
the prevacuolar compartment and the vacuole in yeast or the lysosome in mammalian cells are poorly understood. To learn more about the specificity of this intercompartmental step, we have examined the
subcellular localization of a SEC1 homologue, Vps33p, a
protein implicated to function in transport between the prevacuolar
compartment and the vacuole. Following short pulses, 80-90% of newly
synthesized Vps33p cofractionated with a cytosolic enzyme marker after
making permeabilized yeast cells. However, during a chase, 20-40% of Vps33p fractionated with permeabilized cell membranes in a
time-dependent fashion with a half-time of ~40 min.
Depletion of cellular ATP increased the association rate to a half-time
of ~4 min and caused 80-90% of newly synthesized Vps33p to be
associated with permeabilized cell membranes. The association of Vps33p
with permeabilized cell membranes was reversible after restoring cells
with glucose before permeabilization. The
N-ethylmaleimide-sensitive fusion protein homologue,
Sec18p, a protein with known ATP binding and hydrolysis activity,
displayed the same reversible energy-dependent
sedimentation characteristics as Vps33p. We determined that the
photosensitive analog, 8-azido-[-32P]ATP, could bind
directly to Vps33p with low affinity. Interestingly, excess unlabeled
ATP could enhance photoaffinity labeling of
8-azido-[
-32P]ATP to Vps33p, suggesting cooperative
binding, which was not observed with excess GTP. Importantly, we did
not detect significant photolabeling after deleting amino acid regions
in Vps33p that show similarity to ATP interaction motifs. We visualized
these events in living yeast cells after fusing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p. In
metabolically active cells, the fully functional Vps33p-GFP fusion
protein appeared to stain throughout the cytoplasm with one or two very
bright fluorescent spots near the vacuole. After depleting cellular
ATP, Vps33p-GFP appeared to localize with a punctate morphology, which
was also reversible upon restoring cells with glucose. Overall, these
data support a model where Vps33p cycles between soluble and
particulate forms in an ATP-dependent manner, which may
facilitate the specificity of transport vesicle docking or targeting to
the yeast lysosome/vacuole.
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INTRODUCTION |
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The compartmentalized nature of eukaryotic cells demands high fidelity mechanisms to ensure maintenance of each organelle's unique content. Organelles of the secretory and endocytic system rely on vesicle-mediated transport pathways not only to move protein cargo throughout the cell but also to preserve their specific protein and lipid compositions.
The SNARE hypothesis describes fundamental molecular mechanisms that give rise to the specificity of vesicle-mediated protein transport in eukaryotic cells (1). The basic tenet of this notion is that interactions between integral membrane proteins in transport vesicles (v-SNAREs) and in their cognate target organelles (t-SNAREs) direct the specificity of all intercompartmental events (2). Although substantial evidence exists to support the existence of many v- and t-SNARE proteins, precisely how they function in vesicle transport specificity is continually being revised as more details are uncovered for different systems (3, 4). However, a fact remaining clear and central to the SNARE hypothesis is that multimeric interactions of v- and t-SNAREs with cytosolic proteins play an essential role in the precision of vesicle-mediated transport (5, 6).
The robust genetics of the yeast, Saccharomyces cerevisiae, has identified four genes whose cytosolic protein products are believed to help specify vesicle docking and/or fusion. Fittingly, one of these, SEC1, not only was the first secretory gene identified through mutant isolation (7) but was also the namesake of this family of proteins that are ubiquitously expressed in eukaryotic organisms (for reviews see Refs. 8-10). The Sec1 group of proteins in yeast consists of Sec1p (11), Vps45p (12, 13), Sly1p (14), and Vps33p (15, 16).
The VPS33 gene was identified in a selection for yeast
mutants with defects in vacuolar protein sorting (17). Loss of
VPS33 function results in three prominent phenotypes; 1)
temperature-sensitive growth (restrictive temperature is 38 °C), 2)
severe missorting of both soluble and membrane vacuolar proteins, and
3) abnormal vacuole morphology. Although the vacuole in
vps33 mutants is very fragmented, it is not as severe as
the other class C vps mutants,
end1/vps11, vps18, and
vps16 (18). The VPS33 gene product, Vps33p, is
691 amino acids long, mostly hydrophilic, and cytoplasmically localized. The primary sequence of Vps33p contains two regions that are
similar to the type A and B (also called Walker A and B) nucleotide
consensus sequences (15), suggesting that it may bind or hydrolyze ATP,
which has never been established.
In this study, we have examined ATP binding in the possible function of Vps33p. Our data indicate that Vps33p can indeed bind ATP and that deletion of a region that includes the type B consensus pattern results in both the loss of ATP binding and a nonfunctional protein. Further, Vps33p reversibly changed its localization from the cytosol to a particulate fraction when ATP was depleted in yeast cells, which was also observed for the NSF1 homologue, Sec18p.
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MATERIALS AND METHODS |
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Media and Strains
Yeast strains were maintained on YPD medium (1% yeast extract, 2% peptone, 2% dextrose, and 2.5% bactoagar). Liquid medium for radiolabeling and plasmid maintenance was Wickerham's minimal proline (19) medium supplemented with 0.5% yeast extract.
Strains
The yeast strains used in this study include TVY3350, LBY317
(15) CPY-Inv-LEU2/leu2-3,112::pBHY11; BGY3300,
MAT a ura3-52 leu2-3,112 his3-200 trp 1-
901
lys2-801 suc2-
9 vps33
3::HIS3 (complete removal of
coding region); SEY6210 (17); TVY614, MAT
ura3-52 leu2-3,112 his3-
200 trp 1-
901 lys2-801 suc2-
9 prc1
::HIS3 prb1
::hisG
pep4
::LEU2; and TVY1, MAT
ura3-52 leu2-3,112 his3-
200 trp 1-
901 lys2-801
suc2-
9 pep4
::LEU2.
Plasmid Construction
A series of green fluorescent protein (GFP) fusion vectors were constructed by inserting a BamHI-BamHI fragment coding for a "super bright" GFP variant, M2 GFP (20) from pCSK-100 (21), kindly provided by Xiaolan Ma and William Margolin (University of Texas Medical School, Houston, TX). into the unique BamHI site of both pRS416 and pRS426 (22, 23). This resulted in four vectors with two orientations of GFP having several multiple cloning sites. These vectors are named pBG416-GFP-SK, pBG416-GFP-KS (cen/ARS), pBG426-GFP-SK, and pBG426-GFP-KS (2µ origin). Two plasmids, both containing a 3.7-kilobase pair SalI-XbaI DNA fragment, were used for expression of the wild-type VPS33 gene. The first plasmid uses pRS416 (cen/ARS) as vector, pJWY33-39; the second uses pRS426 (2µ origin) as vector, pBG33-126. The plasmid, pPRP33-100, is a pBluescript KS vector (Stratagene, La Jolla, CA) with the 3.7-kilobase pair SalI-XbaI VPS33 DNA fragment. To translate the VPS33 gene in vitro, 1600 base pairs were removed between the SalI site and the ATG codon in pPRP33-100, generating pPRP33-101. This placed the VPS33 gene under control of the T3 RNA polymerase promoter.
For the remaining constructs, site-directed mutagenesis (24) was used
to add restriction sites or delete nucleotides. An AatII
site was put at 9 base pairs from the VPS33 ATG codon in pJWY33-39 using the oligonucleotide
5'-CTATTCATCGTGACGTCATTTGATAAAGTTGG-3' to generate pBG33-116. To
delete 57 nucleotides encoding amino acids 661-679 in VPS33
(the type B-like nucleotide interaction region), an oligonucleotide
(5'-TATAGAGTTCATGATCCTTGTGCCATTGATCAACAAATGCTTCATTATAGCTATTTCACCC-3') was used to loop out the corresponding DNA segment in pJWY33-39. The
subsequent VPS33 deletion mutant was subcloned
(SalI-XbaI) into pRS426 to generate pCT33-202.
Oligonucleotides were used to place BamHI sites in
VPS33, in frame to GFP, at amino acids 423 (5'-CAACTCGAGTATAGGATCCTCAATGTCGTT-3') and 691 (5'-CTATCATATAATGGATCCGATATAGAGTTC-3') in pJWY33-39. The resulting
plasmids, pPRP33-302 (BamHI at position 423), and
pPRP33-303 (BamHI at position 691 were used to construct pPRP33-304, which has both BamHI sites by replacing a
1-kilobase pair XhoI-XbaI fragment in
pPRP33-302 with the corresponding XhoI-XbaI fragment from pPRP33-303. To put the M2 GFP mutant under control of
the VPS33 promoter, pBG33-116 was cut with
AatII, blunted, and then cut with SalI and
ligated into pBG426-GFP-KS cut with SalI and SmaI
to generate pBG33pr-2GFP. To make a GFP fusion at the C terminus of
VPS33, a BamHI-BamHI M2 GFP fragment
was ligated into pPRP33-303 at the engineered BamHI site
(pBG33691-GFP). To express this from a 2µ plasmid, the
SphI and SacI fragment in pBG33-126 was replaced
with the SphI and SacI fragment from
pBG33691-GFP, making pBG33691-2GFP. A truncated VPS33-GFP
fusion was made by cutting pPRP33-304 with BamHI and
ligating a BamHI-BamHI M2 GFP fragment, creating
pBG33423-GFP. For expression of this truncated GFP fusion, pBG423-GFP
was cut with SalI and SacI and inserted into
pRS426 to make pBG423-2GFP. To make a Kex2-GFP fusion, a 4-kilobase
pair BamHI-BamHI fragment from pYcP-KX2 (25) was ligated into the BamHI site of pRS416, creating pBG416-KX.
The KEX2 gene was amplified from pBG416-KX via polymerase
chain reaction with SpeI/SacI ends, cut with
SpeI/SacI, and ligated into pBG426-GFP-SK, making
pBGKex2-2GFP. This contains GFP at the full-length C-terminal tail of
Kex2p. A plasmid encoding an alkaline phosphatase-GFP fusion
(Pho8p-GFP) was kindly provided by Greg Odorizzi and Scott Emr
(University of California, San Diego, La Jolla, CA).
Radiolabeling, Immunoprecipitations, and Permeabilized Cell Fractionation
The preparation and radiolabeling of yeast spheroplasts with
Tran35S-label (ICN Radiochemicals, Costa Mesa, CA) used
standard methods previously described (26). The preparation of
permeabilized yeast spheroplasts was also via a standardized protocol
previously described (27). Immunoprecipitations for Vps33p were all
treated with IgGsorb (The Enzyme Center, Waltham, MA) for 20 min before the addition of antiserum (15). Extracts were rocked
overnight at 4 °C and then treated with protein A-Sepharose
(Amersham Pharmacia Biotech). The protein A-Sepharose pellets were
washed sequentially with two 1-ml portions of 50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and
0.5% Tween 20 (Tween 20 buffer), two 1-ml portions of Tween 20 buffer
containing 2 M urea, and two 1-ml portions of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM EDTA. After drying, the immunoprecipitates were
boiled for 4 min in standard Laemmli sample buffer (2× concentration),
and SDS-PAGE was performed as described (28). Most gels were dried and
subjected to autoradiography using Kodak BioMax film. In some cases,
the gels were subjected to phosphor imaging analysis using a STORM
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Fluorescence Microscopy
Cells expressing various GFP fusion constructs were grown in minimal proline medium with 0.5% yeast extract to middle or late log phase. They were concentrated to ~20 A600 units/ml and examined in the same medium. For energy depletion experiments, the cells were washed two or three times in minimal proline medium without glucose and then resuspended in the same medium containing 10 mM NaN3 and 10 mM NaF for periods up to 2 h. To reverse the effects of energy depletion, the cells were washed two or three times in minimal proline medium containing glucose and then incubated for 60-90 min at 30 °C. All micrographs were digitally captured on a Zeiss Axioskop epifluorescence microscope using an Optronics (Goleta, CA) DEI-750 CCD color camera with Adobe Premiere software (Adobe Systems, Mountain View, CA), a TARGA 2000 video board (Truevision, Inc., Santa Clara, CA), and a PowerPC Macintosh 9500 (Apple Computer, Cupertino, CA). All images were edited with Adobe Photoshop software (Adobe Systems).
Photoaffinity Labeling with 8-Azido-ATP
Extract Preparation--
Yeast strain TVY3350 containing
pBG33-126 (wild-type VPS33), pRS426 (vector control), or
pCT33-202 (VPS33 with a 19-amino acid deletion of the type
B-like region) was grown in minimal proline medium to exponential
phase. The cells (25-50 A600 units) were
harvested and converted to spheroplasts. The spheroplasts were
regenerated in YPD plus 1.0 M sorbitol for 60 min at
30 °C, harvested again, and washed twice at 4 °C in 1.0 M sorbitol, 20 mM HEPES-KOH, pH 7.0, 150 mM potassium acetate, and 5 mM magnesium acetate containing sodium azide and sodium fluoride (10 mM
each). The washed cells were resuspended in 10 mM Tris, pH
7.6, 5 mM MgCl2, 0.1 mM
KPO4, 0.1 mM NaF, 0.1 mM
Na2P2O7 (lysis buffer) by adding 15 µl/A600 unit, and a protease inhibitor mixture
was added, which included 2-macroglobulin (125 µg/ml),
1-chloro-3-tosylamido-7-amino-2-heptanone (40 µg/ml),
L-1-tosylamido-2-phenylethyl chloromethyl ketone (80 µg/ml), leupeptin (1 µM), trypsin inhibitor (50 µg/ml), phenylmethylsulfonyl fluoride (0.1 mM), and
pepstatin A (0.7 µg/ml). The spheroplasts were agitated twice with
0.5-mm glass beads in a bead beater (BioSpec Products, Bartlesville,
OK) at 1-min intervals. The lysate was subjected to centrifugation at
4,000 × g for 10 min. The pellet fraction was rinsed
with lysis buffer, combined with the first supernatant, and desalted
using Bio-Gel P-6DG (Bio-Rad). The desalted extract was subjected to
centrifugation at 125,000 × g for 15 min. The pellet
fraction was resuspended in lysis buffer at 10-25 mg/ml. All extracts
were stored at
70 °C.
Photoaffinity Reactions--
Reactions were typically 12.5-25
µl in total volume and consisted of ~125 µg of membrane extract,
20-40 µM 8-azido-[-32P]ATP (ICN
Radiochemicals, Costa Mesa, CA, or RPI Corp., Mt. Prospect, IL). All
reactions were performed in a 96-well microtiter dish. In general, the
reactions were set up and preincubated for 2-10 min prior to UV
irradiation. The microtiter dish was placed ~2 cm below a hand-held
254-nm UV lamp (UVP, Upland, CA, model UVGL-25), and the reactions were
irradiated once for 1 min, followed with a 1-min interval of no
irradiation and then a second irradiation for 1 min. In some
experiments, the filter was removed from the UV light source to
increase the radiation. The reactions were stopped by adding an equal
volume of 4% SDS and 4 mM dithiothreitol. The samples were
transferred to a 1.5-ml tube, and the wells were rinsed with 2% SDS
and 2 mM dithiothreitol and pooled with the first portion.
The samples were heated (65-100 °C) for 2-5 min. The reactions
were then immunoprecipitated for Vps33p, subjected to SDS-PAGE, and
subjected to autoradiography or PhosphorImager analysis. Ten percent of
every reaction was not immunoprecipitated and was analyzed for the
total proteins that were photoaffinity-labeled. Important control
reactions included using creatine phosphokinase as a positive control,
irradiating the 8-azido-[
-32P]ATP before the addition
of the yeast extract, and omitting the irradiation step. Photoaffinity
labeling of creatine kinase with 8-azido-[
-32P]ATP was
consistently competed greater than 80% with 2 mM ATP. We
never observed photoaffinity labeling of Vps33p when the
8-azido-[
-32P]ATP was photolyzed before the addition
of extracts, indicating that secondary reactants were not produced.
Similarly, we never observed labeling of Vps33p when we did not
photoactivate the 8-azido-[
-32P]ATP, indicating that
the azido group on carbon 8 of the adenine ring was the only reactive
moiety. For competition experiments, unlabeled nucleotides were
generally added 2-10 min before the addition of the
8-azido-[
-32P]ATP. When augmenting the reactions with
unlabeled 8-azido-ATP, a mixture of the cold and radioactive
nucleotides was made, and the two were added together.
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RESULTS |
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Vps33p Fractionates with Permeabilized Yeast Cell
Membranes--
When the VPS33 gene was first cloned, its
protein product, Vps33p, was demonstrated to be a soluble cytoplasmic
protein through subcellular fractionation (15). When examining the
biochemical function of Vps33p, we used an in vitro
reconstitution assay that measures intercompartmental transport of
procarboxypeptidase Y to the yeast vacuole in permeabilized cells (27).
The vps33-4 allele has a single L646P change and retains
partial function for vacuolar protein sorting (15, 29, 30).
Spheroplasts from a vps33-4 mutant strain were able to
mature 35-40% of carboxypeptidase Y (CPY) in vivo, while
the majority was secreted as the Golgi-modified p2 precursor (data not
shown). However, even with this capacity for CPY maturation,
vps33-4-permeabilized cell membranes were unable to
transport p2CPY in vitro when incubated with ATP and a
cytosolic extract made from a VPS33 yeast strain (data not
shown). In contrast, 40% maturation of p2CPY was achieved in
VPS33-permeabilized cell membranes under these same
conditions (data not shown). Significant maturation of p2CPY (32%) was
still observed if VPS33-permeabilized cell membranes were
incubated with a cytosolic extract made from a vps33
strain (data not shown). From these results, measuring protein
transport to the yeast vacuole, we concluded that the functional
fraction of Vps33p was associated with permeabilized cell membranes and
not the cytosol. Thus, we examined the fractionation of Vps33p more
carefully in wild-type permeabilized cells to determine if any of the
protein was associated with a particulate fraction.
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Loss of Cellular Energy Increases the Rate and Amount of Vps33p Insolublility-- We suspected that cellular energy played a role in the association/disassociation of Vps33p with membranes. To examine this possibility, cells were treated with agents that deplete cellular ATP. Sodium azide and sodium fluoride inhibit mitochondrial respiration (31) and glycolysis (32), respectively, and thus act to completely stop the cell from maintaining homeostatic ATP concentrations. In yeast, NaN3 and NaF are the most potent compounds for total and rapid depletion of intracellular ATP (33).
Depletion of intracellular ATP with NaN3 and NaF had a pronounced affect on the fractionation behavior of Vps33p. After a 15-min pulse with Tran35S-label, ~85% of the newly synthesized Vps33p cofractionated with the cytosol (Fig. 1B, lanes 1 and 2) as observed previously (Fig. 1A, lanes 1 and 2). However, if NaN3 and NaF (10 mM each) were added during the chase, Vps33p rapidly and completely fractionated with permeabilized cell membranes (Fig. 1B, lanes 3-12). The apparent half-time for this association was 3-4 min, which was 8-9-fold faster than the rate observed in the absence of energy poisons in vivo (Fig. 1B). The effect of energy depletion was most likely specific to properties associated with Vps33p, because the cytosolic enzyme, G6PDH, remained soluble throughout the course of NaN3/NaF treatment. If energy poisons were withheld from the chase, nearly 90% of the newly synthesized Vps33p remained soluble and cofractionated with G6PDH (Fig. 1B, lanes 13 and 14). We tested whether the action of NaN3 and NaF was reversible and extended this analysis to two other proteins, Sec18p and Vps45p. The NSF homolog, Sec18p, was examined because it exists in soluble and particulate forms (34) with putative ATP binding activity like mammalian NSF (35, 36). The Sec1 homolog, Vps45p, also exists in soluble and particulate forms (12) and is 22% identical to Vps33p (8). However, unlike NSF/Sec18p, Vps45p shows no sequence similarity to proteins that bind or hydrolyze ATP (12, 13). If spheroplasts were fractionated after pulse/chase radiolabeling in glucose medium, >80% of Vps33p and Sec18p and ~55% of Vps45p cofractionated with the cytosolic marker G6PDH after 30 min of chase (Fig. 2A, lanes 1 and 2). In contrast, treatment with NaN3 and NaF for 30 min caused >85% of both Vps33p and Sec18p to fractionate with permeabilized cell membranes, but the amount of Vps45p that was membrane-associated did not increase significantly (Fig. 2A, lanes 3 and 4). After the cells were washed several times in medium to remove the NaN3 and NaF, replenished with fresh glucose, and incubated for another 30 min, >90% of Vps33p and >70% of Sec18p fractionated again with the cytosol (Fig. 2A, lanes 5 and 6). This suggested that the effects of energy depletion were reversible. Although not shown, the fractionation behavior of Vps33p was the same when cycloheximide (100 µg/ml) was added to the cells during energy regeneration (after NaN3 and NaF treatment), indicating that new protein synthesis was not required to displace the membrane-associated pool of Vps33p. We also examined the ability of cells to reverse intercompartmental protein transport to the vacuole after energy depletion. After a 15-min pulse with [35S]Met/Cys, ~18% of CPY was present as the mature vacuolar form, while the remaining ~85% represented the p1 and p2 precursor forms (Fig. 2B). Nearly complete maturation of CPY occurred in the presence of glucose but was completely blocked when NaN3 and NaF were added during a 30-min chase period (Fig. 2B). If the energy poisons were washed free of the cells, >95% maturation of CPY took place during a further 30-min chase in glucose (Fig. 2B). Overall, these results suggest that yeast spheroplasts can withstand the deleterious effects of extreme energy depletion, which argued that the insolubility of Vps33p and Sec18p were not due to irreversible toxicity.Vps33p Aggregates after Energy Depletion but Can Be Dissociated with ATP and Cytosol-- To examine in more detail the biochemical nature of the Vps33p particulate association, various treatments were used to solubilize the protein. After 30 min in NaN3 and NaF, permeabilized cell membranes were obtained from yeast spheroplasts and treated with KCl (1 M), Triton X-100 (1%), urea (2 M), and sodium carbonate, pH 11 (0.1 M). Only sodium carbonate was effective at releasing a significant amount (33%) of Vps33p from the membranes (Fig. 3A, lanes 9 and 10), while the other reagents released less than 10% (Fig. 3A, lanes 3-8). This result suggested the interaction between Vps33p and permeabilized cell membranes after energy depletion was strong enough to resist disruption with high salt, nonionic detergent, or denaturing reagents. One possibility to explain this behavior is protein aggregation after energy depletion.
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Vps33p Can Be Labeled with Photoactive ATP-- Several possibilities could explain the effect of energy starvation and rapid loss of Vps33p localization from the cytosol to a particulate fraction. One prospect could be that Vps33p is a reversible substrate for a modifying enzyme like a protein kinase or phosphatase. The lack of ATP in the cell may trap the phosphorylation status of Vps33p and this could result in its aggregation. Indeed, Vps33p is a phosphoprotein in vivo, but we have not observed a difference in its fractionation properties dependent on phosphorylation (data not shown). More importantly, treatment of cells with NaN3 and NaF did not change the phosphorylation status of Vps33p (data not shown). A simpler explanation for the energy-dependent solubility might be that Vps33p directly binds ATP, and this binding is necessary to keep the protein cytosolic.
Covalent cross-linking to a photosensitive ATP analog, 8-azido-ATP was examined to test the simplest model for localization of Vps33p. A high speed membrane fraction was made from a yeast strain deleted for the chromosomal copy of VPS33 but expressing the wild-type gene from a multicopy plasmid (pBG33-126). To demonstrate specificity of the 8-azido-ATP ligand, we examined all of the proteins in the extract. Over a dozen abundant, well resolved polypeptides existed in the high speed pellet fraction (Fig. 5A). However, only three bands were detected after irradiation with 8-azido-[
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Deleting the Putative Nucleotide Hydrolysis Region from Vps33p Abolishes ATP Binding, Energy-dependent Cytosol Distribution, and Vacuolar Protein Sorting-- We examined if the regions of Vps33p that show similarity to proteins that bind or hydrolyze ATP were involved in photoaffinity labeling with 8-azido-ATP. Using site-directed mutagenesis to change conserved amino acid residues within these motifs was avoided because Vps33p does not strictly conform to the known consensus sequence motifs. Moreover, changing aspartate 678 to asparagine (D678N) did not show defective phenotypes (data not shown). Therefore, we deleted 19 amino acids, residues 661-679, because this encompasses all of the region that shows strong similarity to proteins with demonstrated ATP binding or hydrolysis activity like NSF/Sec18p (36), Vps4p (40), Escherichia coli SecA (41), adenylate kinase (42), Hsp70 (43), and Hsp90 (44). A Vps33p mutant lacking these 19 amino acids showed an 18-fold decrease in binding to 8-azido-ATP, which was unchanged in the presence of excess ATP (Fig. 6A, lanes 3 and 4). This suggested that these 19 amino acids were directly or indirectly critical for forming an ATP binding site or were important in maintaining the stability of Vps33p (see below). Removing the 19-amino acid putative ATP hydrolysis region in Vps33p also abolished the ability of the protein to function in vacuolar protein sorting. Maturation of carboxypeptidase Y was not detectable even after a 90-min chase (Fig. 6B).
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VPS33p Is Localized to Punctate Structures after Energy Depletion in Vivo-- To examine the intracellular location of Vps33p, fusion proteins were constructed with the jellyfish green fluorescent protein. This approach was chosen over immunofluorescence because it allowed for a dynamic assessment of intracellular localization in living cells. Three GFP fusion constructs were made to the VPS33 gene all under control of the VPS33 promoter. One was made to the promoter region alone with no coding sequence (VPS33pr-GFP), a second was made to the first 423 amino acids (Vps33p423-GFP), and a third was made to the full length protein (Vps33p691-GFP). Both hybrid proteins had GFP at their C termini. Importantly, the Vps33p691-GFP construct complemented all phenotypes associated with loss of VPS33 function, including growth at 38 °C, proper sorting of vacuolar proteins, and normal vacuole morphology. The truncated fusion, Vps33p423-GFP, did not complement any of these phenotypes when expressed in a yeast strain deleted for the entire VPS33 coding region. This suggested that a functionally important polypeptide tract of Vps33p was in the C-terminal third of the protein between amino acids 423 and 691.
The Vps33p691-GFP fusion protein was localized as a haze throughout the cytoplasm of cells in glucose medium (Fig. 8, Vps33p691-GFP panel). This pattern was consistent with the fractionation experiments described earlier (cf. Figs. 1 and 2). In addition to the cytoplasmic haze, the Vps33p691-GFP fusion protein was also localized to one or two bright spots per cell in glucose, which were usually near the vacuole (Fig. 8, Vps33p691-GFP panel). Significantly, these perivacuolar spots were not observed in cells expressing the truncated Vps33p423-GFP fusion protein or GFP alone (Fig. 8, Vps33p423-GFP and VPS33pr-GFP panels). The exclusive cytoplasmic localization of these two control constructs argued that the C-terminal third of Vps33p might contain information that caused the protein to associate with a specific subcellular organelle or structural complex. Furthermore, quantitation of the fluorescence in several cells with image analysis software (NIH Image) revealed that the cytoplasmic haze accounted for ~80-85% of the total signal. The remaining 15-20% of the signal comprised the one or two fluorescent spots. These values correlate well with the fractionation data of Vps33p, where 80-85% of the protein was cytosolic and 15-20% was particulate under steady state conditions.
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DISCUSSION |
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In these studies, we have presented data to provide evidence for a new biochemical function of a previously identified gene product. Our work establishes Vps33p as a low affinity ATP binding protein that shows energy-dependent cytosolic localization. This conclusion rests on several different types of experimental data. Depleting cellular ATP results in a rapid change of Vps33p from a cytosolic protein to one that fractionates as a particulate protein. Restoring cellular energy with glucose reverses this effect. In glucose medium, a full-length Vps33p-GFP fusion was distributed throughout the cytoplasm. After energy depletion, the GFP fluorescence was localized to punctate structures, which returned to the cytoplasm with fresh glucose. The particulate form of Vps33p after energy depletion required both ATP and a cytosolic extract for efficient release to a soluble form in vitro. The ATP analog, 8-azido-ATP, could bind directly to Vps33p in a saturable manner. Deletion of a 19-amino acid region in Vps33p, similar to many ATP binding proteins and ATPases, prevented photoaffinity labeling with 8-azido-ATP. The deletion mutant protein was also particulate even under energy rich conditions and did not have the ability to support vacuolar delivery of CPY. Overall, the data suggest a model where Vps33p may function in vesicle traffic by cycling between soluble and particulate forms dependent on ATP binding or hydrolysis.
The first clue that Vps33p may interact with ATP was its sequence similarity to proteins that bind or hydrolyze this nucleotide (15). The most common and easily identified sequences are the type A (putative triphosphate-binding), and type B (adenine-binding), consensus regions (47, 48). Frequent renditions of the type A and B sequences are (A/G)XXXXGK(T/S) and (H/R/K)X(5-8)-HyXHyHy(D/E) (where Hy is hydrophobic), respectively (49). Although Vps33p does not precisely match the type A sequence (15), the putative type B sequence in Vps33p is 666KKGINKRFIIIAD678 and matches the consensus pattern more closely except for an alanine residue. The 19-amino acid deletion (residues 661-679) encompassing the type B-like sequence, prevented Vps33p from efficiently binding to 8-azido-ATP and resulted in a nonfunctional protein. This suggests that ATP binding may be necessary for Vps33p function. Furthermore, the deletion of the type B region causes Vps33p to aggregate in ATP-rich conditions, arguing that ATP binding or hydrolysis is required for solubility. Reconstituting the release of Vps33p from membranes with ATP and cytosol in vitro supports this conclusion and suggests that other protein factors contribute to cytosolic localization.
The binding characteristics of Vps33p indicate that it has a low affinity for ATP under our experimental conditions. From the data, we estimate that half-maximal saturation occurs at ~0.5 mM, which is well within the physiological range of 2-5 mM (50). With this low affinity, the binding site would most likely not be constitutively occupied, and this affinity suggests that binding to ATP may serve a regulatory role in Vps33p function. Multimeric interactions with other proteins or itself may give rise to a conformation of Vps33p with higher affinity for ATP. In support of this possibility, photoaffinity labeling of Vps33p was significantly more efficient in particulate extracts compared with cytosolic extracts. These binding properties of Vps33p imply that it does not interact with ATP in the same manner as proteins like the AAA-type ATPases. For instance, Cdc48p (51), NSF/Sec18p (34, 52), and Vps4p (40) all have 50-60 amino acids between the conserved lysine and aspartate residues of their type A and B consensus sequences. The 183-residue stretch separating lysine 494 and aspartate 678 in Vps33p is considerably longer, suggesting a different structure for the functional nucleotide binding fold. Although deletion of 19 residues encompassing the type A-like sequence also reduced binding of 8-azido-ATP to Vps33p, the resulting protein had nearly wild-type function.2
The ATP-specific enhancement in photoaffinity labeling of Vps33p with 8-azido-ATP insinuates a component of positive cooperativity in its nucleotide binding. This is best illustrated with the data demonstrating a 90% decrease in 8-azido-ATP with 1 mM ATP, a slight increase in 8-azido-ATP binding with 2.5 mM and 5.0 mM ATP, and an 80% decrease with 10 mM ATP (Fig. 5E). Although one possibility for this property would be differences in binding affinity between ATP and 8-azido-ATP, another explanation is positive cooperativity. For example, the activity of cAMP phosphodiesterase is stimulated over 10-fold with 1-10 µM cGMP but inhibited nearly 10-fold with 100-1000 µM cGMP (53). Since we are measuring only nucleotide binding and not enzymatic activity in our assays, a cooperative interaction is difficult to establish. At present, we do not know if Vps33p is an ATPase, and this issue is best reconciled with purified protein. The ATP binding characteristics of Vps33p are very atypical, especially if viewed from the standpoint of a single protein or nucleotide binding site. The extracts that we used for ATP binding were very crude and most likely contain other proteins that could interact with Vps33p. Positive cooperativity in ATP binding may happen if a second site on another protein needed to be saturated or occupied causing a conformational change in Vps33p to form its ATP binding site. This type of cooperative binding occurs in the rubisco activase complex (54, 55), CTP synthetase (56), and E. coli GroEL-GroES chaperone complex (57, 58). This idea gives rise to an intriguing possibility that the type B-like sequence in Vps33p interacts with another protein in such a way as to form an ATP binding site. The heteromultimeric formation of a specific nucleotide binding pocket would provide unique specificity to a vesicle docking or transport reaction.
Although the precise molecular details of ATP binding to Vps33p are unknown, the reliance on sequence specificity conventions or "consensus" sequences should not be overestimated in establishing these features. The case of Hsp90 is an excellent example where it was concluded not to bind ATP after using ATP-agarose chromatography, 8-azido-ATP photoaffinity labeling, fluorescence nucleotide analogs, and sequence analyses (49). However, the recent structure of an Hsp90 N-terminal domain co-crystallized with ATP and/or ADP refutes these biochemical results (44). Moreover, the first 220 amino acids of Hsp90 used for the crystallization do not contain a type A or B consensus sequence. Likewise, synapsins I and II were recently shown to bind ATP, and both of these proteins also lack the typical consensus sequences (59). This indicates that many different ATP binding folds exist and that primary amino acid sequence data are often over- or misinterpreted (60). The interaction of Vps33p with ATP does not conform to characteristics of a conventional nucleotide binding site. Molecular details of the interaction may be resolved when putative protein partners of Vps33p are identified.
The energy-dependent localization of Vps33p most likely reflects an ATP-driven conformational change in the protein. Such alterations in protein structure are well documented in oligomeric proteins that bind or hydrolyze ATP such as heat shock chaperones (61) and the N-ethylmaleimide sensitive fusion protein, NSF (36, 62). The oligomeric nature of Vps33p is unknown. The fractionation behavior of both Vps33p and the NSF homolog, Sec18p, but not Vps45p, most likely reflects an energy-driven cycle that continually localizes these proteins between soluble and particulate forms with homeostatic ATP concentrations.
The behavior of Vps33p in living yeast cells under ATP-rich and ATP-depleted conditions provides new observations for interpretation of its cellular location. As predicted from permeabilized cell preparations, 75-85% of Vps33p is cytosolic, with the remaining portion localizing to fluorescent spots, which appeared perivacuolar. This localization pattern is reminiscent of the prevacuolar compartment in yeast, which is aberrantly exaggerated in class E VPS mutants (63-65). However, our data with equilibrium sucrose density gradients suggest that the 15-20% of Vps33p that is particulate does not cofractionate with the prevacuolar compartment as defined by the presence of pro-CPY (26). Therefore, the perivacuolar fluorescent spots may be a novel organelle or subcellular structure. Recently, a comprehensive study has shown genetic and physical interactions among the four yeast VPS class C genes and gene products (30). The VPS18 and VPS11 gene products form a major hetero-oligomer with minor amounts of associated Vps16p and Vps33p. This complex fractionates as both a dense insoluble protein complex and as a peripheral component of the vacuole (30). The sedimentation characteristics of the Vps11p-Vps18p insoluble fraction are very similar to what we have observed with Vps33p after energy depletion. This suggests that ATP is required to dissociate Vps33p from the Vps11p-Vps18p complex.
The many SEC1 family members suggest that these proteins function at every vesicle-mediated step in eukaryotic cells (8, 10). However, the precise molecular mechanism by which Sec1-like proteins execute their role in vesicle docking/fusion is unknown. The most recent findings suggest that Sec1-like proteins play a negative regulatory role in SNARE complex interactions. The Drosophila Sec1 homologue, Rop1, can negatively regulate neurotransmitter release (66), and yeast Sly1p can prevent v-SNARE/t-SNARE interactions in endoplasmic reticulum to Golgi transport (67). On the other hand, a rat Sly1 homologue, rSly1p, can positively influence transport between the endoplasmic reticulum and Golgi (68), so it seems likely that Sec1-like proteins do not perform a common function at different vesicle-mediated steps along the secretory and endocytic pathways. From this perspective, Vps33p may display a unique activity not provided by any of the other SEC1 family members, since they are not predicted to bind or hydrolyze ATP.
Future studies on Vps33p will focus on establishing the details of ATP binding in the molecular mechanism of transport between the prevacuolar compartment and lysosome/vacuole. In particular, we will determine if Vps33p can hydrolyze ATP after purification of a recombinant protein. The possible multimeric nature of Vps33p will be resolved with gel filtration analysis and sedimentation through glycerol gradients. The recently described genetic interaction between VPS33 and the vacuolar t-SNARE, VAM3, suggests that their protein products may directly interact (29). We will test this with protein cross-linking to determine if Vam3p is associated with Vps33p after energy depletion. Examination of the full-length Vps33p-GFP fusion protein in a class E VPS mutant background stained with the vital endocytosis marker, FM 4-64, should further clarify whether or not Vps33p localizes to the prevacuolar compartment. Reconstitution of intercompartmental transport to the vacuole will be the definitive system to dissect the molecular details of ATP binding and Vps33p function. We are poised to exploit this system with new temperature-sensitive VPS33 alleles that show a rapid block in vacuolar protein transport at elevated temperatures.2 Biochemical complementation of vps33 mutants will aid in proving or disproving if ATP binding or hydrolysis functions to cycle Vps33p from the cytosol to a membrane or membrane complex, allowing vesicular transfer to the yeast vacuole.
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ACKNOWLEDGEMENTS |
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We thank Xiaolan Ma and William Margolin for providing the M2 GFP mutant; Kurt Eakle, William Wickner, and Bruce Horazdovsky for antisera; Bob Fuller for plasmids; and Greg Odorizzi and Scott Emr for the alkaline phosphatase-GFP construct. We also thank Ashok Chavan for valuable advice on photoaffinity labeling with 8-azido-ATP, Sidney (Wally) Whiteheart for sharing results prior to publication, and Roger Barber for helpful discussions of ligand binding.
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FOOTNOTES |
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* This work was supported by NIGMS, National Institutes of Health, Grant GM52092 (to T. V.).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.
To whom correspondence should be addressed. Tel.: 713-500-7445;
Fax: 713-500-7455; E-mail: tvida{at}farmr1.med.uth.tmc.edu.
1 The abbreviations used are: GFP, green fluorescent protein; G6PDH, glucose-6-phosphate dehydrogenase; CPY, carboxypeptidase Y; NSF, N-ethylmaleimide-sensitive fusion protein; PAGE, polyacrylamide gel electrophoresis.
2 B. Gerhardt, T. J. Kordas, C. M. Thompson, P. Patel, and T. Vida, unpublished results.
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