From the Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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Analysis of the cytosolic requirements for in vitro intra-Golgi transport led to the characterization of three proteins: N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment protein (SNAP), and p115, all involved in the docking and fusion of transport vesicles to their target membranes. In the course of determining the minimal cytosolic requirements for intra-Golgi transport in vitro, we identified three additional factors that are sufficient to replace crude cytosol. We describe here the purification and characterization of one of these factors, a novel 16-kDa protein, p16, an essential factor for intra-Golgi protein transport. Based on transport activity, this purification procedure resulted in ~1,400-fold enrichment of p16 to apparent homogeneity. The activity of p16 could be observed in the absence of vesicle formation, suggesting that it may participate in the docking and fusion processes.
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INTRODUCTION |
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Transport of proteins between membrane-bound organelles in eukaryotic cells is a multistage process utilizing soluble and membrane proteins. The molecular machinery mediating this process has been explored biochemically and genetically, leading to the identification and characterization of numerous transport factors (1-3).
Several cytosolic transport factors were purified by the utilization of
an in vitro intra-Golgi transport assay. These include N-ethylmaleimide-sensitive factor
(NSF)1 (4);
-,
-, and
-soluble NSF attachment proteins (SNAPs) (5); and
p115 (6). Genetic studies have identified the yeast homologs of NSF,
SNAP, and p115 as Sec18p (7, 8), Sec17p (9), and Uso1p (10-12),
respectively. NSF and SNAP are considered part of the general
docking/fusion apparatus that functions at several transport stages
along the secretory pathway including endosome-endosome fusion (13),
vacuolar sorting (14), transcytosis (15), and synaptic vesicle fusion
(16).
p115 was isolated as a cytosolic factor required for intra-Golgi transport in vitro (6) and was suggested to act together with NSF and SNAP in direct Golgi-Golgi fusion (17). p115 is a peripheral membrane protein localized predominantly in the Golgi apparatus (6) but has also been identified as a component of transcytotic vesicles (15). Recently, p115 has been implicated, together with NSF and SNAP, in the process of reassembly of post-mitotic Golgi fragments into Golgi cisternae (18, 19). Uso1p, the yeast homolog of p115, is required for assembly of the endoplasmic reticulum-Golgi SNARE complex (20). Other cytosolic factors such as Rab proteins and their effectors were also shown to be involved in this process (21-23). It appears, however, that the amount of Rab proteins present on the membrane is sufficient to promote the transport reaction in vitro.
It was demonstrated originally by Clary and Rothman (24) that in addition to NSF and the SNAPs, several other cytosolic factors were required for reconstituting the SNAP-dependent transport assay. The need for additional cytosolic transport factors was demonstrated further for the p115-dependent assay (6) as well as for direct fusion between Golgi stacks (17). Thus, identification and isolation of these novel factors are essential for understanding the exact molecular machinery of intracellular protein traffic. In the present study we describe the purification of a novel 16-kDa transport factor, p16, from bovine brain on the basis of the in vitro intra-Golgi transport assay. Our data suggest that p16 is a transport factor that participates in the docking/fusion reaction.
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MATERIALS AND METHODS |
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General Procedures
Vesicular stomatitis virus (VSV)-G protein-containing donor Golgi membranes from 15B cells and acceptor membranes from wild-type Chinese hamster ovary cells were prepared as described (25). Protein concentration was determined with the Bio-Rad protein assay. The pH values were determined at room temperature. All fractionations were performed at 4 °C. All cytosolic fractions tested in this assay were dialyzed to 25 mM Tris-HCl, pH 7.4, 50 mM KCl, and 1 mM dithiothreitol (dialysis buffer) before their addition to the transport assay.
Cis- to Medial-Golgi Transport Assay
The standard assay mixture (25 µl) contained 0.4 µCi of UDP-N-[3H]acetylglucosamine (America Radiolabeled Chemical), 5 µl of a 1:1 mixture of donor and acceptor Chinese hamster ovary cellular Golgi membrane, and crude bovine brain cytosol as described (25).
Preparation of Cytosolic Factors
Bovine brain cytosol was prepared by the method of Malhotra
et al. (26). Recombinant His6-NSF and
His6-SNAP were prepared as described (27). Fraction I
was obtained by chromatography of 500 ml of 40% ammonium sulfate
precipitate on a 400-ml Fast Flow Q column (Pharmacia Biotech Inc.)
equilibrated with 25 mM Tris-HCl, pH 7.4, 100 mM KCl, 10 mM
-mercaptoethanol, and 10% glycerol. The column was washed with 800 ml of the same buffer and then
eluted with a 0.1-0.5 M KCl gradient in 1,200 ml. Nine-ml fractions were collected, dialyzed to reduce the KCl concentration to
50 mM, and assayed for transport activity in the presence
of 0.5 µg of p115, 5 ng of His6-NSF, 60 ng of
His6-
SNAP, and 5 µl of Golgi membranes. Typically, a
peak of transport activity was eluted at 0.22-0.30 M KCl,
and the peak fractions were pooled and concentrated by ultrafiltration
using Amicon PM-10 filter. The concentrated pool, designated
, was
dialyzed against dialysis buffer and had a protein concentration of
about 30 mg/ml. p115 was purified from bovine liver cytosol as
described previously (6).
Development of an Intra-Golgi Cell-free Transport Assay for Novel Cytosolic Components
The assay is a modification of the one described by Waters
et al. (6). The 25-µl assay contained 0.4 µCi of
UDP-N-[3H]acetylglucosamine, 5 µl of a 1:1
mixture of donor and acceptor Chinese hamster ovary cellular Golgi
membrane, 100 µg of I, 0.5 µg of p115, 5 ng of recombinant NSF,
60 ng of recombinant SNAP, 10 µM palmitoyl-coenzyme A,
ATP and UTP regeneration systems, and 10 µl of the various cytosolic
fractions as indicated in the figure legends. The transport reactions
were incubated at 30 °C for 2 h.
N-[3H]Acetylglucosamine incorporated into
VSV-G protein was determined as described previously (25).
Purification of p16
Preparation of Bovine Brain Cytosol--
Bovine brains were
obtained immediately after slaughtering and placed on ice-cold 25 mM Tris-HCl, pH 7.4, 340 mM sucrose. The tissue
(600 g) was placed on a glass Waring blender, which was then
filled with 800 ml of homogenization buffer containing 25 mM Tris-HCl, pH 7.4, 500 mM KCl, 250 mM sucrose, 2 mM EGTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 µM 1,10-phenantroline, 2 µM pepstatin A, 2 µg/ml aprotonin, and 0.5 µg/ml leupeptin. The homogenate was
centrifuged at 7,500 × g in a Sorvall GS3 rotor for
1 h at 4 °C. The supernatants were pooled and centrifuged at
120,000 × g in a Beckman Ti-45 rotor for 1 h at
4 °C. The supernatants were pooled and dialyzed against 25 mM Tris-HCl, pH 7.4, 100 mM KCl, and 10 mM -mercaptoethanol (buffer A). The dialyzed material
was collected and clarified by centrifugation at 7,500 × g in a Sorvall GS3 rotor for 1 h at 4 °C. This
material was designated bovine brain cytosol and had a protein
concentration of about 8 mg/ml.
Polyethylene Glycol Precipitation-- The protein concentration of bovine brain cytosol was adjusted to 7.5 mg/ml by dilution with fresh dialysis buffer, and KCl was added to a final concentration of 0.5 M. Polyethylene Glycol 4000 was added slowly to a final concentration of 12.5%. The solution was stirred for 30 min at 4 °C and then centrifuged in a Sorvall GS3 rotor at 8,500 rpm for 30 min at 4 °C. The supernatant was discarded, and the pellets were resuspended in a 100-ml Dounce homogenizer. The insoluble material was removed by centrifugation at 120,000 × g for 6 min at 4 °C, the protein concentration was adjusted to 7 mg/ml and the salt concentration to 0.1 M KCl. This material was termed polyethylene glycol precipitate.
Fast Flow Q Chromatography--
The polyethylene glycol
precipitate was loaded onto a 400-ml Fast Flow Q column equilibrated
with buffer A containing 10% glycerol at 3 ml/min. The column was
washed with 400 ml of the equilibration buffer and then eluted with a
1,200-ml gradient of KCl at a range of 0.1-0.5 M. Two
active fractions were detected, one in the unbound material and the
other in fractions eluted from 0.2 to 0.5 M KCl. All
unbound protein was pooled and termed flow-through Q; it had
a protein concentration of 0.4 mg/ml. The active fractions eluted from
the column were termed (see above).
Isoelectric Precipitation--
The flow-through Q pool was
transferred to Spectra/por 3 dialysis bags and dialyzed against 10 mM potassium phosphate, pH 6.6, and 10 mM
-mercaptoethanol. The insoluble material was removed by
centrifugation in a Sorvall SS34 rotor at 7,800 × g
for 20 min at 4 °C. The supernatant, which had a protein
concentration of about 0.21 mg/ml, was concentrated about 10-fold by
ultrafiltration on an Amicon PM3 filter.
Superdex 75 Chromatography--
The concentrated isoelectric
supernatant was centrifuged for 10 min in a microcentrifuge to remove
insoluble material, and the supernatant was chromatographed on a 24-ml
Superdex 75 HR 10/30 column (Pharmacia) equilibrated in 25 mM Tris-HCl, pH 7.4, 150 mM KCl, 10 mM -mercaptoethanol, and 10% glycerol at 0.3 ml/min. Fractions of 0.7 ml were collected and tested for transport activity. A
single peak of activity was detected at an elution volume corresponding to a molecular mass of 14-16 kDa as determined by low molecular weight
calibration kit (Pharmacia). This material had a protein concentration
of about 0.08 mg/ml.
Mono S Chromatography--
The Superdex 75 pool was adjusted by
dialysis to 10 mM phosphate buffer, pH 6.5, 10 mM -mercaptoethanol, and 10% glycerol and loaded onto a
1-ml Mono S HR 5/5 (Pharmacia) column equilibrated by the same buffer
at 0.5 ml/ml. The column was washed with the equilibration buffer and
eluted with 25 ml of a 0-400 mM KCl gradient. Fractions (1 ml) were collected, and aliquots were analyzed for transport activity
and by electrophoresis followed by Coomassie Blue staining. The
fraction purified in this way was referred to as p16.
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RESULTS |
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Identification of Novel Cytosolic Factors Involved in Intra-Golgi
Transport--
The cell-free system that reconstitutes intra-Golgi
transport has been used in recent years for the characterization and
isolation of several factors involved in intracellular trafficking (2). Proteins such as NSF, SNAPs, and p115 were isolated by this transport assay in which the activity of each factor could be assessed
specifically. One such an experimental design is a complementation
assay where saturating levels of different crude cytosolic fractions
are added to the transport assay, and a signal is observed only upon
the addition of the protein of interest. To identify novel soluble factors required to reconstitute intra-Golgi transport in
vitro, we analyzed the transport activity of different cytosolic
fractions in the presence of saturating levels of NSF, SNAP, and p115.
Fig. 1A describes the
fractionation of 12.5% polyethylene glycol precipitate of bovine brain
cytosolic protein on a Q-Sepharose anion exchange column. This
chromatography step separated two soluble factors with considerable
transport activity (Fig. 1B). A peak of transport activity
could be detected in the unbound material and was tentatively termed
; another factor that showed transport activity was eluted as a
single peak between 0.22 and 0.30 M KCl and was tentatively termed
(Fig. 1B). Each of these cytosolic factors
reconstituted only part of the transport activity observed in the
presence of crude cytosol, whereas both factors together recovered the
full transport activity (Fig. 1C).
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Characterization of a II-dependent Assay--
In
the present study we focused on the purification of the transport
factor present in II
. To assure that II
acts as part of the known
transport machinery, we tested whether the signal obtained with the
different factors was NSF-, SNAP- and p115-dependent. Golgi
membranes treated with N-ethylmaleimide were tested for NSF-dependent transport activity.
II
-dependent signals could be observed only in the
presence of recombinant NSF (Fig.
3A). Furthermore, in the
absence of either
SNAP or p115, no significant II
-dependent transport activity could be observed,
indicating that II
is acting in conjunction with the known transport
factors.
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Purification of a Novel 16-kDa Protein from Pool II--
Using
the II
-dependent assay a 16-kDa protein, p16, was
purified from bovine brain cytosol by the following steps: (i) ammonium sulfate precipitation; (ii) Q-Sepharose anion exchange chromatography; (iii) CM-Sepharose cation exchange chromatography; (iv) gel filtration on a Superdex 75; and (v) Mono S cation exchange chromatography. Fractions obtained from each chromatography step were tested in the
II
-dependent assay. The quantitation of p16 purification is summarized in Table I. This procedure
resulted in the purification of a 16-kDa polypeptide (on
SDS-polyacrylamide gel electrophoresis) to apparent homogeneity.
Protein profile and the transport activity of the last purification
step are depicted in Fig. 4. A typical purification resulted in about 0.3 mg of the 16-kDa protein with an
~1,400-fold increase in specific activity and 4% activity yield. We
have tentatively termed this protein p16.
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Involvement of p16 in Fusion--
It has been demonstrated
previously that the signal in the intra-Golgi transport system could be
mediated by coated vesicles (29), and as such, it is sensitive to
GTPS. In the absence of the coat proteins, the Golgi cisternae fuse
directly with each other, and the assay becomes resistant to GTP
S
(17, 28). Fig. 5 demonstrates that
transport observed in the presence of crude cytosol is inhibited by
GTP
S, whereas p16-dependent assay is not. This result is
consistent with the finding that the II
-dependent assay
is resistant to brefeldin A. Taken together, these results suggest that
p16 is likely to be involved in docking or fusion rather than in
vesicle production. The possibility that p16 affects glycosylation of
VSV-G protein rather than the transport process per se was
ruled out because we found no difference in the rate of
[3H]UDP-GlcNAc uptake into the Golgi lumen (30) in the
presence versus the absence of purified p16 (2 ± 0.06 pmol/min/mg compared with 1.9 ± 0.04 pmol/min/mg,
respectively).
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DISCUSSION |
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We have utilized the well characterized intra-Golgi cell-free transport assay to detect yet unidentified cytosolic transport factors. This work describes the identification of three cytosolic factors and the purification of a novel 16-kDa protein required for intra-Golgi transport.
In the process of identifying novel cytosolic factors required for
intra-Golgi protein transport in vitro, we characterized three different protein pools each exhibiting low transport activity in
the presence of NSF, SNAP, and p115, yet together they reconstituted the full transport activity observed with crude cytosol. Previously, Waters et al. (6) identified two crude cytosolic factors
required to reconstitute intra-Golgi transport in vitro in
addition to NSF, SNAP, and p115. Factors and
described in the
present study could be related to these factors. In this study we were able to separate further the
factor into two distinct activities. Pool
significantly stimulated the signal of either I
or II
, whereas combining I
and II
in the absence of
resulted in only a slight increase in the assay signal (Fig. 2C).
Conceivably, both
factors could share the active protein component.
However, further purification of I
based on its transport activity
revealed that the active component in this pool was a 56-kDa
polypeptide,2 whereas in the
present study we show that the transport activity of II
was
attributed to p16. It could still be possible that both crude fractions
in the early stages of the purification might include small amounts of
reciprocal activity.
We described here a purification procedure based on a functional
intra-Golgi transport assay, which led to the isolation of a novel low
molecular weight protein, p16. Using this purification procedure, p16
was enriched by about 1,400-fold to apparent homogeneity. Similar
enrichment of transport activity was required for the other soluble
transport factors, such as SNAP and p115, indicating that the
activity of p16 in the cytosol is comparable to these other transport
factors. Amino acid sequence analysis of five different tryptic
peptides derived from the pure 16-kDa protein indicates that it is a
novel protein (data not shown). We have recently cloned the p16
encoding cDNA from bovine brain, and all sequences of the peptides
obtained from the endogenous protein are represented in the putative
cDNA p16 amino acid
sequence.3
This clearly demonstrates that the 16-kDa protein in the pure fraction
is a single polypeptide. We also found that a recombinant p16 was
active in the intra-Golgi transport cell-free assay, strongly supporting the notion that the 16-kDa polypeptide described here is the
active component of the pure
fraction.4
Several well defined steps are required for intracellular vesicular
protein traffic, including budding, targeting, docking, and fusion of
vesicles with their target membranes, each requiring a different set of
cytosolic factors. It has been demonstrated previously that ARF-1 and
the coatomer are the only cytosolic factors required for the production
of Golgi-derived COPI (31). Vesicle targeting involves interaction
between integral membrane proteins, the v-SNAREs located on vesicles
and the t-SNAREs present on the target membrane (32, 33). The
v-SNARE·t-SNARE complex then binds SNAP and NSF which, in turn,
catalyze the disassembly of the SNARE complex, thus initiating fusion
(32). This core machinery of protein transport probably requires the
participation of additional accessory factors. Our data suggest that
all three factors described in this study, I,
, and p16, are part
of the targeting and fusion machinery. Analysis of the intra-Golgi
transport assay revealed that in the absence of the coat proteins the
assay measures mainly fusion between the Golgi cisternae (17). This uncoupled fusion reaction requires known components such as NSF, SNAP,
and p115 and is resistant to GTP
S. We demonstrated here that the
p16-dependent assay was not inhibited by GTP
S or
brefeldin A, indicating that it represents an uncoupled fusion. Based
on these results we suggest that p16 plays a role in docking or fusion of vesicles rather than being part of the budding apparatus.
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ACKNOWLEDGEMENTS |
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This work was initiated in the laboratory of Dr. James E. Rothman in Sloan-Kettering Cancer Center, New York. We thank Haim Great for technical assistance, Tony Futerman, Shulamit Michaeli, and Yigal Avivi for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by the Israel Science Foundation and the Israel Cancer Foundation.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.
Incumbent of the Sholimo and Michla Tomarin Career Development
Chair of Membrane Physiology. To whom correspondence should be
addressed: Tel.: 972-8-934-3682; Fax: 972-8-934-4112; E-mail: bmzevi{at}weizmann.weizmann.ac.il.
1
The abbreviations used are: NSF,
N-ethylmaleimide-sensitive fusion protein; SNAP, soluble
NSF attachment protein; SNARE, SNAP receptor; VSV, vesicular stomatitis
virus; GTPS, guanosine
5
-3-O(thio)triphosphate.
3 Y. Sagiv, A. Legesse-Miller, and Z. Elazar, manuscript in preparation.
4 It should be noted that affinity-purified anti-p16 antibodies directed against the recombinant protein specifically inhibited the cell-free intra-Golgi transport assay in the presence of crude cytosol, hence demonstrating that p16 is required for intra-Golgi transport under conditions in which the cytosolic factors coatomer and ARF are present.
2 A. Porat and Z. Elazar, unpublished data.
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REFERENCES |
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