Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro--factor from
the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in
the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi
membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to
Golgi transport in yeast semi-intact cells depends on
COPII proteins (components of a membrane coat that
drive vesicle budding from the ER). Thus, membrane
fusion is coupled to vesicle formation in ER to Golgi
transport even in the presence of saturating levels of
purified fusion factors. Manipulation of the semi-intact
cell assay is used to distinguish freely diffusible ER-
derived vesicles containing pro-
-factor from docked
vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking
phase, but are required for efficient fusion of ER-
derived vesicles with the Golgi complex. Surprisingly,
elevated levels of Sec23p complex (a subunit of the
COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering
experiments using the dilution resistant intermediate
and reversible Sec23p complex inhibition indicate
Sec18p action is required before LMA1 function.
IN eukaryotic cells, transport between many distinct
membrane-bound compartments proceeds through a
vesicular intermediate that buds from one membrane
and fuses selectively with another. A framework for this
transport process has emerged whereby soluble coat proteins are recruited to the donor membrane forming vesicles that uncoat before fusion with an acceptor compartment (Rothman, 1994 Using the yeast Saccharomyces cerevisiae as a model eukaryote for the investigation of intracellular transport, a
cell-free assay that faithfully reproduces ER to Golgi transport has been developed based on the well-characterized
processing and maturation of pro- To characterize the molecular events associated with intracellular membrane fusion, the long-term goal is to reconstitute this event with a minimal set of membrane
bound and soluble proteins. In this report, the soluble factors required for cell-free docking and fusion of ER-
derived vesicles to the Golgi complex have been defined.
This vesicle fusion reaction in the presence of ATP and GTP requires purified Sec18p, Uso1p, and a protein complex referred to as LMA1, a heterodimer composed of
thioredoxin and IB2 (Xu and Wickner, 1996 Strains and Reagents
Strains RSY607 (MAT Plasmid Construction
The c-myc epitope was fused to the COOH terminus of Uso1p by modification of a 2 µm -URA3 plasmid containing the USO1 gene pSK47
(Sapperstein et al., 1995 Cytosol and Membrane Preparations
Strain RSY607 was grown to mid-log phase (OD600 = 1) (determined with
a spectrophotometer; Beckman Instruments, Inc., Fullerton, CA) in 15 liters of yeast extract, peptone, dextrose (YPD) medium. The cells were
harvested and washed once with cold H2O, and once with buffer 88 (B88)
(20 mM Hepes, pH 7.0, 0.25 M sorbitol, 0.15 M KOAc, 5 mM MgOAc).
Approximately 80 g (wet wt) of cells were recovered from this procedure
and are resuspended with 15 ml of B88, and then quick frozen by drop-wise addition to liquid nitrogen. The frozen cells were mixed with liquid
nitrogen in a Waring blender for 10 min to prepare a cell lysate (Dunn and
Wobbe, 1989 Cytosol used for transport reactions or for fractionation on Mono Q
was prepared from thawed aliquots of MSS by centrifugation at 89,000 g
for 15 min (50,000 rpm in a TLA120.2 rotor; Beckman Instruments, Inc.).
The resulting supernatant fluid was recovered and mixed with two volumes of B88 containing 1 mM DTT and 1 mM PMSF (B88D/P), and then
centrifuged at 175,000 g for 15 min (70,000 rpm in a TLA120.2 rotor). The
clarified portion of this supernatant fraction was removed with a pipette
and frozen in liquid nitrogen for storage at Acceptor membranes were prepared from the same MSS as above, except after the first centrifugation step (at 89,000 g) the resulting membrane pellet was processed. First, loosely sedimented membranes were aspirated from this pellet, leaving a compact translucent pellet. This pellet
was resuspended by dounce homogenization in B88D/P to a volume equal
the initial MSS volume. The membranes were collected again by centrifugation at 89,000 g and the B88D/P wash was repeated. This final pellet was
resuspended by dounce homogenization in B88D/P with one-fifth of the
initial MSS volume, and aliquots were frozen in liquid nitrogen and stored
at Fractionation of Cytosol by Mono Q Chromatography
Starting with 7 ml of MSS, 15 ml of cytosol was prepared as described
above and loaded at 0.5 ml/min onto a Mono Q HR 10/10 column (Pharmacia Biotechnology Inc., Piscataway, NJ) that was equilibrated in buffer
A (20 mM Hepes, pH 7.0, 0.15 M KOAc, 1 mM MgOAc, 0.1 mM DTT, 0.1 mM PMSF, and 0.01 mM ATP). Fractions (2 ml) were collected from the
Mono Q flowthrough. The column was washed with 15 ml of buffer A, followed by step elutions with 15 ml of buffer A containing 0.75 M KOAc,
and 15 ml of buffer A containing 1.5 M KOAc. The protein peaks were
contained in fraction 6 (flow through [QFT] ~4 mg/ml), fraction 20 (intermediate ionic strenth [Q.75] ~10 mg/ml), and fraction 27 (high ionic strength [Q1.5] ~3 mg/ml). These fractions were dialyzed against B88D/P, frozen
in liquid nitrogen, and then stored at Protein Purification
Proteins contained in the Mono Q fractions were resolved on a Superose 6 HR 10/30 column (Pharmacia Biotechnolgy Inc.), equilibrated, and then
eluted with 20 mM Hepes, pH 7.0, 150 mM KOAc, 1 mM MgOAc. Samples (0.2 ml) were loaded at a flow rate of 0.3 ml/min, and 0.5-ml fractions
were collected after the initial 5 ml had been discarded. The void volume
for this column corresponds to 7.5 ml. The c-myc-tagged version of Uso1p
was purified from strain CBY324 as follows. 1 liter of cells was grown under selective conditions (yeast nitrogen base, 2% dextrose, and supplements minus uracil), and then transferred to 12 liters of YPD for growth to an OD600 = 1.0 and processed as for the preparation of cytosol from
RSY607. Proteins contained in this cytosol were bound to the Mono Q
HR 10/10 column as above except a 20-ml linear gradient was delivered
from 0.75 to 1.5 M KOAc instead of a step elution. The peak of activity
eluting from the Mono Q column was applied to a Superose 6 HR 10/30
column and developed as described above.
Bacterially expressed Sec18p-6His was purified as previously described
(Whiteheart et al., 1994 Preparation of ER-derived Vesicles Containing
35S-labeled, Core-glycosylated Pro- Vesicles were synthesized from a microsomal preparation (Wuestehube
and Schekman, 1992 Vesicle Fusion Assay
The fusion of purified, ER-derived vesicles with the Golgi compartment
was reproduced in vitro by incubation of acceptor membranes (12 µg),
vesicles (containing ~4,000 cpm of 35S-labeled glyco-pro (gp)- ER to Golgi Transport Assays
Yeast semi-intact cells were prepared from log phase cultures of strain
RSY607 (Baker et al., 1988 Fractionation of Cytosolic Components Required for
Vesicle Fusion
The formation of transport vesicles from the ER has been
reproduced in a cell-free assay using purified soluble factors (Sar1p, Sec13p complex, and Sec23p complex) and
washed membranes. The isolated vesicle intermediates
produced in this reaction are distinct from the donor membrane fraction and are competent for fusion with the Golgi
complex (Barlowe et al., 1994 Cytosol was bound to an anion exchange resin and three
fractions were obtained: the flow through (QFT), a spectrum of proteins that elute at an intermediate ionic strength
(Q.75), and proteins that elute at a high ionic strength
(Q1.5). The individual fractions were dialyzed and tested
in a cell-free assay for promotion of vesicle fusion (Fig. 1).
Addition of individual fractions revealed that none of the
fractions alone could drive vesicle fusion as efficiently as a
crude cytosol, though a significant signal could be detected
by adding saturating amounts of the Q1.5 fraction alone
(Fig. 1, columns 1-4). A maximal signal (comparable to cytosol) could be obtained by combining all three of the fractions, and in fact, omission of either fraction resulted in a
fusion efficiency below crude cytosol (Fig. 1, columns 5-8).
Since a number of the yeast proteins involved in intracellular transport have been identified, specific antibodies
that recognize these species were used in an attempt to
guide further purification efforts. An immunoblot characterizing some of the species contained in these fractions is
shown in Fig. 2. A requirement for Sec7p in vesicle transport has been reported (Lupashin et al., 1996
Sec18p Replaces the Q.75 Fraction
Sec18p/NSF is required for several intracellular transport
steps and is considered a "general fusion factor" that uses
ATP hydrolysis in fulfilling a role in membrane fusion (Rothman, 1994
Uso1p Replaces the Q1.5 Fraction
Resolution of proteins contained in the Q1.5 fraction by
gel filtration chromatography on a Superose 6 column indicated a single peak of activity that migrated at ~800 kD
could support vesicle fusion when combined with purified
Sec18p and the QFT fraction (data not shown). Two previously characterized proteins could behave in this manner
and have been implicated in ER to Golgi transport: Sec7p
(Franzusoff and Schekman, 1989
To confirm that Uso1p was the active component and to
guide purification efforts, a version of Uso1p was constructed whereby the COOH terminus of the protein contains an additional 11 amino acid residues comprising a
c-myc epitope recognized by mAb 9E10 (Evan et al.,
1985
LMA1 Replaces the QFT
Addition of saturating amounts of Sec18p and Uso1p to
vesicle fusion reactions resulted in a fusion efficiency below that catalyzed by crude cytosol, yet addition of the
QFT fraction stimulated to near cytosolic levels (data not
shown). Fractionation of QFT by Superose 6 chromatography revealed a broad peak of activity in the ~20-40 kD
range however, maximal activity could not be recovered
perhaps because of extensive dilution through these combined purification steps. Immunoblot analysis of these
fractions suggested the presence of Ypt1p, Sec19p (GDI),
and thioredoxin: a subunit of a heteroligomeric complex
termed LMA1 (low molecular weight activity 1) required
for vacuole membrane fusion (Xu et al., 1997
These purified protein fractions were titrated to obtain
an optimal concentration for fusion efficiency and each
component was then tested alone and in various combinations for support of vesicle fusion (Fig. 7 A). Neither of the
fractions alone provided significant levels of vesicle fusion
activity, whereas addition of Sec18p and Uso1p resulted in
half the maximal level of vesicle transport but could be
stimulated by addition of LMA1. In Fig. 7 B, vesicle fusion
catalyzed by crude cytosol or the purified factors was monitored over time at relevant temperatures. The reconstituted reaction showed time and temperature dependence that was very similar to a reaction driven by crude cytosol.
Therefore in this fusion assay, purified Sec18p, Uso1p, and
LMA1 together represent a minimal set of proteins required to catalyze the fusion of ER-derived vesicles with
the Golgi in a manner indistinguishable from crude cytosol. Requirements for these individual factors now allow
for further characterization in the processes of vesicle docking and membrane fusion.
Requirements for COPII and Fusion Factors in ER to
Golgi Transport
The initial cell-free assay for ER to Golgi transport (Baker
et al., 1988
Elevated concentrations of COPII inhibit ER to Golgi
transport (Fig. 8 A). One interpretation of this result is
that under high concentrations of COPII, ER-derived vesicles retain bound COPII proteins thus hindering access to
the Golgi docking and/or fusion machinery. Indeed, under
conditions where the COPII coat remains locked on ER-derived vesicles because of inhibition of the Sar1p GTPase, vesicles fail to fuse with the Golgi complex (Barlowe
et al., 1994 Uso1p Catalyzes Vesicle Docking
Semi-intact yeast cells incubated with COPII proteins produce freely diffusible, ER-derived vesicles that remain in
the supernatant fraction after centrifugation at 18,000 g for
3 min (Fig. 8). Upon addition of purified fusion factors, it
was observed that a significant fraction of these vesicles
(>50%) cosedimented with semi-intact cells and were not
contained in the supernatant fraction. This likely reflects
vesicles docked and/or fused with the Golgi complex that
sediment with semi-intact cells under the conditions of this
assay. To discern the roles of various budding and fusion factors in this process, the level of 35S-labeled gp- The addition of Uso1p alone significantly reduced the
amount of freely diffusible vesicles contained in the supernatant fraction in the presence of COPII proteins (Fig. 9,
compare black bars, columns 9 and 12). In the absence of
COPII proteins, there was residual budding and it is interesting to note that the level of diffusible vesicles under this
condition was also reduced upon addition of Uso1p (Fig.
9, compare black bars, columns 1 and 4). These results
may be explained if Uso1p were tethering ER-derived vesicles to the Golgi complex allowing for sedimentation of
vesicles with semi-intact cells. Alternatively, Uso1p could
be cross-linking vesicles together generating an intermediate of significant size to pellet at the applied g force. This
possibility was tested by incubating freely diffusible vesicles contained in a medium speed supernatant fraction (generated under the conditions shown in Fig. 9, column 9)
with purified Uso1p in the presence or absence of washed
semi-intact cells. Both Uso1p and semi-intact cells are required to pellet ER-derived vesicles contained in the medium speed supernatant fraction (data not shown). Therefore,
the most likely explanation for these observations is that
Uso1p tethers ER-derived vesicles to the Golgi compartment.
Although Uso1p reduces the level of freely diffusible
vesicles produced by COPII, vesicle fusion as measured by
Golgi specific glycosylation under this condition was modest (Fig. 9, hatched bar, column 12) and does not account
completely for the decrease in diffusible vesicles. These
observations suggest that vesicles dock, but require additional factors to promote bilayer fusion. Indeed, the addition of LMA1 resulted in a significant increase in vesicle
fusion (Fig. 9, hatched bar, column 14) that was largely dependent on Uso1p (Fig. 9, hatched bar, compare to column
10). Addition of Sec18p has a minor effect on vesicle fusion in this assay that may reflect ample levels of endogenous Sec18p associated with semi-intact cell membranes.
The effects of inhibitory amounts of Sec23p complex on
vesicle docking and fusion were also determined. As documented in Fig. 8 B, Sec23p complex is a potent inhibitor of
ER to Golgi transport at high concentrations. Upon addition of this inhibitor, transport was blocked (Fig. 9, hatched
bar, column 17) but the reduction in freely diffusible vesicles was quite similar to conditions of optimal targeting
(Fig. 9, compare to column 16). Thus, Sec23p complex appears to interfere with some aspect of membrane fusion, perhaps binding to SNARE molecules, but does not affect
vesicle docking. Together, these results suggest that components involved in Uso1p mediated vesicle attachment
are distinct from the components involved in vesicle fusion.
Ordering Uso1p, Sec18p, and LMA1 Requirements
Ordering of the Uso1p, Sec18p and LMA1 requirements
was pursued in the reconstituted vesicle fusion assay because the semi-intact cell assay does not show a strict dependence on exogenously added Sec18p. If Uso1p tethers
ER-derived vesicle to the Golgi compartment, the action
of Uso1p may produce a dilution resistant intermediate. Indeed, results shown in Fig. 10 support this concept. In
this experiment, the concentrations of vesicles, acceptor
membranes and purified proteins were lowered (see Fig.
10, figure legend) to manipulate this intermediate. The
maximal fusion efficiency was reduced under these conditions but a clear sensitivity to 10-fold dilution was observed (Fig. 10,
To determine the temporal requirements for Sec18p and
LMA1, the Uso1p-tethered intermediate was generated in
the presence of Sec18p or LMA1 for 20 min followed by
dilution with the missing factor (Fig. 11). In column 3, incubation in the first stage with Uso1p and LMA1, followed
by dilution with Sec18p resulted in a low fusion efficiency
whereas preincubation with Uso1p and Sec18p, and then
dilution with LMA1 (Fig. 11, column 4) resulted in a significant fusion signal. These results suggest that Sec18p
function precedes the LMA1 requirement. Further support
for this idea is shown in Fig. 11, columns 5-8 using a Sec23p
blocked intermediate. In column 5, potent inhibition of
vesicle fusion upon the addition of Sec23p complex was
observed in the reconstituted reaction as was found in the
semi-intact cell assay (Figs. 8 and 9). Dilution of this inhibited species with buffer alone or buffer containing Sec18p resulted in a modest increase in fusion efficiency. Strikingly, the addition of diluent containing LMA1 reverses the
Sec23p stalled intermediate and promotes vesicle fusion to
near maximal levels (Fig. 11, column 7). This result indicates Sec23p inhibition allows for Uso1p-mediated docking
and Sec18p action but prevents LMA1 function that leads to
vesicle fusion. In summary, these experiments demonstrate
that Sec18p function precedes the LMA requirement.
A cell-free assay that measures vesicle fusion has been reconstituted with Golgi membranes, purified ER-derived
vesicles and three soluble proteins: Sec18p, LMA1, and
Uso1p. In the presence of ATP and GTP, these proteins
fully substitute for a crude yeast cytosol in catalyzing vesicle fusion. In an overall transport reaction performed in
yeast semi-intact cells, COPII proteins, and fusion factors
are required for efficient transport. Even with saturating
levels of budding factors (COPII) or fusion factors (Uso1p,
Sec18p, and LMA1), transport remains coupled COPII produces freely diffusible ER-derived vesicles
from semi-intact cells (Salama et al., 1993 Chase of Uso1p-docked, dilution-resistant intermediates indicates a temporal order for the Sec18p and LMA1
requirements. Sec18p function precedes the LMA1 requirement. This was observed whether the dilution-resistant intermediate was accumulated in the presence of
Uso1p and Sec18p or through Sec23p complex inhibition.
These observations may be summarized in a model for
vesicle docking and fusion with the Golgi complex as follows. First, Uso1p tethers freely diffusible vesicles to the acceptor compartment in a reaction that is independent of
Sec18p and LMA1. Second, Sec18p activates SNARE
molecules contained on the acceptor and/or vesicle membranes. Third, LMA1 action leads to specific SNARE associations that are prevented by elevated Sec23p complex
concentrations. The features of Uso1p, Sec18p, and LMA1
are further discussed below in the context of this proposed
model.
A requirement for purified Uso1p and a role in vesicle
docking is consistent with much of the current literature
(for review see Pfeffer, 1996 Based on biochemical and rotary shadowing electron
microscopy experiments, p115 forms an extended homodimer with two globular NH2-terminal domains and a
parallel coiled-coil rod domain (Sapperstein et al., 1995 A requirement for Sec18p, the yeast homologue of NSF,
was expected from genetic experiments and from analyses
of several cell-free membrane fusion reactions that require
Sec18p/NSF (Block et al., 1988 A requirement for LMA1 in the fusion of ER-derived
vesicles to the Golgi was unexpected. However, this affect
is very reproducible and LMA1 activity appears to depend
on Sec18p function. LMA1 was initially discovered by fractionation of a yeast cytosol required to drive homotypic
vacuole fusion in vitro, and is composed of two polypeptides Genetic and biochemical approaches have implicated
additional soluble factors in the vesicle fusion stage of ER
to Golgi transport in yeast including Ypt1p (Rexach and
Schekman, 1991 The isolated fusion factors represent some, but not all of
the genetic requirements for vesicle fusion established by
analyses of secretion defection cells. Some of these factors
(such as Sly1p and Ypt1p) are peripherally bound to membranes and have required schemes designed to selectively
inactivate their function. In addition to the soluble factors
and peripherally bound proteins, several of the genetically
defined components of vesicle fusion encode integral membrane proteins such as Sec22p, Bet1p, Bos1p, and Sed5p
(Dascher et al., 1991). With respect to the membrane
fusion step, a set of general factors including N-ethylmaleimide-sensitive fusion protein (NSF)1 and soluble NSF attachment protein (SNAP) are required at many intracellular compartments (Block et al., 1988
; Eakle et al., 1988
;
Clary and Rothman, 1990
; Clary et al., 1990
). These general factors are used in concert with compartment specific
factors such as v/t-SNAP receptors (SNAREs) and small
GTPases that appear to be structurally related, yet impart
specificity in membrane docking and fusion. The basic
mechanisms of intracellular membrane fusion appear to
be conserved within a species and from yeast to mammals
(for review see Pfeffer, 1996
). Although several of the
molecules that catalyze specific intracellular membrane
fusion reactions have been identified through biochemical
and genetic approaches, a molecular description of this
process is incomplete.
-factor, the precursor
of a secreted pheromone in yeast (Baker et al., 1988
). The
application of an in vitro transport assay, together with genetic analyses of sec mutant strains has allowed the division of ER to Golgi transport into three distinct steps: vesicle
budding, vesicle targeting, and membrane fusion (Kaiser
and Schekman, 1991; Rexach and Schekman, 1991
). The
first step in this sequence, vesicle budding, has been reconstituted in vitro with a set of soluble factors (Sar1p, Sec23p
complex, and Sec13p complex) that collectively form a
membrane coat that drives budding from the ER. The vesicles formed with these purified protein fractions, termed
COPII-coated vesicles, are competent for fusion with the
Golgi apparatus in a reaction that requires cytosol and
ATP (Barlowe et al., 1994
). This cytosol provides multiple
protein components required for fusion, some of which are
defined by known secretion defective mutants (Lupashin
et al., 1996
).
; Xu et al.,
1997
). Although these purified factors are sufficient for
vesicle targeting and fusion to the Golgi complex, overall
ER to Golgi transport in yeast semi-intact cells is dependent on the addition of COPII proteins. Thus, six purified,
soluble proteins are shown to catalyze anterograde ER to
Golgi transport and are ordered in distinct steps of vesicle
budding, docking, and fusion.
Materials and Methods
leu2-3,112 ura3-52 pep4::URA3), RSY919 (MAT
ura3-1 mnn1 mnn2), RSY949 (MAT
lys2-801 trp1 ura3-52 uso1-1), CBY300 (MAT
his3
200 leu2
1, lys2
202 ura3-52 trp1
63 uso1-1), and
CBY324 (MAT
his3
200 leu2
1, lys2
202 ura3-52 trp1
63 uso1-1 pUSO1-Myc) were used in these studies. Rabbit antiserum specific for
-1,6-mannose linkages was prepared by intravenous injection of heat-treated RSY919 cells as previously described (Ballou, 1970
). Antibodies
directed against Sec7p (Franzusoff et al., 1991
), Sec13p (Salama et al.,
1993
), Sec18p (Haas and Wickner, 1996
), Sec19p (Haas et al., 1995
), Sec23p
(Hicke and Schekman, 1989), Sec26p (Duden et al., 1995), Trx1p (kindly
provided by Z. Xu, Dartmouth Medical School, Hanover, NH), Ypt1p
(Rexach et al., 1994
), and monoclonal 9E10 anti-myc (Evan et al., 1985
)
were used in these studies. In vitro-translated pre-pro-
-factor was synthesized (Baker et al., 1988
) using translation grade [35S]methionine (Amersham Corp., Arlington Heights, IL). Methods for SDS-PAGE (Laemmli,
1970
), silver staining (Bloom et al., 1987
), immunoblotting (Towbin et al.,
1979
), and immunodetection by enhanced chemiluminescence (Amersham Corp.) have been described. Deuterium oxide, Nycodenz, ATP, GTP,
creatine kinase, and phosphocreatine were purchased from Sigma Chemical Co. (St. Louis, MO).
). Oligonucleotides U1 (CGGGATCCAT GGACATCATT CAAGGACTG) and U2 (CCGAGCTCTC ACAAGTCTTC
TT-CAGAAATA AGCTTTTGTT CTTCTGCCAC TTGCCCTTCT TCCTC G) were used to amplify a portion of the USO1 gene, which was then
digested with the restriction enzymes XbaI and SacI and inserted into
pBluescript (Stratagene, La Jolla, CA) digested with XbaI and SacI. After
confirmation of this sequence, the 1.4-kb XbaI-SacI fragment was inserted into pSK47 digested with SacI and partially digested with XbaI. The resulting construct (pUSO1-myc) fully complements an uso1-1 strain (CBY300) for growth at 37°C.
). This lysate was thawed on ice and diluted with 10 ml of B88
and adjusted to final concentrations of 1 mM DTT and 1 mM PMSF. A
medium speed supernatant (MSS) fraction was prepared by centrifugation
at 25,000 g for 15 min (SS34 rotor; Sorvall, Newtown, CT), and then decanted from centrifuge tubes carefully excluding loosely sedimented
membranes. Aliquots of this MSS fraction were quick frozen in liquid nitrogen and stored at
75°C.
75°C. This cytosol fraction
serves as the starting material for fractionation procedures and was ~5
mg/ml protein, using BSA as a standard (Bradford, 1976
).
75°C. This preparation of acceptor membranes was ~3 mg membrane
protein/ml.
75°C.
) but after isolation, the peak fractions were
pooled and dialyzed against B88 that contained 0.1 mM DTT, 0.1 mM
PMSF, and 0.05 mM ATP. During dialysis, the solution turned turbid, and
the precipitate was removed by centrifugation at 20,000 g for 10 min. A
majority of Sec18p remained soluble after this dialysis step and was stored
in aliquots at
75°C. Purified LMA1 was a gift of Z. Xu (Xu et al., 1997
).
-factor
) with purified Sar1p, Sec23p complex, and Sec13p
complex (Barlowe et al., 1994
). First, posttranslational translocation of
pre-pro-
-factor into microsomal membranes was performed in 0.4 ml of
B88 containing 0.7 mg of membrane protein, 35S-labeled pp-
-factor (5 × 106 cpm), and an ATP regeneration system (Baker et al., 1988
) at 10°C for
15 min. After chilling on ice, membranes were diluted in B88, centrifuged
at 12,000 g for 3 min, and then washed twice with B88 by gentle resuspension in buffer before centrifugation. The final pellet was resuspended in
0.1 ml of B88 and mixed with 5 µg each of purified Sar1p, Sec13p complex, and Sec23p complex in 0.6 ml total vol of B88 containing the ATP regeneration system and 0.1 mM GTP. Budding reactions were incubated at
20°C for 10 min, and then placed on ice for 5 min. Vesicles were separated from microsomal membranes by centrifugation at 14,000 g for 5 min, and
0.55 ml of the supernatant fluid-containing vesicles was removed and
mixed with 0.8 ml of a 60% Nycodenz (wt/vol) in D2O containing 20 mM
K-Hepes, 150 mM KOAc, and 5 mM MgOAc. This mixture was layered
on the bottom of a SW60 tube (344062; Beckman Instruments Co.) followed by 0.9-ml layers of 25% Nycodenz, and 20% Nycodenz in the same
D2O-containing buffer. A final layer of B88 (1 ml) was placed on top, and
the tube was centrifuged at 50,000 rpm in a SW60 rotor for 3 h. The top 0.8 ml
was discarded, and then 0.15-ml fractions were taken and the vesicle peak
was determined by scintillation counting. Typically, the vesicle peak was
recovered in 0.3 ml and contained 35S-labeled, core-glycosylated pro-
-factor (~2,000 cpm/µl) that was protease protected, and precipitable with
concanavalin A-Sepharose beads (Pharmacia Biotechnology Inc.).
-factor),
and protein fractions (as indicated in figures) in a 30-µl reaction volume.
Assays were performed at 23°C in B88, which contained an ATP regeneration system and GTP (0.1 mM). After specified times (standard reactions were 60 min), 50 µl of SDS (2%) was added and tubes were heated to
95°C for 4 min. 1 ml of IP buffer (25 mM Tris-Cl, 150 mM NaCl, 1% Triton X-100) was added, followed by 15 µl of anti-
-1,6-mannose-specific serum and 35 µl of a 20% vol/vol solution of protein A-Sepharose (Pharmacia Biotechnology Inc.). Immunoprecipitation reactions were gently
rotated at room temperature for 2 h, and immune complexes were processed as previously described (Baker et al., 1988
). Vesicle fusion measured in these experiments reflects the amount of 35S-labeled gp-
-factor
that acquired the Golgi-specific
-1,6-mannose outer-chain modification
(cpm immunoprecipitated) as a percentage of the total 35S-labeled gp-
-factor, as determined by precipitation with concanavalin A linked to
Sepharose beads. Data points represent the average of duplicate determinations, where each duplicate set varied by <10%.
). Spheroplasts were perforated using low osmotic support buffer and ER to Golgi transport reactions performed in
two stages as described (Rexach and Schekman, 1991
). After stage I
(translocation of 35S-labeled pre-pro-
-factor into ER membranes), cells
were placed on ice, centrifuged at 15,000 g for 2 min in a refrigerated centrifuge (5417; Eppendorf Inc., Madison, WI). Cells were then gently resuspended and washed three times in B88. Transport reactions (30 µl) containing semi-intact cells, GTP, ATP regeneration system, and various
protein fractions were incubated at 23°C for 45 min, and then chilled on
ice for 5 min. A 5-µl aliquot was removed and treated with trypsin followed by solubilization in 1% SDS and precipitation with concanavalin
A-Sepharose to determine total protease protected 35S-labeled gp-
-factor (Baker et al., 1988
). Transport to the Golgi is expressed as the percentage of this concanavalin A-precipitable, 35S-labeled gp-
-factor that has
acquired Golgi-specific outer chain modification determined by precipitation with anti-
-1,6-mannose serum as described above for the vesicle fusion assay. Parallel (but separate) tubes were processed to quantify the
amount of freely diffusible, ER-derived vesicles present under each condition. After 5 min on ice, the samples were spun at 18,000 g for 3 min in a
refrigerated centrifuge (5417; Eppendorf Inc.). Aliquots (15 µl) were
taken from the resulting supernatant fluid (medium-speed supernatant)
and the amount of 35S-labeled gp-
-factor was determined after protease
treatment and precipitation with concanavalin A-Sepharose (Rexach and
Schekman, 1991
). Budded vesicles are expressed as the percentage of total 35S-labeled gp-
-factor that is contained in the medium-speed supernatant fraction. Data points represent the average of duplicate determinations, where each duplicate set varied by <10%.
Results
). This serves as a starting
point for the isolation of soluble factors contained in a
yeast cytosol preparation that catalyze vesicle fusion in an
assay that is independent of factors required for vesicle
budding. A facile and reproducible assay to measure this
process is established in this report whereby the components of this assay (purified ER-derived vesicles, yeast cytosol, and Golgi membranes) could be prepared in large
quantities and stored at
75°C for extended periods of
time with minimal loss of activity.
Fig. 1.
Separation of fusion factors by Mono Q anion
exchange chromatography.
Isolated ER-derived vesicles
containing 35S-labeled gp-
-factor and acceptor membranes were incubated in a
30-µl reaction with the following protein fractions: 50 µg of cytosol (CYT), 50 µg of
the Q flowthrough (QFT), 50 µg of the 0.75 M eluate
(Q.75), or 15 µg of the 1.5 M
eluate (Q1.5). The percent
vesicle fusion represents the
amount of 35S-labeled gp-
-factor that has been modified by the addition of Golgi
specific
-1,6-mannose residues. In this experiment, the amount
of background fusion (complete reaction minus cytosol) was
2.7%.
[View Larger Version of this Image (13K GIF file)]
), and this
protein was detected in the Q1.5 eluate. Sec23p, a subunit of the COPII complex required for vesicle budding (Hicke
and Schekman, 1989) was also contained in the Q1.5 fraction. Sec26p, the
-COP subunit of yeast COPI complex
(Duden et al., 1994
) elutes at ~0.73 M KOAc (Hosobuchi
et al., 1992
) and was found in both the Q.75 and the Q1.5
fractions. Sec18p, the yeast homologue of NSF, was found
exclusively in the Q.75 fraction; however, yeast thioredoxin, a protein involved in homotypic vacuolar membrane fusion (Xu and Wickner, 1996
) was not bound to
this anion exchange resin under these conditions and was
contained in the flow through fraction. Together, these results indicate the column was not overloaded with cytosolic protein, and that several protein species were effectively resolved. In addition to the proteins shown in Fig. 2,
Sec19p, which is the yeast GDP dissociation inhibitor (GDI) (Garrett et al., 1994
), and the small GTPase, Ypt1p, were
detected in both the QFT and the Q.75 fractions and monitored throughout the following purification procedures.
Fig. 2.
Immunoblot analysis of Mono Q fractions.
Equal volumes of the load
fraction (L), flow-through
fraction (QFT), proteins
eluting at 0.75 M KOAc
(Q.75), and proteins eluting
at 1.5 M KOAc (Q1.5) obtained as described under the
methods section, were resolved by SDS-PAGE, followed by immunoblot for
Sec7p, Sec26p, Sec23p, Sec18p,
and thioredoxin (Trx1p).
[View Larger Version of this Image (74K GIF file)]
). Mutant sec18 yeast strains are defective for
ER to Golgi transport in vivo and in vitro although this
block has not been readily restored in various cell-free assays (Rexach and Schekman, 1991
; Lupashin et al., 1996
).
Recombinant forms of NSF and Sec18p that contain six
His residues at their COOH termini have been constructed, which may be overproduced in Escherichia coli
(Whiteheart et al., 1994
), and appear to be fully functional
in cell-free assays (Whiteheart et al., 1994
; Haas et al.,
1996). Since Sec18p plays a central role in intracellular
membrane fusion reactions, the six His-tagged protein was
isolated in a buffer compatible with this assay and tested
for promotion of vesicle fusion to the Golgi complex. Sec18p could completely substitute for the Q.75 fraction (Fig. 3 A)
at a concentration similar to that contained in a saturating
amount of this fraction (see Fig. 6 B). Furthermore, the
purified Sec18p alone was not sufficient for fusion of ER-derived vesicles but required the addition of both the QFT
and the Q1.5 for a maximal signal in the vesicle fusion assay (Fig. 3 A).
Fig. 3.
Sec18p replaces the Q.75 fraction. (A) Vesicle fusion
was measured (as in Fig. 1) with various fractions in the presence
or absence of purified Sec18p-6His (50 ng) in 30 µl. (B) Vesicle fusion in the presence of QFT and Q1.5 plus increasing amounts of Sec18p-6His. In this experiment, a plus cytosol control yielded 20% vesicle fusion.
[View Larger Version of this Image (16K GIF file)]
Fig. 6.
LMA1 replaces the
QFT fraction. In A, isolated vesicles and acceptor membranes
were incubated with Sec18p (50 ng) and Uso1p (75 ng) alone
(column 2) or with increasing
amounts of purified LMA1 (columns 3-6) in 30-µl reactions. In
B, immunoblots with antithioredoxin and anti-Sec18p are shown
to compare 10 ng of purified
LMA1 and Sec18p-6His with 5 µg
of crude cytosol (Load) and other
column fractions as described in
Fig. 2. The pure proteins loaded
represent ~1/5 of the amount required for saturation and the
crude cytosol shown (Load) represents ~1/10 the amount required for saturation.
[View Larger Versions of these Images (42 + 15K GIF file)]
; Franzusoff et al., 1991
;
Franzusoff et al., 1992
; Lupashin et al., 1996
) and Uso1p
(Nakajima et al., 1991
; Seog et al., 1994
; Lupashin et al.,
1996
; Sapperstein et el., 1996). A series of experiments were performed to determine if either or both of these
proteins are functional components of the Q1.5 fraction.
An antibody that recognizes Sec7p (Franzusoff et al., 1992
)
was used to analyze the fractions eluting from the Superose 6 column, and a broad peak of Sec7p immunoreactivity was detected that was offset from the activity peak (not
shown). However, there was clearly Sec7p immunoreactivity contained in the peak activity fraction raising the possibility that Sec7p plus additional factors are provided from
the Superose 6 activity peak. To exclude this possibility, a
cytosol was prepared from a mutant yeast strain that is
rendered temperature sensitive due to an amber mutation
in the USO1 gene (Seog et al., 1994
). Cytosol prepared
from the mutant strain was directly compared to a wild-type cytosol by fractionation on a Mono Q column (see
Materials and Methods). In this experiment (Fig. 4), a linear gradient of increasing ionic strength was used to elute
proteins from 0.75 to 1.5 M KOAc instead of the step gradient used to prepare the Q1.5 fraction. Under these conditions, two observations strongly suggest that Uso1p
alone is the active component present in the Q1.5 fraction.
First, assay of individual fractions across the elution profile of a wild-type cytosol reveal a single peak of activity
when combined with purified Sec18p and QFT fractions
while cytosol prepared from an uso1-1 strain did not contain detectable activity eluting at this ionic strength (Fig.
4). Second, immunoblot analysis of wild-type fractions indicated Sec7p immunoreactivity was resolved from the
peak of vesicle fusion activity on this gradient and eluted
before the activity peak (not shown).
Fig. 4.
An uso1-1 strain
lacks Q1.5 activity. Cytosols
from a wild-type and uso1-1
strain were bound and eluted
from a Mono Q column. Individual fractions eluting
from the 0.75 to 1.5 M KOAc
gradient were tested (2 µl)
for stimulation of vesicle fusion in the presence of QFT
and Sec18p in a 30-µl reaction volume. Data represents fusion above the QFT
and Sec18p level that was
5.2% in this experiment.
[View Larger Version of this Image (15K GIF file)]
). Expression of Uso1p-myc from a multicopy vector
in a uso1-1 temperature sensitive strain fully complements when grown at 37°C indicating the c-myc extension does
not interfere with Uso1p function. This strain was used to
overproduce and purify Uso1p-myc by a two-step procedure using anion exchange and gel filtration chromatography as described under Materials and Methods. The final
purification step (elution from the Superose 6 column) is
shown in Fig. 5. The peak of fusion activity that elutes
from the Superose 6 column at 800 kD coincided with a
single 210-kD polypeptide species observed on SDS-polyacrylamide gel and c-myc immunoreactivity. These fractionation properties are consistent with previous studies
suggesting Uso1p forms a nonglobular oligomer due to an
extended coiled-coil rod structure similar to a related protein in mammalian cells, termed p115 (Waters et al., 1992
; Seog et al., 1994
; Sapperstein et al., 1995
). The peak fractions eluting from the Superose 6 column were pooled
(16-17) for use in later experiments, and neither Sec7p nor
Sec26p (
-COP) could be detected in this pooled fraction
by immunoblot analysis.
Fig. 5.
Purification of functional Uso1p-myc. Fractions eluting
from a Superose 6 column were analyzed by SDS-PAGE and silver stained (A), immunoblotted with anti-myc mAb (B), and
then assayed for stimulation of vesicle fusion in the presence of
QFT and Sec18p-6His (C). Fraction 17 contains the peak of activity and anti-myc immunoreactivity. Activity data represents vesicle
fusion above the QFT and Sec18p level that was 7% in this experiment.
[View Larger Versions of these Images (11 + 61K GIF file)]
). Addition of
fractions enriched in Ypt1p or Sec19p did not stimulate
nor replace the QFT requirement when combined with
purified Uso1p and Sec18p (not shown). However, the addition of purified LMA1 stimulated the cell-free vesicle fusion reaction and when saturating levels of protein were
added, fusion efficiencies at or above cytosolic levels were
obtained (Fig. 6 A). The amounts of Sec18p and LMA1 required for saturation are comparable to levels contained in
a crude cytosol (Fig. 6 B). Saturating levels of LMA1 alone
did not support vesicle fusion in the absence of Uso1p and
Sec18p (Fig. 7 A).
Fig. 7.
Reconstitution of vesicle fusion with purified factors.
(A) Isolated, ER-derived vesicles containing 35S-labeled gp--factor and acceptor membranes (12 µg) were incubated with saturating amounts of individual purified factors (Sec18p [50 ng], Uso1p
[75 ng], and LMA1 [50 ng]) or various combinations in 30-µl reactions. (B) Time course of vesicle fusion in the presence of purified
proteins at 4°C (
), at 23°C (
), or with crude cytosol (50 µg) at
23°C (
).
[View Larger Version of this Image (16K GIF file)]
) required semi-intact yeast cells and cytosol. If the vesicle fusion assay used in this current study represents an authentic subreaction of the overall transport process, these isolated fusion factors should be required for
overall transport. Furthermore, if vesicle budding is a prerequisite for membrane fusion, the isolated fusion factors
should be most effective in the presence of the COPII proteins (Sar1p, Sec23p complex, and Sec13p complex) that
drive vesicle formation (i.e., fusion is coupled to budding).
Washed semi-intact cells were first incubated with levels
of COPII proteins that promote maximal budding efficiencies in the presence of purified fusion factors. Overall
transport could not be detected above background under
these initial conditions. However, a titration revealed that
high concentrations of COPII proteins inhibit transport
whereas lower concentrations of the coat constituents
stimulate transport, even though suboptimal for vesicle
budding (Fig. 8 A). Transport of 35S-labeled gp-
-factor to
the Golgi was maximally 21% efficient in the reconstituted
reaction compared to a background of 3%. As observed in
Figs. 8 and 9, both COPII (2 ng/µl) and purified fusion factors were necessary for overall ER to Golgi transport in
semi-intact cells indicating vesicle budding is a prerequisite for membrane fusion. To determine if the production
of vesicle intermediates simply satisfies a spatial separation of the ER and Golgi present in semi-intact cells, a
similar experiment was performed using diffusible ER
(microsomes) and acceptor membranes incubated with fusion factors in the presence or absence of COPII (2 ng/µl)
as in Fig. 8 A. Under this condition, addition of both fusion
factors and COPII were again required for a maximal stimulation of transport (not shown), similar to that observed
in semi-intact cells. These results suggest that COPII budding
activates factor(s) involved in membrane fusion and that
direct fusion of ER with Golgi membranes is not efficient.
Fig. 8.
Excess COPII proteins inhibit ER to Golgi transport.
(A) Budding and transport assays with semi-intact yeast cells in
the presence of purified factors. Budding of 35S-labeled gp--factor into freely diffusible vesicles (
) was determined in the presence of increasing amounts of COPII proteins alone (no fusion
factors added). COPII concentrations reflect individual protein
concentrations such that the 2 ng/µl COPII condition is 2 ng/µl
Sar1p, 2 ng/µl Sec23p complex, and 2 ng/µl Sec13p complex.
Transport (
) was quantified in the presence of fusion factors
(Sec18p [50 ng], Uso1p [75 ng], and LMA1 [50 ng]) and varying
concentrations of COPII proteins in 30-µl reactions. (B) Transport in the presence of fusion factors (as in A) and COPII proteins (2 ng/µl), plus 8 ng/µl of Sar1p (S), or 8 ng/µl Sec13p complex (13), or 8 ng/µl Sec23p complex (23).
[View Larger Version of this Image (21K GIF file)]
Fig. 9.
Purified fusion factors mediate distinct steps in vesicle
docking and fusion. Saturating amounts of fusion factors (Sec18p [50 ng], Uso1p [75 ng], LMA1 [50 ng]) and 2 ng/µl COPII proteins were mixed in various combinations with semi-intact cells.
35S-labeled gp--factor contained in freely diffusible vesicles (black
bars) and Golgi modified forms (hatched bars) were determined after 45 min at 23°C. The incubation on the far right (+) contains an additional 8 ng/µl of Sec23p complex.
[View Larger Version of this Image (30K GIF file)]
). Interestingly, only one of the COPII components, the Sec23p complex, was required for potent inhibition of vesicle fusion (Fig. 8 B). The inhibition caused by
elevated concentrations of Sec23p complex could be at
vesicle docking or bilayer fusion. A method to monitor these
separate events was devised in the following experiment.
-factor
contained in freely diffusible vesicles and outer chain modified forms were quantified under various conditions (Fig. 9).
). Preincubation of vesicles and acceptor
membranes with Uso1p alone generated a dilution resistant species that may be chased upon addition of Sec18p
and LMA1 (Fig. 10,
). In contrast, incubation with
Sec18p or LMA1 alone does not produce dilution resistance even when adequate levels of the missing fusion factors are supplied in the diluent. These results again suggest
Uso1p function is independent from and precedes Sec18p
and LMA1 action. Incubation with Uso1p alone followed
by dilution with Sec18p and LMA1 (Fig. 10,
) was not
the same as that observed for the complete reaction diluted with buffer containing ATP (Fig. 10,
). This was
due to dilution of the Sec18p and LMA1 proteins under
the latter condition whereas active concentrations of these
fusion factors were maintained under the former. Therefore at early time points, Uso1p-docked vesicles efficiently
chase due to maintenance of LMA1 and Sec18p throughout the second incubation but docked vesicles for the complete reaction (Fig. 10,
) were not chased as efficiently
because of dilution of Sec18p and LMA1. At later times
(20 and 30 min), the fusion efficiency of the Uso1p-docked intermediate was not as efficient as that observed with the
complete reaction. This may be because of lability of the
docked intermediate in the absence of Sec18p and LMA1.
Fig. 10.
Uso1p action generates a dilution resistant intermediate. Isolated vesicles were mixed on ice with acceptor membranes
and individual fusion factors Uso1p (U), LMA1 (L), and Sec18p
(18), or the set of fusion factors together (U/L/18). In these experiments, the concentrations of acceptor membranes, vesicles,
and fusion factors were half of the standard condition (described
in Fig. 7). After incubation at 23°C for various times, reactions
were diluted 10-fold with one of the following: buffer containing
ATP, LMA1, and Sec18p (L/18); buffer containing ATP, Uso1p,
and Sec18p (U/18); buffer containing ATP, Uso1p, and LMA1
(U/L); or buffer containing ATP alone (B88). Diluents contained
purified proteins at levels that produce initial concentrations of
each indicated species. Each tube was incubated at the reaction
temperature for a total of 90 min. In this experiment, background
fusion (vesicles, acceptor membranes, and ATP, undiluted) was
1.7% and maximal fusion (vesicles, acceptor membranes, fusion
factors, and ATP, undiluted) was 12%.
[View Larger Version of this Image (26K GIF file)]
Fig. 11.
Sec18p is required before LMA1 action. Reaction
conditions are as described in Fig. 10 except Sec23p (6 ng/µl) was
added to indicated reactions. After a 20-min incubation at 23°C,
reactions were left undiluted (NA) or diluted 10-fold with buffer
containing ATP (B88), buffer containing ATP and Sec18p (18),
buffer containing ATP and LMA (L), or buffer containing ATP,
LMA, and Sec18p (L/18). Each tube was incubated at the reaction temperature for a total of 90 min. In this experiment, background fusion was 2.3% and maximal fusion was 19%.
[View Larger Version of this Image (20K GIF file)]
Discussion
budding
must precede membrane fusion. Because direct membrane
fusion of ER with the Golgi is not efficient in vitro, it is
probable that vesicle formation activates components involved in fusion as well as bridging a spatial barrier separating these organelles. This result contrasts reconstituted
intra-Golgi transport where membrane fusion can be
driven in the absence of COPI, the membrane coat that
forms Golgi-derived vesicles (Malhotra et al., 1989
; Elazar et al., 1994
).
) or microsomal
membranes (Barlowe et al., 1994
). In this report, it is
shown that the addition of Uso1p in the presence of vesicles and Golgi acceptor members reduces the level of diffusible vesicles and produces a docked, dilution-resistant
intermediate. LMA1 and Sec18p are then required for the
fusion of Uso1p docked vesicles. Elevated concentrations of the Sec23p complex are found to reversibly inhibit vesicle fusion but do not interfere with Uso1p-mediated docking. Normally, the Sec23p complex acts in vesicle formation and is proposed to bind cargo and targeting molecules
for packaging into COPII-coated vesicles (Schekman et al.,
1995
; Campbell and Schekman, 1997
). Therefore, this inhibitory effect may be due to competition between Sec23p
complex and component(s) of the fusion machinery. For
example, Sec23p is detected in a specific complex with ER
to Golgi v-SNARES, including Sec22p (Kuehn et al.,
1997
) and Bet1p (Barlowe, C., unpublished observation).
These observations suggest reasonable sites of inhibition
to test, however there may be other unknown components
of the fusion machinery that interact with the Sec23p complex and prevent vesicle fusion.
) but has not been directly demonstrated until this report. The function of Uso1p as a general fusion factor is not well established, but evidence indicates that a related protein in mammalian cells (termed
p115) participates in multiple transport processes including intra-Golgi transport (Waters et al., 1993; Sapperstein
et al., 1995
), the formation of Golgi cisternae after mitotic
disassembly (Rabouille et al., 1995
), and in transcytotic membrane traffic (Barroso et al., 1995
). In yeast, genetic
and biochemical evidence clearly implicates Uso1p in ER
to Golgi transport, placing the requirement after vesicle
formation but before SNARE complex assembly (Nakajima et al., 1991
; Lupashin et al., 1996
; Sapperstein et al.,
1996
). Although a role for Uso1p in other transport processes cannot be excluded, the essential role of this protein
appears to be in early stages of the yeast secretory pathway since overproduction of specific ER to Golgi SNARE
proteins are able to suppress uso1-1 temperature-sensitive
mutations as well as an uso1 deletion (Sapperstein et al.,
1996
).
).
Uso1p is a 206-kD protein that shares overall amino acid
identity with p115 in both the NH2-terminal globular domains as well as portions of the coiled-coil domains and
electron microscopy experiments indicate Uso1p is arranged in a similar parallel homodimer (Yamakawa et al.,
1996
). Uso1p possesses a predicted coiled-coil rod domain
about twice the size of p115. Interestingly, p115 is reported
to act in conjunction with a Golgi matrix protein (termed GM130) whose sequence predicts extended coiled-coil structures (Nakamura et al., 1995
). It has been suggested that
Uso1p represents a fusion of p115 and GM130 although
this possibility has not been explored (Nakamura et al.,
1997
). Because a soluble form of Uso1p is required for
vesicle docking in vitro, binding sites for Uso1p are likely
contained on purified ER-derived vesicles; the identification of protein(s) comprising this binding site are currently
under investigation.
; Beckers et al., 1989
; Diaz
et al., 1989
; Rexach and Schekman, 1991
; Söllner et al., 1993
;
Mayer et al., 1996
). Sec18p/NSF is a homo-oligomeric ATPase that promotes bilayer fusion through interactions with
membrane-bound SNAP (soluble NSF attachment protein)
and SNAREs. While a role for Sec18p/NSF activity in separating SNARE protein complexes seems clear (Söllner
et al., 1993
; Sogaard et al., 1994
; Otto et al., 1997
), the
placement of this activity in the context of membrane
docking and fusion is debated. Sec18p/NSF function may
be required to activate SNARE molecules before formation of v/t-SNARE complexes (Morgan and Burgoyne,
1995
; Mayer et al., 1996
; Otto et al., 1997
), or to drive
membrane fusion after assembly of the v/t-SNARE complex (Söllner et al., 1993
; Sogaard et al., 1994
). In this report, Sec18p function is not required for vesicle docking
and acquisition of dilution resistance, however, Uso1p docking may be independent of SNARE protein function.
In support of this notion, experiments with thermosensitive
sed5-1 and sly1-1 membranes in the reconstituted assay
demonstrate Uso1p-mediated docking does not depend on
Sed5p or Sly1p function whereas vesicle fusion requires
their action (Cao, X., and C. Barlowe, manuscript in preparation). Thus, the status of ER to Golgi SNARE proteins during different stages of this reconstituted reaction need
to be determined before Sec18p/NSF function in activation or bilayer fusion can be unequivocally assigned.
thioredoxin plus IB2 (Xu et al., 1996). In cell extracts IB2 can be found in association with thioredoxin
(LMA1) or as a monomeric species (LMA2), each capable
of stimulating vacuolar membrane fusion (Xu et al., 1997
).
Addition of LMA1 and Sec18p replaces the requirement
for crude cytosol in a fusion reaction between salt washed
vacuoles. Results from this system indicate the action of
LMA1 and Sec18p are coupled with a Sec18p requirement
that precedes LMA1 function (Xu et al., 1997
). Experiments with ER to Golgi transport described in this report
indicate a similar order for Sec18p and LMA1. Both subunits of LMA1 are needed for normal vacuole inheritance
in vivo although neither subunit is essential for cell viability, nor are there detectable delays of secretory protein
transport in strains lacking the IB2 subunit (Barlowe, C.,
unpublished observation). However, there is a potential open reading frame (YHR138c) contained in the yeast genome that shares significant amino acid identity with IB2
and may compensate for the loss of IB2 function. Further
in vivo analyses can address this question. Regardless, the
involvement of LMA1 in ER to Golgi transport and homotypic vacuolar membrane fusion using very different cell
free assays in addition to a functional relationship with Sec18p suggests a general role for this factor in trafficking.
), Sly1p and Sec7p (Lupashin et al., 1996
).
Furthermore, ADP-ribosylation factor (ARF) and COPI
are proposed to operate at this stage in mammalian cells
(Aridor et al., 1995
), and there is increasing evidence that
the protein machinery of ER to Golgi transport is highly conserved between yeast and mammals (Orci et al., 1991
;
Kuge et al., 1994
; Shaywitz et al., 1995
; Dascher and Balch,
1996
; Hay et al., 1997
). Requirements for Ypt1p, Sly1p,
Sec7p, ARF, and COPI are not detected through the biochemical approach used here either because of ample peripherally bound species on Golgi membrane preparations
or the cell-free assay may not reproduce all of the in vivo
requirements. Alternatively, these proteins may not be directly required for vesicle fusion. For Ypt1p, and Sly1p, it seems likely that sufficient protein is contained on acceptor membranes since >90% of these species sediment with
the membrane fraction (Cao, X., and C. Barlowe, unpublished observation). In vivo experiments with a version of
Ypt1p that contains a transmembrane segment on the
COOH terminus, which converts the protein to an integral
membrane species, is functional, and suggests that a soluble form of this protein is not essential for membrane fusion (Ossig et al., 1995
). Significant pools of soluble Sec7p
(Franzusoff et al., 1991
) and COPI subunits (Hosobuchi et
al., 1992
; Stirling et al., 1992) are found in yeast cells.
These proteins may cooperate in the production of COPI-coated vesicles since recent data indicates that Sec7p homologues encode guanine nucleotide exchange factors for
ARF GTPases (Chardin et al., 1996
; Morinaga et al., 1996
; Peyroche et al., 1996
) and it is this activated GTP-bound
form of ARF that triggers assembly of COPI vesicles. In
mammalian cells, experiments suggest there is a sequential
coupling between COPI and COPII vesicle coats in ER to
Golgi transport such that after budding from the ER, the
COPII coat is shed and an additional round of coating and
uncoating by COPI is required to complete transport of
secretory proteins to the cis-Golgi (Aridor et al., 1995
;
Rowe et al., 1996
). Purified, ER-derived vesicles from
yeast are not enriched in ARF or COPI subunits (Barlowe
et al., 1994
). If COPI is required for sequential assembly
onto purified, ER-derived vesicles, it must be provided
from the acceptor membrane fraction in reconstitution experiments such as shown in Fig. 7 of this report, or there
may be no direct requirement for COPI in this assay. Regardless, an acceptor membrane preparation depleted of COPI, ARF, and Sec7p will be required to rigorously address this issue.
; Newman et al., 1990
; Shim et al., 1991
;
Hardwick et al., 1992). To advance the long-term goal of
reconstituting this process with pure protein and lipid fractions, the solubilization and reconstitution of ER-derived
vesicles will be required.
Received for publication 9 May 1997 and in revised form 30 September 1997.
I thank A. Haas, S. Sapperstein, G. Waters, and W. Wickner for gifts of plasmids, antibodies, and reagents. I am indebted to Zuoyu Xu for his advice.This work was supported by a grant from the National Institute for General Medical Sciences.
ARF, ADP-ribosylation factor; B88, buffer 88; GDI, GDP dissociation inhibitor; LMA1 and LMA2, low molecular weight activity 1 and 2; MSS, medium speed supernatant; NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; YPD, yeast extract, peptone, dextrose.
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