Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes

Abstract

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.

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The Journal of Cell Biology, Volume 149, Number 1, April 3, 2000 55–65
http://www.jcb.org 55
Asymmetric Requirements for a Rab GTPase and SNARE Proteins in
Fusion of COPII Vesicles with Acceptor Membranes
Xiaochun Cao and Charles Barlowe
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
Abstract.
Soluble NSF attachment protein receptor
(SNARE) proteins are essential for membrane fusion
in transport between the yeast ER and Golgi compart-
ments. Subcellular fractionation experiments demon-
strate that the ER/Golgi SNAREs Bos1p, Sec22p,
Bet1p, Sed5p, and the Rab protein, Ypt1p, are distrib-
uted similarly but localize primarily with Golgi mem-
branes. All of these SNARE proteins are efficiently
packaged into COPII vesicles and suggest a dynamic
cycling of SNARE machinery between ER and Golgi
compartments. Ypt1p is not efficiently packaged into
vesicles under these conditions. To determine in which
membranes protein function is required, temperature-
sensitive alleles of
BOS1
,
BET1
,
SED5
,
SLY1
,
and
YPT1
that prevent ER/Golgi transport in vitro at re-
strictive temperatures were used to selectively inacti-
vate these gene products on vesicles or on Golgi mem-
branes. Vesicles bearing mutations in Bet1p or Bos1p
inhibit fusion with wild-type acceptor membranes, but
acceptor membranes containing these mutations are
fully functional. In contrast, vesicles bearing mutations
in Sed5p, Sly1p, or Ypt1p are functional, whereas ac-
ceptor membranes containing these mutations block fu-
sion. Thus, this set of SNARE proteins is symmetrically
distributed between vesicle and acceptor compart-
ments, but they function asymmetrically such that
Bet1p and Bos1p are required on vesicles and Sed5p
activity is required on acceptor membranes. We pro-
pose the asymmetry in SNARE protein function is
maintained by an asymmetric distribution and require-
ment for the Ypt1p GTPase in this fusion event. When
a transmembrane-anchored form of Ypt1p is used to re-
strict this GTPase to the acceptor compartment, vesi-
cles depleted of Ypt1p remain competent for fusion.
Key words: trafficking • Golgi apparatus • endoplas-
mic reticulum • secretion • membrane fusion
Introduction
A related family of integral membrane proteins, termed
SNAREs (soluble NSF attachment protein receptors),
1
mediate a variety of intracellular membrane fusion reac-
tions in eukaryotic cells (for review see Gotte and Fischer
von Mollard, 1998). For example, SNARE proteins func-
tion in heterotypic fusion reactions including the fusion of
synaptic vesicles with presynaptic membranes (Robinson
and Martin, 1998) as well as homotypic fusion reactions
that underlie organelle inheritance and dynamics (Nichols
et al., 1997; Patel et al., 1998; Rabouille et al., 1998). Stud-
ies on the synaptic fusion machinery were the first to iden-
tify SNARE proteins, namely syntaxin 1, SNAP25 (synap-
tosome-associated protein of 25 kD) and VAMP (vesicle-
associated membrane protein, also known as synaptobrevin)
that form a stable 7S complex (Bennett et al., 1992). Based
largely on sequence comparisons, intracellular SNAREs
have been divided into two general categories, designated
v-SNARE for vesicle-SNARE (related to synaptobrevin)
and t-SNARE for target-SNARE (related to syntaxin 1 or
SNAP25). However, these designations can be difficult in
some instances because v- and t-SNAREs are likely to
have evolved from a common ancestral gene and belong to
the same superfamily (Weimbs et al., 1997, 1998). Both v-
and t-SNARE proteins possess 50–60 amino acid stretches
with the potential to form a coiled-coil structure that are
typically near their COOH-terminal transmembrane do-
mains. Indeed, structural analyses on neural SNARE pro-
teins indicate that v- and t-SNAREs form parallel het-
erodimers (Lin and Scheller, 1997) and heterotetramers
(Poirier et al., 1998; Sutton et al., 1998). The formation of
this stable SNARE bundle has been proposed to create a
fusion pore bridging opposed membranes (Sutton et al.,
Address correspondence to Charles Barlowe, Department of Bio-
chemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH
03755. Tel.: (603) 650-6516. Fax: (603) 650-1353. E-mail: barlowe@
dartmouth.edu
1
Abbreviations used in this paper:
gp-
␣
F, glyco-pro-
␣
factor; NSF,
N
-ethylmaleimide–sensitive factor; SNAP25, synaptosome-associ-
ated protein; SNARE, soluble NSF attachment protein receptor; t- or
v-SNARE, target or vesicle SNARE, respectively.

The Journal of Cell Biology, Volume 149, 2000 56
1998; Weber et al., 1998). Although SNARE complexes
are thought to be an essential feature of intracellular
membrane fusion events, their precise function in bilayer
fusion reactions remains obscure. It is generally accepted
that the ATPase activity of NSF/Sec18p disrupts SNARE
complexes (Wilson et al., 1992; Sollner et al., 1993b). How-
ever, different models have been proposed such that NSF
drives bilayer fusion as SNARE proteins are disassembled
(Sollner et al., 1993a) or, alternatively, that separation of
SNARE complexes is a priming event that enables reas-
sembly of SNARES in trans to promote bilayer fusion
(Chamberlain et al., 1995; Mayer et al., 1996). Additional
debate centers on the function of SNARE proteins in
membrane fusion events; specifically, do these proteins
form a fusion pore (Sutton et al., 1998; Weber et al., 1998)
or are their associations part of a reaction series leading to
bilayer fusion but do not constitute a fusion pore (Coors-
sen et al., 1998; Ungermann et al., 1998).
In addition to SNARE proteins, GTPases of the Rab/
Ypt family appear to impart another layer of specificity to
SNARE-dependent membrane fusion events through the
concerted activity of distinct GTPases with specific sets of
SNAREs. Rab/Ypt proteins appear to act before SNARE
protein function (Dascher et al., 1991; Sogaard et al., 1994;
Lupashin and Waters, 1997), and allow fusion partners to
pair before engaging SNARE proteins (Cao et al., 1998).
Because single SNARE proteins are capable of participat-
ing in multiple fusion events (Fischer von Mollard et al.,
1997; Spang and Schekman, 1998), upstream regulation by
Rab/Ypt members may be crucial determinates in specify-
ing membrane fusion.
In
Saccharomyces cerevisiae
, genetic and biochemical
approaches have implicated the SNAREs Sec22p, Bet1p,
Bos1p, Ykt6, and Sed5p (Newman and Ferro-Novick,
1987; Newman et al., 1990; Dascher et al., 1991; Hardwick
et al., 1992; Lian and Ferro-Novick, 1993; McNew et al.,
1997) in transport between the ER and the Golgi complex.
All of these SNAREs coprecipitate in a complex with
Sed5p when isolated from a
sec18
mutant strain (Sogaard
et al., 1994). In addition to these proteins, Sly1p, Sft1p, and
p28 are found in this complex (Sogaard et al., 1994; Ban-
field et al., 1995). A similar cast of proteins has been char-
acterized in mammalian ER/Golgi transport and desig-
nated syntaxin 5, rsly1, rsec22, rbet1, membrin, and GOS28
(Dascher et al., 1994; Nagahama et al., 1996; Subramaniam
et al., 1996; Hay et al., 1997, 1998; Xu et al., 1997; Zhang et
al., 1997). The subcellular distributions of Sed5p and its ho-
mologue syntaxin 5 have been investigated, and evidence
indicates these proteins localize to Golgi membranes but
cycle between the ER, intermediate compartment, and the
Golgi complex (Hay et al., 1998; Rowe et al., 1998; Wood-
ing and Pelham, 1998). A similar observation was docu-
mented for the neuronal plasmalemma t-SNARE syntaxin
1, which distributes between synaptic vesicles and the cell
surface (Walch-Solimena et al., 1995). Thus, a strict dis-
tribution of some SNAREs to donor membranes and
t-SNARES to acceptor membranes does not seem to be a
general feature of these heterotypic fusion reactions. The
question arises as to how directionality is imparted to trans-
port processes if SNARE proteins are cycling between
compartments such as the Golgi and ER.
In this report, we first investigate the subcellular distri-
bution of ER/Golgi SNARE proteins in yeast and mea-
sure their incorporation into COPII-coated vesicles. The
localization of Bet1p, Bos1p, Sed5p, and Ypt1p have been
documented (Newman et al., 1992; Hardwick and Pelham,
1992; Preuss et al., 1992; Lian and Ferro-Novick, 1993);
however, we sought to directly compare the level of colo-
calization among these and other proteins involved in
transport between the ER and the Golgi complex. We used
a reconstituted ER/Golgi transport assay to determine the
compartmental requirements for specific SNAREs in fu-
sion of ER-derived vesicles with Golgi membranes. Our
approach is through selective inactivation of protein func-
tion on isolated ER-derived vesicles or on isolated accep-
tor membranes. Although the SNARE molecules appear
to be symmetrically distributed between vesicle and accep-
tor compartments, the functional requirements are asym-
metric such that Bet1p and Bos1p are required on vesicles
and Sed5p activity is required on acceptor membranes. A
requirement for Sec22p activity in anterograde transport
was not detected. We propose the asymmetry in SNARE
protein function is maintained by an asymmetric distribu-
tion and requirement for Ypt1p in this fusion event.
Materials and Methods
General Materials and Techniques
Yeast strains used in this study are CBY267
(MAT
␣
trp1-1 ade2-1 ura3-1
leu2-3,112 can1-100
), CBY268 (
MAT
␣
trp1-1 ade2-1 ura3-1 leu2-3
,
112
can1-100 sly1-ts
), CBY263
(MAT
␣
trp1-1 ade2-1 ura3-1 leu2-3,112 can1-
100 sed5-1
), RSY255 (
MAT
␣
ura3-52 leu2-3,112
), RSY944 (
MATa ura3-
52 lys2-801 bet1-1
), RSY954 (
MATa leu2-3,112 lys2-801 bos1-1
), and
CBY474 (
MAT
␣
trp1-1 ade2-1 ura3-1 leu2-3,112 can1-100 ypt1-3
), and
they have been previously described (Cao et al., 1998). The Ypt1-TM2p
strain (ROH713-10A;
MAT
␣
his3 leu2 ypt1
⌬
::HIS3
with a
CEN-
YPT1TM2-LEU2
plasmid) and isogenic wild-type strain (ROH713-10B;
MAT
␣
his3 leu2
) have been described previously (Ossig et al., 1995). The
yeast strain containing a myc-tagged version of Sly1p, CBY73 (
MAT
␣
ura3-52 lys2-801 ade2-101 trp1
⌬
63 his3-
⌬
200 leu2-
⌬
1 sly1
⌬
::HIS3
with a
CEN-LEU2-SLY1-MYC
plasmid) was constructed as described below.
Strains were grown in rich medium (1% bactoyeast extract, 2% bactopep-
tone, and 2% glucose), and converted to semi-intact cells as described by
Baker et al. (1988). Antibodies directed against
␣
-1,6-mannose linkages
(Barlowe, 1997), Ypt1p (Rexach et al., 1994), Sec61p (Stirling et al., 1992),
GDPase (Berninsone et al., 1995), Sec12p (Powers and Barlowe, 1998),
Sec22p (Bednarek et al., 1995), Bet1p (Rexach et al., 1994), Bos1p
(Sogaard et al., 1994), Sed5p (Cao et al., 1998), Emp47p (Schroder et al.,
1995), Sec23p (Hicke and Schekman, 1989), and the c-myc epitope (Evan
et al., 1985) have been described previously. Polyclonal antibodies pre-
pared against Ykt6p were generated against a hexahistidine-tagged ver-
sion of recombinant Ykt6p (McNew et al., 1997) as described (Un-
germann et al., 1999). For immunoblots, samples were resolved by
SDS-PAGE (Lammeli, 1970), transferred to nitrocellulose (Towbin et al.,
1979) and filter-bound secondary antibodies were detected by peroxidase-
catalyzed chemiluminescence (Amersham).
Plasmid Construction
The plasmid YEP51 containing the
SLY1
gene (Dascher et al., 1991) was
a gift from H.D. Schmitt (Max Plank Institute, Gottingen). The restriction
sites BamHI and EcoRV were used to subclone the 3
⬘
end of
SLY1
(nu-
cleotides 2,550–3,557) into the BamHI and EcoRV sites of pBluescript
SK
⫹
(Stratagene) to construct pXC1. The restriction sites SpeI were used
to subclone the 5
⬘
end of
SLY1
(nucleotides 728–2,834) into the SpeI sites
of pXC1 to produce pXC2 (pBS-
SLY1
). The peptide sequence EEQKLI-
SEEDLHHHHHH (c-myc epitope and hexahistide tag) was fused to the
COOH terminus of Sly1p as follows. Two complementary oligonucle-
otides A25635 (5
⬘
-GAGGAGCAGAA ATTAATCAG CGAAGAGGAC
CTCCTCAGGA AGAGGCATCA CCATCACCAT CACTAAGATA
TCTGCA
-
3
⬘
) and A25636 (5
⬘
-GATATCTTAG TGATGGTGAT GGT-

Cao and Barlowe
Asymmetric Requirements for ER/Golgi SNAREs
57
GATGCCTCT TCCTGAGGAG GTCCTCTTCG CTGATTAATT
TCTGCTCCTC TGCA-3
⬘
) were annealed to produce a DNA fragment
flanked by ends compatible with sites generated by an NsiI digest. The an-
nealed oligonucleotides were inserted into the NsiI site of pXC2 (a unique
restriction site directly before the stop codon of
SLY1
) to produce pXC4
(pBS-SLY1-MYC-6HIS). This construct was sequenced to confirm faith-
ful insertion of the fusion sequences. Finally, the restriction sites XhoI and
XbaI of pXC4 were used to subclone
SLY1-MYC-6HIS
into the XhoI and
XbaI sites of pRS315 (Sikorski and Hieter, 1989) to produce pXC5r
(
pRS315-SLY1-MYC-6HIS
).
Strain Construction
The
SLY1
locus was targeted for disruption with the
HIS3
gene (Baudin
et al. 1993). PCR was used to amplify a
HIS3
disruption fragment flanked
by sequences immediately before and after the
SLY1
open reading frame,
using the primers C35571 (5
⬘
-ATATATATAT ATTAGTCTAT CGT-
CATTGGG GCTAGATGCC AATTAGCGCG CCTCGTTCAG AATG-
3
⬘
) and C33626 (5
⬘
-GTCATTGCCA GTTGCTAAGT ATCTTTGACC
AAAAATCACA ACATCGGCCT CCTCTAGTAC ACTC-3
⬘
). The
HIS3
homologous regions are underlined. The resulting product has the
HIS3
gene flanked by 45 bp of the
SLY1
gene directly before the ATG
start and directly after the stop codon. Strain YPH501 (Sikorski and Hie-
ter, 1989) was transformed with this PCR product, and histidine prototro-
phs were screened by colony PCR using the primer SLY15 (5
⬘
-
CCGTTTCCCT CTTCGCG-3
⬘
; 142 bp upstream of the
SLY1
start
codon) and an internal
HIS3
primer (5
⬘
-GGCTCATCCAAAGGCGC-
3
⬘
). Several colonies were identified that contained a
HIS3
disruption of
SLY1
. One strain, designated CBY69, was heterozygous at the
SLY1
lo-
cus and used in further studies. CBY69 was transformed with pXC5r
(
pRS315-SLY1-MYC-6HIS
) and grown under conditions to induce sporu-
lation. Viable haploid disruptants of
SLY1
containing pXC5r were identi-
fied, and exhibited growth properties that were indistinguishable form
wild-type strains. One strain (CBY73) was used in the following studies.
Subcellular Fractionation
Membrane organelles prepared from cell lysates were resolved on 22–
60% sucrose density gradients (Antebi and Fink, 1992) with minor modifi-
cations as described by Powers and Barlowe (1998). Gradient fractions
were collected and diluted 1:1 with SDS-PAGE sample buffer and immu-
noblotted for Sec61p (ER marker), Emp47p (Golgi marker), Bet1p,
Bos1p, Sec22p, Sed5p, Sly1p, Ypt1p, and Ykt6p. Relative levels of specific
proteins in each fraction were quantified by densitometric scanning of im-
munoblots. GDPase activity (Golgi marker) was determined as described
(Yanagisawa et al., 1990) using CDP to subtract nonspecific phosphatase
activity. Sucrose concentrations of individual fractions were determined
by measuring the refractive index with an Abbe refractometer (American
Optical).
In Vitro Budding and Transport Assays
Microsomes were isolated from the CBY73 strain and incubated in the
presence or absence of proteins required for reconstitution of vesicle for-
mation as described in Barlowe et al. (1994). A 30-
␮
l aliquot of the total
reaction and 300
␮
l of a supernatant fluid containing vesicles released
from budding reactions were centrifuged at 100,000
g
in a TLA100.3 rotor
(Beckman Instruments) to collect membranes. This resulting membrane
pellet was solubilized in 30
␮
l of SDS-PAGE sample buffer, and 10
␮
l was
resolved on 12.5% polyacrylamide gels and immunoblotted for various
proteins as indicated in the figure legends.
For in vitro fusion assays, ER-derived vesicles and acceptor mem-
branes were isolated from indicated strains as follows. Semi-intact yeast
cells were prepared (Baker et al., 1988) from wild-type,
bet1-1
,
bos1-1
,
sed5-1
,
sly1-ts
,
and
ypt1-3
strains that were grown at 24
⬚
C. To prepare ER-
derived vesicles that contained [
35
S]glyco-pro-
␣
factor (gp-
␣
F), semi-
intact cells were thawed quickly and washed three times with buffer 88 (25
mM Hepes, pH 7.0, 150 mM potassium acetate, 250 mM sorbitol, and 5
mM magnesium acetate) to remove cytosol. Each wash was followed by
centrifugation at 15,000
g
(12,000 rpm) in an Eppendorf refrigerated cen-
trifuge (model 5417). gp-
␣
F was posttranslationally translocated into the
ER of semi-intact cells at 10
⬚
C for 10 min in the presence of an ATP re-
generation system (Baker et al., 1988). Vesicles were synthesized from
semi-intact cells by incubation with purified COPII proteins (Sar1p,
Sec23p complex, and Sec13p complex) at 20
⬚
C for 10 min as previously
described (Barlowe, 1997). After budding, the reactions were chilled on
ice and centrifuged for 5 min at 20,000
g
(14,000 rpm in an Eppendorf re-
frigerated centrifuge). The supernatant fluid, containing budded vesicles,
was collected and used in subsequent fusion assays.
To prepare acceptor membranes, semi-intact cells were thawed and
washed three times as described above. Fusion reactions were performed
by incubating isolated vesicles (containing
ⵑ
5,000 cpm of [
35
S]gp-
␣
F) with
washed semi-intact cells in the presence of Uso1p and LMA1 at 23
or 29
⬚
C
(Barlowe, 1997). Under these conditions, washed semi-intact cells provide
the acceptor membrane for vesicle fusion. After 90-min incubations,
transport reactions were stopped by the addition of SDS to a final concen-
tration of 1% and heated to 95
⬚
C for 2 min. Solubilized membranes were
diluted 20-fold with IP buffer, followed by the addition of anti–
␣
-1,6-man-
nose-specific serum and protein A–Sepharose (Pharmacia Biotech).
Outer chain–modified forms of [
35
S]gp-
␣
F (reflecting delivery to a Golgi
compartment) were precipitated at room temperature for 2 h and pro-
cessed as described (Baker et al., 1988). The percent transport is the
amount of outer chain–modified [
35
S]gp-
␣
F divided by the total amount of
protease-protected ConA-precipitable [
35
S]gp-
␣
-factor contained in iso-
lated vesicles. For each figure, multiple data sets were obtained, and a rep-
resentative experiment plotting the mean and range of duplicate samples
is shown.
Results
Subcellular Distribution of SNAREs between the ER
and Golgi Membranes
One model for vesicle-mediated transport suggests that t-
and v-SNAREs reside on separate compartments (Soll-
ner et al., 1993a; Rothman and Wieland, 1996), although
there is experimental evidence indicating these species are
not strictly compartment localized in heterotypic systems
(Walch-Solimena et al., 1995; Rowe et al., 1998). Beyond
these cellular localization studies, little has been done to dis-
tinguish in which vesicle or a membrane compartment dis-
tinct v- and t-SNARE proteins are functionally required.
Here, we address these questions in a simplified system of
membrane fusion between ER-derived vesicles and Golgi
membranes. First, we examined the subcellular distribu-
tion of the SNARE proteins involved in this fusion event.
The fractionation behavior of Bet1p, Bos1p (Shim et al.,
1991; Newman et al., 1992; Lian and Ferro-Novick, 1993),
and Sed5p (Hardwick et al., 1992) have been documented
by various methods; however, we sought to directly com-
pare the level of colocalization among these and other
proteins involved in transport the between ER and Golgi.
To determine their steady state distribution, a standard
procedure was employed (Antebi and Fink, 1992) to re-
solve ER and Golgi compartments by velocity sedimenta-
tion on sucrose density gradients (Fig. 1). The resolution
of Golgi membranes and the ER by this method was con-
firmed through the analysis of Golgi-localized markers,
GDPase and Emp47, which peaked in fraction 7 and the
ER marker, Sec61p, which peaked in fraction 13. Immuno-
blot analysis of these same gradient fractions with antibod-
ies specific for Bos1p, Bet1p, Sec22p, Sed5p, Sly1p, Ypt1p,
and Ykt6p are shown in Fig. 1 (A–H). Notably, Bos1p,
Bet1p, and Sed5p displayed similar distribution patterns
with major peaks that were coincident with Golgi and ER
markers. This method was performed multiple times with
different wild-type yeast strains, and very similar fraction-
ation profiles were observed, such that these SNARE pro-
teins distributed between ER and Golgi compartments
with a majority (
ⵑ
60–80%) localizing with Golgi mem-
branes. Sly1p is peripherally associated with membranes,
in part, through binding to Sed5p, and we observed signifi-

The Journal of Cell Biology, Volume 149, 2000 58
cant overlap between Sly1p and Sed5p. Ypt1p followed a
similar distribution with a majority localized to Golgi
membrane fractions, however, some Ypt1p migrated with
ER membranes. A fraction of Ykt6p colocalized with ER
and Golgi markers, but the overall pattern was distinct
from other ER/Golgi SNARE, proteins and may reflect a
role for this protein in fusion events later in the secretory
pathway (Ungermann et al., 1999). In summary, the steady
state distributions of ER/Golgi SNAREs (Bet1p, Bos1p,
Sec22p, and Sed5p) are similar and do not appear to be re-
stricted to single membrane compartments. These obser-
vations on Sed5p are consistent with data suggesting the
t-SNARE Sed5p/Syn5 cycles between the ER and Golgi
compartments (Rowe et al., 1998; Wooding and Pelham,
1998).
Incorporation of SNAREs and SNARE Regulatory
Proteins into COPII-coated Vesicles
The primary route of protein transport from the ER is
thought to be via COPII-coated transport vesicles (Schek-
man and Orci, 1996). Methods have been established to
isolate in vitro synthesized COPII vesicles (Barlowe et al.,
1994; Rexach et al., 1994), and we sought to determine the
efficiency with which distinct SNARE and SNARE regu-
latory proteins are packaged into isolated COPII vesicles
from microsomes. For these studies, microsomes were pre-
pared from a yeast strain (CBY73) containing an epitope-
tagged version of Sly1p (see Materials and Methods).
Incubation of microsomes with purified Sar1p, Sec23p
complex, and Sec13p complex, in the presence of ATP and
GTP, produced COPII vesicles that were separated from
larger ER membranes by differential centrifugation. The
COPII vesicles contained in the supernatant fraction were
collected by centrifugation at 100,000
g
. Fig. 2 shows the
content of specific proteins contained on these isolated
vesicles. First, resident ER proteins such as Sec61p and
Sec12p were not packaged into COPII-coated vesicles as
has been previously observed (Barlowe et al., 1994; Rex-
ach et al., 1994), indicating a faithful reproduction of sort-
ing during in vitro budding, and that the integrity of ER
membranes was preserved though this procedure. Sec-
ond, GDPase (a Golgi resident) was not efficiently pack-
aged into COPII-coated vesicles, suggesting a selectivity
of the COPII coat for ER membranes even though iso-
lated microsomes contain a significant amount of Golgi
membranes. Finally, the ER to Golgi SNARE proteins
monitored (Bos1p, Bet1p, Sec22p, and Sed5p) and the
t-SNARE–associated protein (Sly1p) were specifically
packaged into COPII-coated vesicles. Subsequent repeti-
tions of this experiment produced qualitatively similar re-
sults, and the efficiency of their incorporation into COPII-
Figure 1. Subcellular distribution of ER/Golgi proteins by su-
crose gradient. Lysed spheroplasts made from CBY409 cells were
loaded on a 20–60% sucrose gradient. The gradients were centri-
fuged at 35,000 rpm in an SW40 rotor for 2.5 h at 4⬚C. Fractions
from the gradient were resolved on SDS-PAGE and immuno-
blotted for Sec61p (ER marker), Emp47p (Golgi marker),
Sec22p, Bet1p, Bos1p (v-SNARE), Sed5p (t-SNARE), and the
t-SNARE–associated protein, Sly1p. The relative abundance
contained in each fraction was determined by densitometry using
NIH image. The GDPase assay measures Ca2⫹-dependent GDP-
ase activity and serves as a marker for Golgi membranes.
Figure 2. SNARE proteins are incorporated into ER-derived
vesicles. COPII-coated vesicles were synthesized from ER mem-
branes and collected by centrifugation. Lanes labeled “Total” are
membranes from one-tenth of a total reaction (containing both
vesicles and ER). Lanes labeled “⫹Recon” are vesicles produced
under conditions of reconstituted vesicle formation by addition
of COPII proteins. Lanes labeled “⫺Recon” are those produced
in the absence of COPII proteins. Samples were resolved on
12.5% SDS-PAGE, transferred to nitrocellulose, and blotted
with antibodies specific for indicated proteins.

Cao and Barlowe
Asymmetric Requirements for ER/Golgi SNAREs
59
coated vesicles from the experiment shown in Fig. 2 is
listed in Table I. Interestingly, the percentage of Bet1p in-
corporated into the vesicles was reproducibly twofold
higher than the other SNAREs monitored in these experi-
ments (Sec22p, Bos1p, Bet1p, and Sed5p). In addition, the
level of the GTPase Ypt1p packaged into the COPII vesi-
cles was quite low, and suggested this protein was not a
constituent of these transport intermediates, an observa-
tion that is consistent with previous experiments (Barlowe
et al., 1994; Rexach et al., 1994). A similar result was ob-
tained in analysis of Ykt6p, as this protein was not effi-
ciently packaged into COPII-coated vesicles. Together
with the subcellular fractionation results, these observa-
tions are consistent with the notion that specific sets of
SNARE proteins cycle between the ER and Golgi instead
of a hypothesis that v-SNAREs are enriched on transport
vesicles and t-SNAREs localize to acceptor membranes
(Rothman and Wieland, 1996). In contrast, the small GTP-
ase Ypt1p was not efficiently packaged into COPII vesi-
cles, even though the subcellular distribution of Ypt1p is
similar to ER/Golgi SNARE proteins.
Localized Requirements for SNARE Protein Function
The above experiments indicate that ER/Golgi SNAREs
are similarly distributed between these compartments, and
are efficiently packaged into ER-derived vesicles. How-
ever, these results do not indicate in which compartments
their activities are required, although there is ample evi-
dence indicating that Bet1p, Bos1p, and Sed5p function in
the fusion of ER-derived vesicles with Golgi membranes
(Newman et al., 1992; Lian and Ferro-Novick, 1993; Cao et
al., 1998). To test the functional requirements for these ac-
tivities on vesicles and acceptor membranes, we used
bet1-1
,
bos1-1
,
sed5-1
, and sly1-ts mutant strains that allow for se-
lective inactivation of specific proteins on vesicles or ac-
ceptor membranes. Our previous experiments have estab-
lished that the mutated versions of Bet1p, Bos1p, Sed5p,
and Sly1p cause thermosensitive blocks in cell-free vesicle
fusion assays (Cao et al., 1998). Here, we employ these
same mutations to inactivate SNARE proteins on vesicles
or acceptor membranes and monitor fusion efficiency.
Transport between the ER and Golgi may be repro-
duced with washed semi-intact cells incubated with puri-
fied COPII proteins, Uso1p, and LMA1, and is monitored
by the processing of [35S]gp-␣F. COPII produces freely
diffusible vesicle intermediates containing [35S]gp-␣F that
then tether to Golgi membranes in the presence of Uso1p.
Fusion of tethered vesicles requires the activities of
Sec18p and LMA1 (Barlowe, 1997). With this refined
transport assay, we can isolate freely diffusible vesicles
from membranes incubated with COPII and, in a second
stage, incubate vesicles with Golgi membranes from
washed semi-intact cells in the presence of fusion factors.
The percentage of vesicles that fuse with acceptor mem-
branes was quantified by determining the amount of
[35S]gp-␣F that had acquired the Golgi-specific outer chain
␣-1,6-mannose modification. An example is shown in Fig.
3 (A), where incubation of wild-type vesicles containing
[35S]gp-␣F with the membranes from the bet1-1 mutant
strain resulted in fusion and outer chain modification of
[35S]gp-␣F at permissive or nonpermissive temperatures.
However, incubation of vesicles from the bet1-1 mutant
strain with wild-type acceptor membranes at a restrictive
temperature–blocked vesicle fusion, indicating that the ac-
tivity of Bet1p is required on ER-derived vesicles. Similar
experiments were performed using components from the
bos1-1 and sed5-1 mutant strains to determine the com-
partmental requirements for Bos1p and Sed5p. As shown
in Fig. 3 (B), vesicles carrying mutant Bos1p failed to de-
liver [35S]gp-␣F to wild-type Golgi membranes at 29⬚C.
This result is consistent with previous studies that demon-
strated a requirement for Bos1p on ER-derived vesicles
when neutralizing anti-Bos1p antibodies were used (Lian
and Ferro-Novick, 1993). In contrast, Fig. 4 shows that
vesicles carrying the thermosensitive version of Sed5p
were fully functional for delivery of [35S]gp-␣F to the wild-
type acceptor at both temperatures, whereas acceptor
membranes from the sed5-1 strain were not functional at a
Table I. Efficiency of Protein Packaging into COPII Vesicles
Protein Percent incorporated
(⫺Recon.) Percent incorporated
(⫹Recon.)
Sec61p 0.3 0.4
Sec12p 0.2 0.2
GDPase 0.6 0.9
Ypt1p 0.8 1.0
Ykt6 0.7 1.1
Sec22p 0.6 9.9
Bos1p 0.4 10.3
Bet1p 0.4 19.4
Sed5p 0.6 8.8
Sly1p 0.8 4.9
Values represent the percentage of each protein in vesicles compared to total mi-
crosomes and were determined by densitometric scanning of blots shown in Fig. 2.
Figure 3. The v-SNAREs
Bet1p and Bos1p are func-
tionally required on ER-
derived vesicles. COPII vesi-
cles, which were synthesized
in vitro from wild-type or
mutant ER membranes at
20⬚C, were incubated in a
second stage with wild-type
or mutant Golgi membranes
at 23 or 29⬚C. (A) Vesicle fu-
sion with membrane compo-
nents from a bet1-1 strain or
(B) a bos1-1 strain. Reactions
contained an ATP regenera-
tion system alone (Mem. ⫹
Ves., open bars) or an ATP
regeneration system with
Uso1p and LMA1 (Mem. ⫹
Ves. ⫹ Fusion Factors, black
bars). The percent fusion was
quantified after precipitation
of the outer chain–modified
forms of [35S]gp-␣F.

The Journal of Cell Biology, Volume 149, 2000 60
restrictive temperature. These results indicate that ER/
Golgi SNAREs display spatially distinct requirements in
the fusion of ER-derived vesicles with acceptor mem-
branes. Bet1p and Bos1p functioned on vesicles, whereas
Sed5p was required on acceptor membranes.
From the experiments shown in Figs. 3 and 4, we hy-
pothesized that vesicles prepared from the sed5-1 strain
would fuse with acceptor Golgi membranes prepared from
the bet1-1 strain even at restrictive temperatures. In other
words, simultaneous inactivation of a v-SNARE on the ac-
ceptor and a t-SNARE on the vesicle should not inhibit fu-
sion. Indeed, as shown in Fig. 5, vesicles from the sed5-1
strain and Golgi membranes from the bet1-1 strain fused
at both 23 and 29⬚C. As controls for this experiment, other
combinations of these mutant components showed clear
temperature sensitivity. A similar result was observed
when the experiment was performed with sed5-1 vesicles
and bos1-1 acceptor membranes (not shown), although the
magnitude of the fusion signal was lower than for the
bet1-1 experiment. Taken together, these results indicated
a requirement for Bet1p and Bos1p on vesicles and the
t-SNARE Sed5p on the acceptor compartment.
Asymmetric Requirements for SNARE
Regulatory Proteins
At this point, we have examined the localized require-
ments for SNARE proteins in ER/Golgi transport. Al-
though SNARE proteins are central components, addi-
tional proteins that appear to regulate SNARE protein
function are essential for membrane fusion, and we next
investigated the requirements for two of these regulators,
Ypt1p and Sly1p (Lian et al., 1994; Lupashin and Waters,
1997). Sly1p is an essential 84-kD protein that binds to
Sed5p (Dascher et al., 1991; Sogaard et al., 1994). Our
analyses of Sly1p on sucrose gradients and on COPII vesi-
cles showed that Sly1p distributes between ER and Golgi
similarly to SNAREs, but was incorporated into vesicles
less efficiently than Sed5p or other ER/Golgi SNAREs.
We have previously shown that the sly1-ts mutation pro-
duces a temperature-dependent block in our in vitro trans-
port assay (Cao et al., 1998). As shown in Fig. 6, we found
that the requirement for Sly1p resided specifically on ac-
ceptor membranes, whereas the ER-derived vesicles pre-
pared from this strain were fully functional. Therefore, the
distribution and spatial requirement for Sly1p were similar
to Sed5p and may reflect a linked function for these mole-
cules.
Previous studies have shown that isolated vesicles accu-
mulated in a ypt1 temperature-sensitive mutant were func-
tional for fusion with wild-type Golgi membranes (Rexach
et al., 1994), suggesting functional Ypt1p was not required
on vesicles. This published observation employed a rich
cytosol, which contains some soluble Ypt1p, to drive the
second stage fusion reaction. Therefore, we reevaluated
the requirements for Ypt1p in our cell-free assay under
conditions where soluble Ypt1p or Gdi1p was not pro-
vided. We have previously shown that the ypt1-3 mutation
displays some temperature sensitivity in vitro when the re-
action was driven with purified transport factors; however,
even at permissive temperatures, the transport efficiency
was low in reactions that employ the ypt1-3 mutation (Cao
et al., 1998). As seen in Fig. 7, Golgi membranes from
ypt1-3 cells did not function with wild-type vesicles,
whereas vesicles isolated from the ypt1-3 strain were fully
active for fusion with wild-type acceptor membranes. Un-
der conditions where ypt1-3 acceptor membranes were
used, we detected only modest amounts of transport at
permissive temperatures. These results suggest that Ypt1p
function was restricted to Golgi membranes as was ob-
served for Sed5p and Sly1p and places this set of mole-
Figure 4. The t-SNARE
Sed5p is functionally re-
quired on Golgi membranes.
COPII vesicles synthesized in
vitro from wild-type or sed5-1
ER membranes at 20⬚C were
incubated in a second stage
with wild-type or sed5-1
Golgi membranes at 23 or
29⬚C. Reactions contained an
ATP regeneration system
alone (Mem. ⫹ Ves., open
bars) or an ATP regeneration
system with Uso1p and
LMA1 (Mem. ⫹ Ves. ⫹ Fusion Factors, black bars). The percent
fusion represents the amount of the outer chain–modified forms
of [35S]gp-␣F.
Figure 5. The v-SNARE
Bet1p is required on ER-
derived vesicles, and the
t-SNARE Sed5p is required
on Golgi membranes. Vesi-
cles synthesized from a bet1-1
strain and Golgi membranes
from a sed5-1 strain or vice
versa were incubated in a sec-
ond stage at 23 or 29⬚C. Reac-
tions contained an ATP re-
generation system alone
(Mem. ⫹ Ves., open bars) or
an ATP regeneration system
with Uso1p and LMA1 (Mem. ⫹ Ves. ⫹ Fusion Factors, black
bars). The percent fusion represents the amount of the outer
chain–modified forms of [35S]gp-␣F.
Figure 6. Sly1p function is
required on Golgi mem-
branes. COPII vesicles syn-
thesized in vitro from
wild-type or sly1-ts ER
membranes at 20⬚C were in-
cubated in a second stage
with wild-type or sly1-ts
Golgi membranes at 23 or
29⬚C. Reactions contained an
ATP regeneration system
alone (Mem. ⫹ Ves., open
bars) or an ATP regenera-
tion system with Uso1p and
LMA1 (Mem. ⫹ Ves. ⫹ Fusion Factors, black bars). The per-
cent fusion represents the amount of outer chain–modified forms
of [35S]gp-␣F.

Cao and Barlowe Asymmetric Requirements for ER/Golgi SNAREs 61
cules in a distinct group from Bet1p and Bos1p. However,
we cannot exclude the possibility that wild-type acceptor
membranes transferred membrane-bound Ypt1p to in-
coming vesicles, although we consider this unlikely be-
cause the addition of varying amounts of Gdi1p does not
stimulate this reaction (data not shown).
A Requirement for Ypt1p Can Be Fulfilled When This
GTPase Is Restricted to Acceptor Membranes
As an independent test for localized Ypt1p activity on ac-
ceptor membranes, we used a membrane-anchored form
of this protein (Ypt1-TM2p), which has been previously
described in the literature (Ossig et al., 1995). We rea-
soned that if vesicles do not require Ypt1p bound to their
surface for function, then acceptor membranes containing
Ypt1-TM2p should support vesicle fusion when transfer is
prevented by anchoring. The membrane-anchored form
used in our experiments fuses the transmembrane domain
of Sec22p to the COOH terminus of Ypt1p in a man-
ner that replaces the CAAX sequence of this GTPase.
Ypt1-TM2p behaves as an integral membrane protein and
expression complements a ypt1⌬ strain. However, the
Ypt1p-TM2p must be overproduced (approximately two-
fold) for complementation and, even when overexpressed,
the growth of the Ypt1-TM2p strain was slightly slower
(20%) than a wild-type rate. Regardless, transport of
secretory proteins and Golgi function appeared normal in
the Ypt1-TM2p strain where membrane detachment is
prevented (Ossig et al., 1995).
We first characterized the properties of the Ypt1-TM2
strain in our reconstituted transport assay and observed
that vesicles were synthesized in a COPII-dependent man-
ner, ER-derived vesicles were tethered in an Uso1p-
dependent process, and fusion required Sec18p and
LMA1p (Table II). However, the efficiency of vesicle for-
mation and fusion were reduced in comparison to a wild-
type strain, yet vesicle tethering remained efficient in the
Ypt1p-TM2 strain. These results indicate the Ypt1-TM2p
membranes are not optimal for ER/Golgi transport in our
reconstituted assays, but that [35S]gp-␣F was transported
in a conventional manner and, importantly, anchoring
Ypt1p yields active acceptor membranes. Therefore, ac-
ceptor membranes, which were prepared from this strain
should allow us to test if transfer of Ytp1p from donor
membranes to vesicles is a requirement for their fusion.
The yeast GDP dissociation inhibitor, Gdi1p, is an in-
hibitor of Ypt and Rab GTPases that binds and extracts
the GDP-bound form of these proteins from intracellular
membranes (Garrett et al., 1994). We tested if Ypt1-TM2p
was sensitive to extraction by Gdi1p for two purposes.
First, although the transmembrane form of Ypt1p behaves
as an integral membrane protein, we wanted to directly
demonstrate that the anchored form of Ypt1p was not sol-
ubilized by Gdi1p to exclude the possibility of transfer
from acceptor membranes to vesicles in our reaction. Sec-
ond, vesicles generated from wild-type or the ypt1-3 strain
could contain trace amounts of Ypt1p that may be suffi-
cient for vesicle fusion. We reasoned that if Ypt1-TM2p
was insensitive to Gdi1p extraction, addition of excess
Gdi1p to vesicles should selectively inhibit the activity
of lipid-anchored forms of Ypt1p and not membrane-
anchored Ypt1p. As seen in Fig. 8, wild-type membranes,
which contain the lipid-anchored form of Ypt1p, are sensi-
tive to Gdi1p, and Ypt1p was extracted upon addition
of excess Gdi1p, whereas Ypt1p-TM2p was not extract-
able. In this experiment, Sec23p, a peripherally associ-
ated membrane protein (Hicke and Schekman, 1989),
and Sec61p, an integral membrane protein (Stirling et al.,
1992), served as controls to demonstrate effective separa-
tion of cytosol (supernatant) and membrane (pellet) frac-
tions. The Ypt1-TM2p fusion migrates as a larger species
Figure 7. The small GTPase
Ypt1p is functionally re-
quired on Golgi membranes.
COPII vesicles, which were
synthesized in vitro from
wild-type or ypt1-3 mem-
branes at 20⬚C, were incu-
bated in a second stage with
wild-type or ypt1-3 Golgi
membranes at 23 or 29⬚C.
Reactions contained an ATP
regeneration system alone
(Mem. ⫹ Ves., open bars) or
an ATP regeneration system
with Uso1p and LMA1 (Mem. ⫹ Ves. ⫹ Fusion Factors, black
bars). The percent fusion represents the amount of the outer
chain–modified forms of [35S]gp-␣F.
Table II. Budding, Tethering and Fusion with Wild-type and
Ypt1p-TM2 Membranes
Diffusible vesicles (%) Transport (%)
Membranes NA COPII COPII/Uso1p NA Recon.
WT 4.5 ⫾ 0.4 36.2 ⫾ 0.4 25.9 ⫾ 0.2 4.6 ⫾ 0.2 22.9 ⫾ 0.5
TM2 4.7 ⫾ 0.2 22.6 ⫾ 0.6 14.7 ⫾ 2.0 4.5 ⫾ 0.9 12.9 ⫾ 1.0
The percentages of freely diffusible [35S]gp-␣ Factor–containing vesicles were deter-
mined in the presence of COPII (budding) and COPII ⫹ Uso1p (tethering) after 30
min at 20⬚C. In separate reactions, the percentage of transport was quantified by pre-
cipitation of the outer-chain modified forms of [35S]gp-␣ Factor in the presence of Re-
con. proteins (COPII, Uso1p and LMA1) after 60 min at 23⬚C. Samples were pro-
cessed as described previously (Cao et al., 1998).
Figure 8. Gdi1p extracts Ypt1p but not Ypt1-TM2p. Wild-type
(WT) or Ypt1-TM2p (TM2) semi-intact cells were incubated
with Gdi1p (50 ␮g/ml) for 20 min at 25⬚C. Total reactions (T)
were centrifuged at 100,000 g to generate the membrane pellet
(P) and supernatant (S) fractions. Fractions were resolved on
12.5% polyacrylamide gels, and immunoblot analyses were per-
formed with specific antibodies to measure the content of Sec23p
(a peripheral membrane protein control), Sec61p (control for in-
tegral membrane protein), and the lipid-anchored or membrane-
anchored form of Ypt1p.

The Journal of Cell Biology, Volume 149, 2000 62
because of the addition of 26 amino acid residues from the
transmembrane domain of Sec22p.
To determine if vesicles could fuse with acceptor mem-
branes when Ypt1p transfer was prevented by membrane
anchoring, we prepared vesicles from the ypt1-3 strain
and acceptor membranes from wild-type or Ypt1-TM2p
strains. As seen in Fig. 9, vesicles incubated with wild-type
acceptor membranes fused at an efficient level, and this fu-
sion was sensitive to Gdi1p. When vesicles were incubated
with acceptor membranes that contained Ypt1-TM2p, ves-
icles fused at a somewhat lower efficiency but, im-
portantly, this reaction was insensitive to Gdi1p. This
experiment indicates that ER-derived vesicles that were
depleted of Ypt1p by ypt1-3 mutation and treated with
Gdi1p still fused with the acceptor when Ypt1-TM2 was
restricted to acceptor membranes. Based on these results,
we propose that the requirement for Ypt1p in the fusion of
ER-derived vesicles with acceptor membranes resides in
the Golgi compartment.
Discussion
Questions concerning the regulation and function of dis-
tinct SNARE proteins in intracellular fusion reactions
have become increasingly complex (Nichols and Pelham,
1998; Gotte and Fischer von Mollard, 1998). For example,
fusion events involving ER and Golgi membranes presum-
ably include fusion of ER-derived vesicles with Golgi
membranes (Rexach and Schekman, 1991; Cao et al.,
1998), fusion of Golgi-derived retrograde vesicles with the
ER (Lewis and Pelham, 1996; Spang and Schekman,
1998), as well as homotypic membrane fusions between
ER (Latterich et al., 1995; Patel et al., 1998), between
Golgi (Rabouille et al., 1998), and between ER-derived
vesicles (Rowe et al., 1998). In all of these examples,
SNARE proteins have been implicated and, in some in-
stances, distinct fusion reactions appear to employ an
identical SNARE (Patel et al., 1998; Spang and Schekman,
1998). How does each of these fusion events determine
which components will be used when, or is there promiscu-
ity such that any given set can operate? To answer these
questions, we believe it will be important to determine
precisely which sets of proteins are capable of mediating
specific membrane fusion events, and on which compart-
ments they are functionally required.
In this manuscript, we report that ER/Golgi SNARE
proteins display similar distributions, but are asymmetri-
cally required in fusion between ER-derived vesicles
and Golgi membranes. Using thermosensitive versions of
SNAREs and SNARE regulatory proteins, we observe
that Bet1p and Bos1p are functionally required on ER-
derived vesicles, whereas the t-SNARE (Sed5p) and
t-SNARE–associated proteins (Sly1p) are specifically re-
quired on the Golgi acceptor compartment. A recently dis-
covered multispanning membrane protein, termed Got1p,
also functions specifically on this acceptor compartment
and appears to facilitate Sed5p activity (Conchon et al.,
1999). These distinct spatial requirements suggest that het-
erotypic fusion events depend on compartment-specific
cues to regulate SNARE protein function. An important
component of this regulation appears to be the small GTP-
ase Ypt1p. Because Ypt1p is asymmetrically enriched and
required on Golgi membranes with respect to vesicles in
our reaction, we propose that Ypt/Rab proteins play an
important role in compartment-specific regulation. One
manner in which this could be accomplished is that upon
the binding of ER-derived vesicles to Golgi membranes, a
process which requires Ypt1p (Cao et al., 1998), this GTP-
ase is activated and signals the Sed5p/Sly1p complex, en-
abling Golgi SNAREs to engage vesicle SNAREs.
We have not detected a requirement for Sec22p in an-
terograde fusion of ER-derived vesicles with Golgi mem-
branes (Cao, X., and C. Barlowe, unpublished observa-
tion), as might be predicted from in vivo studies (Kaiser
and Schekman, 1990). It is possible that the temperature-
sensitive sec22-3 allele remains functional in our in vi-
tro assay, even at restrictive temperatures, although this
seems unlikely because this same allele exhibits sensitivity
in an in vitro assay that measures COPI-dependent retro-
grade transport from the Golgi to the ER (Spang and
Schekman, 1998). Because strains containing a sec22⌬ al-
lele are viable, this SNARE is apparently not essential for
anterograde or retrograde transport (Ossig et al., 1991),
however, in vitro fusion assays with membrane compo-
nents prepared from this deletion strain may allow for a
more rigorous test of Sec22p function. In addition to
Sec22p, the ER-localized t-SNARE, Ufe1p, appears to op-
erate specifically in retrograde transport to the ER and
not in anterograde transport (Lewis and Pelham, 1996;
Lewis et al., 1997; Spang and Schekman, 1998). This is in
contrast to Bet1p, which appears to operate in both an-
terograde and retrograde fusion events (Cao et al., 1998;
Spang and Schekman, 1998).
Several lines of evidence indicate ER/Golgi SNARE
proteins exist in heteromeric complexes under specific
conditions in yeast (Lian et al., 1994; Sogaard et al., 1994;
Lewis et al., 1997; Lupashin and Waters, 1997) and mam-
malian cells (Hay et al., 1998; Rowe et al., 1998). However,
in most instances, these studies were performed with whole
cell extracts, therefore, it is difficult to determine in which
membrane compartment(s) these complexes exist. Experi-
ments with purified proteins indicate that Bos1p and
Sec22p can bind directly to Sed5p (Sacher et al., 1997), and
Bet1p appears to increase the affinity of Bos1p for Sed5p
(Stone et al., 1997). We have detected some Sed5p, Bet1p,
and Bos1p in a complex on ER-derived vesicles through a
cross-linking immunoprecipitation approach (Cao, X., and
Figure 9. Ypt1-TM2p functions
on Golgi membranes. COPII
vesicles synthesized in vitro
from ypt1-3 membranes were in-
cubated in a second stage with
wild-type or Ypt1-TM2p Golgi
membranes. Reactions con-
tained an ATP regeneration sys-
tem alone (No addition, open
bars) or supplemented with
Uso1p and LMA1 (Fusion Fac-
tors, black bars) or Uso1p and
LMA1 and 50 ␮g/ml Gdi1p (Fu-
sion Factors ⫹ GDI, hatched
bars). The percent fusion repre-
sents the amount of outer chain–
modified forms of [35S]gp-␣F.

Cao and Barlowe Asymmetric Requirements for ER/Golgi SNAREs 63
C. Barlowe, unpublished observation), although it will re-
quire further studies to determine the stochiometry and
composition of SNARE complexes contained on COPII
vesicles. Structural studies on neural SNARE proteins in-
dicate the formation of a stable core complex composed of
four parallel coiled-coil domains, such that syntaxin and
synaptobrevin each contribute a single coiled-coil to this
structure, whereas SNAP25 contributes two (Poirier et al.,
1998; Sutton et al., 1998). Previous reports have noted that
Bet1p shares a high degree of sequence identity with
SNAP25 (Weimbs et al., 1997), leading to the prediction
that the role of Bet1p may be comparable to SNAP25
(Stone et al., 1997; Weimbs et al., 1997). Because Bet1p
contains a single ␣-helical domain, it may contribute two
molecules in fulfilling a putative SNAP25 role (Weimbs et
al., 1998). We have observed that the percentage of Bet1p
packaged into COPII vesicles from starting microsomes is
twice that of Bos1p, Sed5p, and Sec22p (Fig. 2 and Table
I). This may reflect the stochiometry of ER/Golgi SNARE
complexes that are packaged into COPII vesicles. Alterna-
tively, Bet1p could simply posses a higher affinity for sub-
units of the COPII coat, resulting in an increased packag-
ing efficiency. However, based on the collective results, we
speculate that a SNARE complex consisting of Sed5p,
Bos1p, and two molecules of Bet1p functions in fusion of
anterograde vesicles, whereas a complex of Ufe1p, Sec22p,
and two molecules of Bet1p would be required for fusion
of retrograde vesicles with the ER.
Our results indicate some parallels with homotypic
membrane fusion reactions, notably the fusion of vacu-
oles. These reactions employ a similar cast of characters
(SNAREs, Rab proteins, Sec18p, etc.) and, in some re-
spects, appear to operate by similar mechanisms such that
membranes first bind in a reaction that requires a Ypt pro-
tein for tethering before engaging SNARE protein ma-
chinery (Ungermann et al., 1998). A distinct difference in
these reactions, however, is a symmetric requirement for
Ypt7p, which is the counterpart of Ypt1p (Wichmann et
al., 1992; Haas et al., 1995). For homotypic fusion of vacu-
oles, Ypt7p is required on both compartments. Perhaps
this indicates a symmetric nature to the tethering reactions
in homotypic membrane pairing. The vacuolar reaction
can be altered to generate a pseudoheterotypic condition
by deleting a v-SNARE from one vacuole and a t-SNARE
from another. Under this condition, fusion proceeds at a
lowered efficiency, but proceeds nonetheless (Nichols et
al., 1997). Interestingly, the requirement for Sec18p now
becomes asymmetric, such that membranes containing the
t-SNARE molecule (Vam3p) depend on Sec18p for fusion
but a ⌬vam3 vacuole does not. This observation suggests
that ER-derived vesicles may not require Sec18p for fu-
sion, whereas the Golgi acceptor would. Sec18p is clearly
required for the fusion of ER-derived vesicles with the
Golgi (Rexach and Schekman, 1991; Barlowe, 1997), al-
though we currently are not able to distinguish if this re-
quirement is refined to acceptor membranes, vesicles, or
both.
Initial models for SNARE protein function suggested
that t-SNARE proteins remain largely associated with tar-
get membranes, and v-SNARE proteins would be found
on both vesicles and target membranes (Sollner et al.,
1993a). However, a strict separation of v- and t-SNARE
proteins does not appear to be a general feature of mem-
brane fusion reactions (Walch-Solimena et al., 1995). With
respect to Sed5p and Syn5, studies in yeast (Wooding and
Pelham, 1998) and mammalian cells (Rowe et al., 1998) in-
dicate that this protein is rapidly cycling between early
compartments of the secretory pathway. In spite of Sed5p
cycling through the ER, we find that this t-SNARE is func-
tionally required on acceptor membranes and not vesicles.
Our findings are not entirely consistent with those re-
ported on the mammalian Syn5 protein, where function
was restricted to ER-derived vesicles. In these experi-
ments, treatment of ER-derived vesicles with neutralizing
anti-Syn5 antibodies prevented the formation of vesicular-
tubular pre-Golgi intermediates, but these same antibod-
ies did not affect the competency of Golgi membranes to
act as an acceptor (Rowe et al., 1998). These disparate ob-
servations are not easily explained, but may be related to
the use of different inhibitors in these experimental ap-
proaches or may reflect differences in organization of the
early secretory pathway in S. cerevisiae and mammals. A
recent morphometric study on the organization of the
yeast secretory pathway indicates many parallels between
S. cerevisiae and mammals (Morin-Ganet et al., 1999). In
both, ER-derived vesicles appear to fuse with or form ve-
sicular tubular clusters that recruit COPI components, and
then fuse with or mature into cis-Golgi networks (Ban-
nykh et al., 1996; Presley et al., 1997; Scales et al., 1997;
Bonfanti et al., 1998; Morin-Ganet et al., 1999). Precisely
where specific outer-chain carbohydrate modifications oc-
cur in this scheme remains to be determined. The yeast
ER/Golgi transport assay measures a heterotypic mem-
brane fusion reaction that results in a mixture of ER-
derived vesicles containing secretory protein (gp-␣F) with
a compartment that contains ␣-1,6-mannosyl transferase
activity (Baker et al., 1988; Rexach et al., 1994). This is
thought to arise from the fusion of ER-derived vesicles
with a cis-Golgi-like compartment. Alternatively, COPI
vesicles containing ␣-1,6-mannosyl transferase could de-
liver this activity to a vesicular-tubular cluster of COPII
vesicles (Pelham, 1998; Lin et al., 1999). A direct require-
ment for COPI in anterograde transport of gp-␣F to the
␣-1,6-mannosyl transferase–containing compartment has
not been established, whereas COPI and COPI assembly
proteins are required for in vitro retrograde transport of
an HDEL-tagged protein from the Golgi to the ER
(Spang and Schekman, 1998; Poon et al., 1999). Therefore,
we speculate that our in vitro assay measures the fusion of
COPII vesicles with a compartment that contains ␣-1,6-
mannosyl transferase and not direct fusion with COPI ves-
icles derived from the Golgi. We cannot exclude the possi-
bility that ER-derived vesicles fuse homotypically because
our in vitro assay would not detect this event. However, if
homotypic fusion of ER-derived vesicles is catalyzed by
Sed5p, this event does not appear to be a requirement for
ER/Golgi transport in our assay. In mammalian cells, such
a homotypic fusion process may be required for further
progress through the early secretory pathway, and could
explain the different Sed5p/Syn5 requirements observed
in these assays.
If Sed5p is not functionally required on ER-derived ves-
icles, why does this protein actively cycle between the ER
and Golgi compartments? Sed5p may be incorporated into

The Journal of Cell Biology, Volume 149, 2000 64
transport vesicles as a consequence of complex formations
with Bet1p, Bos1p, and Sec22p. COPII proteins have been
demonstrated to bind directly to the soluble domains of
Bet1p and Bos1p (Springer and Schekman, 1998) and
Sed5p (Peng et al., 1999). Presumably COPI binds specific
ER/Golgi SNARE proteins for retrograde transport back
to the ER; therefore, Sed5p may be incorporated into
these vesicle carriers either by association with specific
SNAREs or direct interactions with COPI subunits. In
other words, Sed5p may cycle because it is in complex with
proteins that must cycle. Alternatively, Sed5p cycling be-
tween ER and Golgi may be functionally important for
homotypic fusion of ER-derived vesicles (Rowe et al.,
1998) or for other fusion events that are currently unchar-
acterized. In any event, a biochemical dissection of the
mechanisms underlying this spatial regulation should pro-
vide important insights into SNARE-dependent mem-
brane fusion.
We thank Hans Dieter Schmitt and Dieter Gallwitz for providing antibod-
ies and strains used in these studies.
This work was supported by grants from the National Institute of Gen-
eral Medical Sciences (GM52549) and the Pew Scholars Program in the
Biomedical Sciences.
Submitted: 23 August 1999
Revised: 22 February 2000
Accepted: 24 February 2000
References
Antebi, A., and G.R. Fink. 1992. The yeast Ca2⫹-ATPase homologue, PMR1, is
required for normal Golgi function and localizes in a novel Golgi-like distri-
bution. Mol. Biol. Cell. 3:633–654.
Baker, D., L. Hicke, M. Rexach, M. Schleyer, and R. Schekman. 1988. Recon-
stitution of SEC gene product-dependent intercompartmental protein trans-
port. Cell. 54:335–344.
Banfield, D.K., M.J. Lewis, and H.R. Pelham. 1995. A SNARE-like protein re-
quired for traffic through the Golgi complex. Nature. 375:806–809.
Bannykh, S.I., T. Rowe, and W.E. Balch. 1996. The organization of endoplas-
mic reticulum export complexes. J. Cell Biol. 135:19–35.
Barlowe, C. 1997. Coupled ER to Golgi transport reconstituted with purified
cytosolic proteins. J. Cell Biol. 139:1097–1108.
Barlowe, C., L. Orci, T. Yeung, M. Hosobuchi, S. Hamamoto, N. Salama, M.F.
Rexach, M. Ravazzola, M. Amherdt, and R. Schekman. 1994. COPII: a
membrane coat formed by Sec proteins that drive vesicle budding from the
endoplasmic reticulum. Cell. 77:895–907.
Baudin, A., O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, and C.A. Cul-
lin. 1993. A simple and efficient method for direct gene deletion in Saccharo-
myces cerevisiae. Nucleic Acids Res. 21:3329–3330.
Bednarek, S.Y., M. Ravazzola, M. Hosobuchi, M. Amherdt, A. Perrelet, R.
Schekman, and L. Orci. 1995. COPI- and COPII-coated vesicles bud directly
from the endoplasmic reticulum in yeast. Cell. 83:1183–1196.
Bennett, M.K., N. Calakos, T. Kreiner, and R.H. Sheller. 1992. Synaptic vesicle
membrane proteins interact to form a multimeric complex. J. Cell Biol. 116:
761–775.
Berninsone, P., Z.Y. Lin, E. Kempner, and C.B. Hirschberg. 1995. Regulation
of yeast Golgi glycosylation. J. Biol. Chem. 270:14564–14567.
Bonfanti, L., A.A. Mironov Jr., J.A. Martinez-Menarguez, O. Martella, A.
Fusella, M. Baldassarre, R. Buccione, H.J. Geuze, A.A. Mironov, and A.
Luini. 1998. Procollagen traverses the Golgi stack without leaving the lumen
of cisternae: evidence for cisternal maturation. Cell. 95:993–1003.
Cao, X., N. Ballew, and C. Barlowe. 1998. Initial docking of ER-derived vesi-
cles requires Uso1p and Ypt1p but is independent of SNARE proteins.
EMBO (Eur. Mol. Biol. Organ.) J. 17:2156–2165.
Chamberlain, L.H., D. Roth, A. Morgan, and R.D. Burgoyne. 1995. Distinct ef-
fects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and trig-
gering of regulated exocytosis. J. Cell Biol. 130:1063–1070.
Conchon, S., X. Cao, C. Barlowe, and H.R. Pelham. 1999. Got1p and Sft2p:
membrane proteins involved in traffic to the Golgi. EMBO (Eur. Mol. Biol.
Organ.) J. 18:3934–3946.
Coorssen, J.R., P.S. Blank, M. Tahara, and J. Zimmerberg. 1998. Biochemical
and functional studies of cortical vesicle fusion: the SNARE complex and
Ca2⫹ sensitivity. J. Cell Biol. 143:1845–1857.
Dascher, C., R. Ossig, D. Gallwitz, and H.D. Schmitt. 1991. Identification and
structure of four yeast genes (SLY) that are able to suppress the functional
loss of YPT1, a member of the RAS superfamily. Mol. Cell. Biol. 11:872–885.
Dascher, C., J. Matteson, and W. Balch. 1994. Syntaxin 5 regulates endoplasmic
reticulum to Golgi transport. J. Biol. Chem. 269:29363–29366.
Evan, G.I., G.K. Lewis, G. Ramsay, and J.M. Bishop. 1985. Isolation of mono-
clonal antibodies specific for human c-myc proto-oncogene product. Mol.
Cell. Biol. 5:3610–3616.
Fischer von Mollard, G.F., S.F. Nothwehr, and T.H. Stevens. 1997. The yeast
v-SNARE Vti1p mediates two vesicle transport pathways through interac-
tions with the t-SNAREs Sed5p and Pep12p. J. Cell Biol. 137:1511–1524.
Fischer von Mollard, G.F., and T.H. Stevens. 1999. The Saccharomyces cerevi-
siae v-SNARE Vti1p is required for multiple membrane transport pathways
to the vacuole. Mol. Biol. Cell. 10:1719–1732.
Garrett, M.D., J.E. Zahner, C.M. Cheney, and P.J. Novick. 1994. GDI1 encodes
a GDP dissociation inhibitor that plays an essential role in the yeast secre-
tory pathway. EMBO (Eur. Mol. Biol. Organ.) J. 13:1718–1728.
Gotte, M., and G.F. Fischer von Mollard. 1998. A new beat for the SNARE
drum. Trends Cell Biol. 8:215–218.
Haas, A., D. Scheglmann, T. Lazar, D. Gallwitz, and W. Wickner. 1995. The
GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vac-
uoles for the homotypic fusion step of vacuole inheritance. EMBO (Eur.
Mol. Biol. Organ.) J. 14:5258–5270.
Hardwick, K.G., and H.R. Pelham. 1992. SED5 encodes a 39-kD integral mem-
brane protein required for vesicular transport between the ER and the Golgi
complex. J. Cell Biol. 119:513–521.
Hay, J.C., D.S. Chao, C.S. Kuo, and R.H. Scheller. 1997. Protein interactions
regulating vesicle transport between the endoplasmic reticulum and Golgi
apparatus in mammalian cells. Cell. 89:149–158.
Hay, J.C., J. Klumperman, V. Oorschot, M. Steegmaier, C.S. Kuo, and R.H.
Scheller. 1998. Localization, dynamics, and protein interactions reveal dis-
tinct roles for ER and Golgi SNAREs. J. Cell Biol. 141:1489–1507.
Hicke, L., and R. Schekman. 1989. Yeast Sec23p acts in the cytoplasm to pro-
mote protein transport from the endoplasmic reticulum to the Golgi com-
plex in vivo and in vitro. EMBO (Eur. Mol. Biol. Organ.) J. 8:1677–1684.
Kaiser, C.A., and R. Schekman. 1990. Distinct sets of SEC genes govern trans-
port vesicle formation and fusion early in the secretory pathway. Cell. 61:
723–733.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature. 227:680–685.
Latterich, M., K.U. Frohlich, and R. Schekman. 1995. Membrane fusion and the
cell cycle: Cdc48p participates in the fusion of ER membranes. Cell. 82:885–
893.
Lewis, M.J., and H.R. Pelham. 1996. SNARE-mediated retrograde traffic from
the Golgi complex to the endoplasmic reticulum. Cell. 85:205–215.
Lewis, M.J., J.C. Rayner, and H.R.B. Pelham. 1997. A novel SNARE complex
implicated in vesicle fusion with the endoplasmic reticulum. EMBO (Eur.
Mol. Biol. Organ.) J. 16:3017–3024.
Lian, J.P., and S. Ferro-Novick. 1993. Bos1p, an integral membrane protein of
the endoplasmic reticulum to Golgi transport vesicles, is required for their
fusion competence. Cell. 73:735–745.
Lian, J.P., S. Stone, Y. Jiang, P. Lyons, and S. Ferro-Novick. 1994. Ypt1p impli-
cated in v-SNARE activation. Nature. 372:698–701.
Lin, C.-C., H.D. Love, J.N. Gushe, J.J.M. Bergeron, and J. Ostermann. 1999.
ER/Golgi intermediates acquire Golgi enzymes by brefeldin A–sensitive ret-
rograde transport in vitro. J. Cell Biol. 147:1457–1472.
Lin, R.C., and R.H. Scheller. 1997. Structural organization of the synaptic exo-
cytosis core complex. Neuron. 19:1087–1094.
Lupashin, V.V., and M.G. Waters. 1997. t-SNARE activation through transient
interaction with a Rab-like guanosine triphophatase. Science. 276:1255–
1258.
Mayer, A., W. Wickner, and A. Haas. 1996. Sec18p (NSF)-driven release of
Sec17p (␣-SNAP) can precede docking and fusion of yeast vacuoles. Cell.
85:83–94.
McNew, J.A., M. Sogaard, N.M. Lampen, S. Machida, R.R. Ye, L. Lacomis, P.
Tempst, J.E. Rothman, and T.H. Sollner, 1997. Ykt6p, a prenylated SNARE
essential for endoplasmic reticulum-Golgi transport. J. Biol. Chem. 272:
17776–17783.
Morin-Ganet, M.-N., A. Rambourg, S.B. Deitz, A. Franzusoff, and F. Kepes.
1999. Morphogenesis and dynamics of the yeast Golgi apparatus. Traffic.
1:56–68.
Nagahama, M., L. Orci, M. Ravazzola, M. Amherdt, L. Lacomis, P. Tempst,
J.E. Rothman, and T.H. Sollner, 1996. A v-SNARE implicated in intra-
Golgi transport. J. Cell Biol. 133:507–516.
Newman, A.P., and S. Ferro-Novick. 1987. Characterization of new mutants in
the early part of the yeast secretory pathway isolated by a [3H]mannose sui-
cide selection. J. Cell Biol. 105:1587–1593.
Newman, A.P., J. Shim, and S. Ferro-Novick. 1990. BET1, BOS1 and SEC22
are members of a group of interacting yeast genes required for transport
from the endoplasmic reticulum to the Golgi complex. Mol. Cell. Biol. 10:
3405–3414.
Newman, A.P., M.E. Groesch, and S. Ferro-Novick. 1992. Bos1p, a membrane
protein required for ER to Golgi transport in yeast, co-purifies with the car-
rier vesicles and with Bet1p and the ER membrane. EMBO (Eur. Mol. Biol.
Organ.) J. 11:3609–3617.
Nichols, B.J., and H.R.B. Pelham. 1998. SNAREs and membrane fusion in the
Golgi apparatus. Biochim. Biohys. Acta. 1404:9–31.

Cao and Barlowe Asymmetric Requirements for ER/Golgi SNAREs 65
Nichols, B.J., C. Ungermann, H.R. Pelham, W. Wickner, and A. Haas. 1997.
Homotypic vacuolar fusion mediated by t- and v-SNAREs. Nature. 387:199–
202.
Ossig, R., C. Dascher, H. Trepte, H.D. Schmitt, and D. Gallwitz. 1991. The
yeast SLY gene products, suppressors of defects in the essential GTP-bind-
ing Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport. Mol.
Cell. Biol. 11:2980–2993.
Ossig, R., W. Laufer, H.D. Schmitt, and D. Gallwitz. 1995. Functionality and
specific membrane localization of transport GTPases carrying C-terminal
membrane anchors of synaptobrevin-like proteins. EMBO (Eur. Mol. Biol.
Organ.) J. 14:3645–3653.
Patel, S.K., F.E. Indig, N. Olivieri, N.D. Levine, and M. Latterich. 1998. Or-
ganelle membrane fusion: a novel function for the syntaxin homolog Ufe1p
in ER membrane fusion. Cell. 92:611–620.
Pelham, H.R.B. 1998. Getting through the Golgi complex. Trends Cell Biol.
8:45–49.
Peng, R., R Grabowski, A. De Antoni, and D. Gallwitz. 1999. Specific interac-
tion of the yeast cis-Golgi syntaxin Sed5p and the coat protein complex II
component Sec24p of endoplasmic reticulum–derived transport vesicles.
Proc. Natl. Acad. Sci. USA. 96:3751–3756.
Poirier, M.A., W. Xiao, J.C. Macosko, C. Chan, Y.-K. Shim, and M.K. Bennett.
1998. The synaptic SNARE complex is a parallel four-stranded helical bun-
dle. Nat. Struct. Biol. 5:765–769.
Poon, P.P., D. Cassel, A. Spang, M. Rotman, E. Pick, R.A. Singer, and G.C.
Johnston. 1999. Retrograde transport from the yeast Golgi is mediated by
two ARF GAP proteins with overlapping function. EMBO (Eur. Mol. Biol.
Organ.) J. 18:555–564.
Powers, J., and C. Barlowe. 1998. Transport of Axl2p depends on Erv14p, an
ER–vesicle protein related to the Drosophila cornichon gene product. J. Cell
Biol. 142:1209–1222.
Presley, J.F., N.B. Cole, T.A. Schroer, K. Hirschberg, K.J.M. Zaal, and J. Lip-
pincott-Schwartz. 1997. ER to Golgi transport visualised in living cells. Na-
ture. 389:81–85.
Preuss, D., J. Mulholland, A. Franzusoff, N. Segev, and D. Botstein. 1992.
Characterization of the Saccharomyces Golgi complex through the cell cycle
by immunoelectron microscopy. Mol. Biol. Cell. 3:789–803.
Rabouille, C., H. Kondo, R. Newman, H. Hui, P. Freemont, and G. Warren.
1998. Syntaxin 5 is a common component of the NSF- and p97-mediated re-
assembly pathways of Golgi cisternae from mitotic Golgi fragments in vitro.
Cell. 92:603–610.
Rexach, M.F., and R.W. Schekman. 1991. Distinct biochemical requirements
for the budding, targeting, and fusion of ER-derived transport vesicles. J.
Cell Biol. 114:219–229.
Rexach, M.F., M. Latterich, and R.W. Schekman. 1994. Characteristics of endo-
plasmic reticulum-derived transport vesicles. J. Cell Biol. 126:1133–1148.
Robinson, L.J., and T. Martin. 1998. Docking and fusion in neuronsecretion.
Curr. Opin. Cell Biol. 10:483–492.
Rothman, J.E., and F.T. Wieland. 1996. Protein sorting by transport vesicles.
Science. 272:227–234.
Rowe, T., C. Dascher, S. Bannykh, H. Plutner, and W. Balch. 1998. Role of ves-
icle-associated syntaxin 5 in the assembly of pre-Golgi intermediates. Sci-
ence. 279:696–700.
Sacher, M., S. Stone, and S. Ferro-Novick. 1997. The synaptobrevin-related do-
mains of Bos1p and Sec22p bind to the syntaxin-like region of Sed5p. J. Biol.
Chem. 272:17134–17138.
Scales, S.J., R. Pepperkok, and T.E. Kreis. 1997. Visualization of ER-to-Golgi
transport in living cells reveals a sequential mode of action for COPII and
COPI. Cell. 90:1137–1148.
Schekman, R., and L. Orci. 1996. Coat proteins and vesicle budding. Science.
271:1526–1533.
Schroder, S., F. Schimmoller, B. Singer-Kruger, and H. Riezman. 1995. The
Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not
affected by the ret1-1 mutation in alpha-COP. J. Cell Biol. 131:895–912.
Shim, J., A.P. Newman, and S. Ferro-Novick. 1991. The BOS1 gene encodes an
essential 27-kD putative membrane protein that is required for vesicular
transport from the ER to the Golgi complex in yeast. J. Cell Biol. 113:55–64.
Sikorski, R., and P. Hieter. 1989. A system of shuttle vectors and yeast host
strains designed for efficient manipulation of DNA in Sacchromyces cerevi-
siae. Genetics. 122:19–27.
Sogaard, M., K. Tani, R.R. Ye, S. Geromanos, P. Tempst, T. Kirchhausen, J.E.
Rothman, and T. Sollner. 1994. A rab protein is required for the assembly of
SNARE complexes in the docking of transport vesicles. Cell. 78:927–948.
Sollner, T., M.K. Bennett, S.W. Whiteheart, R.H. Scheller, and J.E. Rothman.
1993a. A protein assembly-disassembly pathway in vitro that may corre-
spond to sequential steps of synaptic vesicle docking, activation, and fusion.
Cell. 75:409–418.
Sollner, T., S.W. Whiteheart, M. Brunner, H. Erdjument-Bromage, S. Gero-
manos, P. Tempst, and J.E. Rothman. 1993b. SNAP receptors implicated in
vesicle targeting and fusion. Nature. 362:318–324.
Spang, A., and R. Schekman. 1998. Reconstitution of retrograde transport from
the Golgi to the ER in vitro. J. Cell Biol. 143:589–599.
Springer, S., and R. Schekman. 1998. Nucleation of COPII vesicular coat com-
plex by endoplasmic reticulum to Golgi vesicle SNAREs. Science. 281:698–
700.
Stirling, C.J., J. Rothblatt, M. Hosobuchi, R. Deshaies, and R. Schekman. 1992.
Protein translocation mutants defective in the insertion of integral mem-
brane proteins into the endoplasmic reticulum. Mol. Biol. Cell. 3:129–142.
Stone, S., M. Sacher, Y. Mao, C. Carr, P. Lyons, A.M. Quinn, and S. Ferro-
Novick. 1997. Bet1p activates the v-SNARE Bos1p. Mol. Biol. Cell. 8:1175–
1181.
Subramaniam, V.N., F. Peter, R. Philp, S.H. Wong, and W. Hong, 1996. GS28, a
28-kilodalton Golgi SNARE that participates in ER-Golgi transport. Sci-
ence. 272:1161–1163.
Sutton, R.B., D. Fasshauer, R. Jahn, and A.T. Brünger. 1998. Crystal structure
of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution.
Nature. 395:347–353.
Towbin, H., T. Straelin, and J. Gordon. 1979. Electrophoretic transfer of pro-
teins from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proc. Natl. Acad. Sci. USA. 76:4350–4354.
Ungermann, C., K. Sato, and W. Wickner. 1998. Defining the functions of
trans-SNARE pairs. Nature. 396:543–548.
Ungermann, C., G.F. von Mollard, O.N. Jensen, N. Margolis, T.H. Stevens, and
W. Wickner. 1999. Three v-SNAREs and two t-SNAREs, present in a pen-
tameric cis-SNARE complex on isolated vacuoles, are essential for homo-
typic fusion. J. Cell Biol. 145:1435–1442.
Walch-Solimena, C., J. Blasi, L. Edelmann, E.R. Chapman, G.F. von Mollard,
and R. Jahn. 1995. t-SNAREs syntaxin 1 and SNAP-25 are present on or-
ganelles that participate in synaptic vesicle recycling. J. Cell Biol. 128:637–
645.
Weber, T., B.V. Zelmelman, J.A. McNew, B. Westermann, M. Gmachl, F. Par-
lati, T.H. Sollner, and J.E. Rothman. 1998. SNAREpins: minimal machinery
for membrane fusion. Cell. 92:759–772.
Weimbs, T., S.H. Low, S.J. Chapin, K.E. Mostov, P. Bucher, and K. Hofmann.
1997. A conserved domain is present in different families of vesicular fusion
proteins: a new superfamily. Proc. Natl. Acad. Sci. USA. 94:3046–3051.
Weimbs, T., K. Mistov, S.H. Low, and K. Hofman. 1998. A model for structural
similarity between different SNARE complexes based on sequence relation-
ships. Trends Cell Biol. 8:260–262.
Wichmann, H., L. Hengst, and D. Gallwitz. 1992. Endocytosis in yeast: evi-
dence for the involvement of a small GTP-binding protein (Ypt7p). Cell. 71:
1131–1142.
Wilson, D.W., S.W. Whiteheart, M. Wiedman, M. Brunner, and J.E. Rothman.
1992. A multisubunit particle implicated in membrane fusion. J. Cell Biol.
117:531–538.
Wooding, S., and H.R. Pelham. 1998. The dynamics of Golgi protein traffic vi-
sualized in living yeast cells. Mol. Biol. Cell. 9:2667–2680.
Xu, Y., S.H. Wong, T. Zhang, V.N. Subramaniam, and W. Hong. 1997. GS15, a
15-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment
protein receptor (SNARE) homologous to rbet1. J. Biol. Chem. 272:20162–
20166.
Yanagisawa, K., D. Resnick, C. Abeijon, P.W. Robbins, and C.B. Hirschberg.
1990. A guanosine diphophatase enriched in Golgi vesicles of Saccharomy-
ces cerevisiae: purification and characterization. J. Biol. Chem. 265:19351–
19355.
Zhang, T., S.H. Wong, B.L. Tang, Y. Xu, F. Peter, V.N. Subramaniam, and W.
Hong. 1997. The mammalian protein (rbet1) homologous to yeast Bet1p is
primarily associated with the pre-Golgi intermediate compartment and is in-
volved in vesicular transport from the endoplasmic reticulum to the Golgi
apparatus. J. Cell Biol. 139:1157–1168.