 |
INTRODUCTION |
Membrane-bound compartments in eukaryotic cells can fuse directly
as shown for the endoplasmic reticulum
(ER)1 and mitotic Golgi
fragments as well as endosomal and lysosomal compartments (homotypic
fusion; see Ref. 1). However, vectorial transport between distinct
compartments mainly involves small coated vesicles whose formation from
the donor membrane is mediated by proteinaceous coats, either COPI,
COPII, or clathrin. After uncoating, vesicles fuse selectively with an
acceptor membrane (heterotypic fusion; see Ref. 2). Both homotypic and
heterotypic fusion events rely on specific attachment reactions to
guarantee that only appropriate membranes can mix. The membrane
attachment itself consists of two steps, tethering and docking,
involving different sets of proteins (3, 4). Tethering factors are peripherally membrane-associated protein complexes consisting of up to
10 different subunits, which share little sequence similarity.
The subsequent docking stage involves specific sets of
membrane-anchored proteins, so-called SNARE proteins (SNARE is
soluble NSF (for
N-ethylmaleimide-sensitive fusion protein)
attachment protein receptor) (5-7). SNAREs are
inserted into the membrane either by a C-terminal transmembrane domain
or through lipid moieties attached to C-terminal cysteine residues. In
contrast to the tethering factors, all known SNARE proteins are members
of either of three protein families: the syntaxins, the synaptobrevins
or VAMPs, and the SNAP-25 family members. To induce membrane fusion,
SNARE proteins from apposed membranes must interact in
trans. The formation of a stable four-helix bundle may
generate enough energy to promote mixing of the lipid bilayer
(8-10).
Lipid mixing experiments using SNARE complexes reconstituted into lipid
bilayer vesicles indicated that only cognate SNARE combinations are
able to induce fusion (11). However, SNARE proteins are rather
promiscuous when the formation of the tight SDS or heat-resistant SNARE
complexes is analyzed (12, 13). Moreover, SNARE proteins can be part of
more than one SNARE complex in vivo (14), and some SNARE
proteins can functionally replace each other (15, 16). In
vitro the synaptobrevin/VAMP homologs Snc1p and Snc2p in yeast can
be replaced by two other members of the synaptobrevin family, the
ER-Golgi SNARE Sec22p and the vacuolar SNARE Nyv1p (11). However, these
SNAREs are unable to replace Snc1/2p in vivo (17), probably
because they are retained in their specific compartments. Thus, the
targeting of SNAREs to the right compartment is one way to increase the
specificity of intracellular membrane attachment/fusion events.
We analyzed previously (18) the targeting of the ER-to-Golgi SNARE
Sec22p and show that the correct targeting of Sec22p involves its
recycling from the Golgi to the ER via COPI-coated vesicles. In this
respect, Sec22p as well as Bos1p (19) behave like ER-resident proteins
that carry a KKXX ER-retrieval signal (20). The coat of COPI
vesicles in mammalian cells and yeast consists of seven subunits (
-,
-, '-, 
-, 
-, 
-, and 
-COP) and the small GTPase, ARF1
(21).
The observations made by Letourneur et al. (20) and Cosson
et al. (22) that KKXX-tagged proteins require
COPI components for retrieval from Golgi to the ER provided first
evidence that COPI vesicles mediate this retrograde transport. The same
is true not only for Sec22p but also for other yeast proteins that
recycle from Golgi to ER, for example, Emp47p, a Golgi lectin-like
protein; Erd2p, the HDEL receptor; Sed5p, a Golgi-localized
syntaxin homolog; and Mnn1p, a glycosyltransferase (23-26). How Sec22p
is sorted into COPI vesicles is currently unknown. Moreover, the
function of Sec22p is not entirely understood. The SEC22
gene was first isolated by us as a multicopy suppressor of defects in
the small GTPase Ypt1p involved in ER-to-Golgi transport (named
SLY2; see Ref. 27). Later SLY2 was found to be
identical to SEC22 (28) for which conditional mutant alleles
had been identified by Novick et al. (29). Like several
other SNARE proteins, Sec22p can be a component of more than just one
SNARE complex. Its physical interaction with the SNARE proteins Sed5p,
Bos1p, Bet1p, and other Golgi SNARE proteins argues for a role in
anterograde traffic from ER-to-Golgi (6, 30, 31). Sec22p also
co-precipitates with the ER proteins Ufe1p and Sec20p that function in
retrograde Golgi-ER transport (24, 32, 33). sec22 mutants
lead to a defect in forward traffic (34-37). However, this defect, as
with many other mutants affected in retrograde transport, could be a
secondary effect. In vitro assays performed with
permeabilized mutant cells showed that the sec22-3 mutation
does not slow down forward transport but does inhibit retrograde
transport (38, 39). Membrane fusion reconstituted with liposomes
containing the ER-to-Golgi SNARE Bet1p requires the presence of Sec22p
along with Sed5p and Bos1p on the opposing membranes to drive fusion (11, 40). In mammalian cells the Sec22p homolog sec22b coprecipitates with syntaxin 5 (~Sed5p), rbet1 (~Bet1p), and membrin
(~Bos1p) (41) as well as syntaxin 18, which may be functionally
equivalent to Ufe1p (42). Therefore, the dual function of Sec22p may be conserved throughout evolution.
To obtain additional clues to the function of Sec22p, we used a genetic
approach. SEC22 is not essential for cell viability (27). We
tried to find mutant yeast strains in which SEC22 became essential. A new allele of yeast ORF YNL258c showed
synthetic lethality with sec22
. Mutants in
YNL258c were recently shown to be dependent on a dominant
allele of SLY1, a suppressor of many ER-to-Golgi transport
defects, and the gene was named DSL1 (43). Evidence was
provided for a function of Dsl1p in ER-to-Golgi forward transport.
Genetic interaction of some dsl1 mutants with the
-COP-encoding SEC21 gene also suggested a role of Dsl1p
in retrograde Golgi-to-ER traffic (43). We show that a new allele of
DSL1, dsl1-22, isolated in our screen indeed
affects Golgi-to-ER retrieval of several proteins with only slight
effects on forward transport. dsl1-22 interacts genetically
with factors required for retrograde traffic, and Dsl1p binds coatomer.
Taken together, our data provide strong evidence for a direct role of
Dsl1p in Golgi-to-ER traffic.
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EXPERIMENTAL PROCEDURES |
Yeast Strains, Genetic Techniques, and
Plasmids--
Saccharomyces cerevisiae strains
used are listed in Table I. Cells were
grown in yeast extract/peptone/dextrose or synthetic minimal medium
containing galactose (2%) or glucose (2%) as carbon sources and
supplemented as necessary with 20 mg/liter tryptophan, histidine,
adenine, uracil or 30 mg/liter leucine or lysine. To enhance the
visualization of sectoring colonies, plates with low adenine
concentration (10 mg/liter adenine) were prepared. 5-FOA plates were
prepared as synthetic minimal medium containing 0.1% 5-FOA. Yeast
transformations were performed as described previously (44). Standard
techniques were used for mating of haploid strains, complementation
analysis, sporulation, and the analysis of tetrads (45). The assay to
detect retention defects using Ste2-Wbp1p was described previously (20,
46). The analysis of synthetic lethal effects between the
dsl1-22 mutation and other ER-Golgi defects was performed
with strains derived from the original mutant by three crosses to wild
type strains or a dsl1-22-myc::KanMX strain derived from the original transformant by two crosses to wild
type strains. When possible tetrad analysis was performed 2 or 3 days
after placing diploid cells on potassium acetate plates. The viability
of spores varied considerably. Therefore, the genotype of viable spores
was determined by crosses to tester strains (complementation assays).
The dsl1-22-myc::KanMX carrying spores
were identified by their resistance to G418. 98% of the possible
dsl1-22, sec23-1, mutants, 80% of the possible
dsl1-22, bet1-1 double mutants, 64% of the dsl1-22,
sec27-1, 47% of the dsl1-22, sec22-3, 36% of the dsl1-22, sed5-1, and 28% of the dsl1-22, bos1
(sec31-1) double mutants could form colonies. The viability
of dsl1-22 single mutants in these tetrads was higher than
90%. No double mutants were obtained when we tried to combine the
dsl1-22 defect with the sec20-1, sec21-1, tip20-5, ret1-1, and
ret1-1. An unexpected result was the very low viability of
all dsl1-22 spores derived from a diploid heterozygous for
dsl1-22 and sly1ts.
Genomic tagging of the DSL1 gene was achieved as described
by De Antoni and Gallwitz (47) using the oligonucleotides UA1 (5'-AAA
CTG AAA AAA AGA CAA CTT ACG CAT ACG TAA TAC AAG ATG TAC ACT ATA GGG AGA
CCG GCA GAT C-3') and UA2 (5'-GCC ATT GAT GAT ATT TAC GAA ATT AGA GGC
ACT GCT CTA GAT GAT TCC CAC CAC CAT CAT CAT CAC-3'), whereas the
oligonucleotides UA1 and UA3 (5'-ATG TTT TAC AAT GGG GAT TTT TAT CTT
TTT GCG ACA GAC GAA CTA ATC TCC CAC CAC CAT CAT CAT CAC-3') were used
for tagging the dsl1-22 mutant. Plasmids used in this work
are listed in Table II.
Synthetic Lethality Screen--
Mutants synthetically lethal
with sec22 were isolated using the ade2/ade8,
red/white sectoring system (48). The SLA28-6C and SUA1-12D strains were
red after transformation with pHDS228 on selective plates but gave
white sectors under non-selective conditions due to plasmid loss. After
mutagenesis with ethyl methane sulfonate 15 non-sectoring colonies
could be identified among 200,000 screened. 10 clones were not able to
lose the plasmid (SEC22, URA3) on 5-FOA plates, and 5 of
these did not grow at 37 °C. Three of these allowed the displacement
of SEC22 by sec22-3. They were transformed with a
LEU2/CEN-based yeast genomic library, and one strain showed
transformants that sectored and did not contain SEC22. The
complementing gene was identified by sequencing the ends of the insert
and expression of the single ORF.
Sequencing the lsd1-1 (dsl1-22) Mutation--
The
lsd1-1 (dsl1-22) mutation was cloned by the
gap-repair method (49). A 3-kilobase pair fragment
(XhoI-BglII) containing YNL258c was
inserted into the XhoI-BamHI sites of pRS315
(LEU2, CEN6), and a BamHI-SnaBI
fragment was removed. The resulting plasmid, which includes DNA that
flanks 5'- and 3'-coding regions of DSL1, was transformed
into the yeast strain YUA1-9C (MAT, leu2,
dsl1-22). Plasmid DNA was isolated from the yeast
transformant, amplified in Escherichia coli, and subjected
to automated sequencing.
Antibodies--
The monoclonal anti-c-Myc antibody 9E10
(50) and a polyclonal anti-c-Myc antibody (A-14) were obtained
from Santa Cruz Biotechnology. Rabbit antibodies against Sec22p
(kindly provided by R. Ossig and R. Grabowski), Emp47p (51),
Bet1p, Bos1p, Sed5p, Ypt1p, Sec24p, and BiP/Kar2p were used (52). The
polyclonal anti-Ufe1p and anti-coatomer antibodies were gifts from M. Lewis and R. Duden, respectively (Cambridge, UK). Horseradish
peroxidase-coupled secondary anti-rabbit or anti-mouse antibodies and
cyanine-(Cy2TM or Cy3TM)-conjugated secondary
antibodies were purchased from The Jackson Laboratories.
Protein Extraction and Immunoblotting--
Western blotting
analysis was performed as described by Boehm et al. (53).
Aliquots (1 A600 = 1.7 × 107
cells) of transformed cells were lysed in 2 M NaOH, 5%
mercaptoethanol and proteins precipitated with 10% trichloroacetic
acid, neutralized with 1.5 M Tris base, and dissolved in
SDS sample buffer. Proteins were resolved on 12% SDS-PAGE.
Purification of Recombinant Proteins and Affinity Binding
Assay--
E. coli and S. cerevisiae strains
expressing GST fusion proteins were lysed, and proteins were
solubilized in lysis buffer (20 mM Hepes, pH 6.8, 150 mM KOAc, 5 mM Mg(OAc)2, 1 mM dithiothreitol, 1% Triton X-100, protease inhibitor
mix). GST fusion proteins were immobilized on glutathione-Sepharose 4B
and washed 5 times with 10 volumes lysis buffer. Proteins bound to GST
fusion proteins expressed in yeast were separated by SDS-PAGE and
analyzed by immunoblotting. E. coli proteins immobilized on
glutathione-Sepharose 4B were incubated at 4 °C for 2 h with
100,000 × g supernatant of yeast cell lysate. The
beads were washed 5 times, and proteins were separated by SDS-PAGE
followed by immunoblot analysis.
Subcellular and Sucrose Gradient Fractionation--
Yeast cells
were harvested at mid-logarithmic phase. The cell pellet was washed
twice with water and once with B88 (20 mM Hepes, pH 6.8, 250 mM sorbitol, 150 mM KOAc, 5 mM
Mg(OAc)2), resuspended in a minimal volume of B88
containing EDTA-free protease inhibitor mix (Roche Molecular
Biochemicals), and pipetted into liquid nitrogen. Cells were ground up
in a mortar. The cell powder was resolved in B88 (supplemented with
EDTA-free protease inhibitor mix) and centrifuged twice at 500 × g for 5 min to remove cell debris, and the clear lysate was
centrifuged at 10,000 × g for 15 min to obtain the P10
pellet. The S10 fraction was then subjected to centrifugation at
100,000 × g at 4 °C for 1 h to obtain P100 and
S100. To investigate the membrane localization of Dsl1p, the supernatant of the cell lysate after a 500 × g
centrifugation was divided into different portions that were treated
for 30 min on ice with either 5 M urea, 1% Triton X-100,
or 1 M NaCl. The 500 × g lysate was also
subjected to sucrose density gradient centrifugation.
For fractionation experiments, lysates were loaded on sucrose density
gradients (51) and spun at 4 °C in a Beckman SW40 rotor at 37,000 rpm for 2.5 h. 1-ml fractions were taken, and the last fraction
was adjusted to 1 ml with B88. Each fraction was mixed with 1 ml of
SDS-PAGE sample buffer (8 M urea, 50 mM Tris-HCl, pH 8.0, 2% SDS, 0.1 mg/ml bromphenol blue) and incubated at
50 °C for 10 min prior to analysis by SDS-PAGE and immunoblotting.
Protein Labeling, Immunoprecipitation, and Invertase
Assay--
For detection of CPY processing cells were shifted to
37 °C for indicated times, pulse-labeled for 5 min with
Tran35S-label (ICN) and chased for 30 min. The labeled
proteins were immunoprecipitated using specific antibodies and
separated by SDS-PAGE. After incubating the gel with Amplify (Amersham
Pharmacia Biotech) for 45 min, the proteins were detected by exposing
the gels to X-Omat AR (Eastman Kodak Co.) at 
80 °C. Invertase
activity staining was carried out as described previously (54).
Fluorescence and Electron Microscopy--
Indirect
immunofluorescence was performed as described by Schröder
et al. (51) using rabbit polyclonal anti-Kar2p and
monoclonal mouse c-Myc epitope (9E10) antibodies.
Cy2TM-conjugated goat anti-rabbit or anti-mouse F(ab') 2 fragment (Jackson ImmunoResearch) served as secondary antibody. DNA was
stained with 4',6-diamidino-2-phenylindole (DAPI). Cells expressing GFP fusion proteins were grown in SD medium at 25 °C to mid-log phase and placed onto a slide. A coverslip was added, and cells were examined
immediately. DAPI staining was achieved after fixing cells in methanol
at 20 °C for 10 min, washing with acetone at 20 °C, and
washing three times with ice-cold PBS, pH 7.4. Confocal images were
obtained with a TSC SP1 confocal laser-scanning microscope (Leica). For
electron microscopy, yeast cells at mid-logarithmic phase were fixed
and stained with permanganate to enhance visualization of membrane
structures (54).
| |
RESULTS |
Identification of Mutants for Which SEC22 Is Essential--
To
find proteins that can substitute for Sec22p or to identify factors
that prevent these proteins from functioning normally, we performed a
synthetic lethality screen. Mutants inviable in the absence of
SEC22 were isolated by using a colony sectoring assay (48).
sec22 mutants that carry a functional SEC22
gene on the centromeric plasmid pHDS228 were mutagenized. In addition to SEC22 this plasmid contains the following two markers
required for pyrimidine and purine biosynthesis: URA3 as
selectable marker and ADE8, which can serve as a color
marker in yeast strains carrying mutated versions of the
ADE2 and ADE8 genes on the chromosomes. The
ade8 mutation is epistatic to ade2 and prevents
the formation of the red color typical for ade2 mutants.
Therefore, cells expressing ADE8 from a plasmid are red,
whereas those that lost the plasmid turn white. As expected, on rich
media sec22, ade8, ade2, ura3 cells containing
pHDS228 could form white sectors since neither SEC22,
ADE8, nor URA3 are essential. After mutagenesis,
we screened for non-sectoring colonies (for details see "Experimental
Procedures"). To confirm that the non-sectoring phenotype in fact
reflects a positive selection for the presence of the
SEC22-carrying plasmid, all mutants were tested
for their ability to lose the second plasmid-encoded marker,
URA3. This test makes use of the drug 5-FOA (5-fluoroorotic acid), which is toxic to Ura+ cells (55). In fact, most of
the non-sectoring mutants were sensitive to 5-FOA and only these
mutants were analyzed further. In addition to these two phenotypes,
five mutants obtained in two independent screens were also
temperature-sensitive for growth. Genetic analysis showed that the
mutations are recessive and that the inability to lose the
SEC22 gene is tightly linked to the growth defect at
37 °C (see Fig. 1A). They
belong to three different complementation groups that we called
"LSD1, -2, and -3" (lethal with
SEC22 deletion).
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Fig. 1.
Growth characteristics of
dsl1-22 mutant cells. A,
sec22::HIS3/sec22::HIS3
DSL1/dsl1-22, ade2/ade2, ade8/ade8 heterozygous diploid
cells carrying ADE8 and SEC22 on a plasmid
(pHDS228) were sporulated; spores were separated, and the segregants
were incubated on rich medium. Tetrads were replica-plated to low
adenine plates and incubated either at 25 or 37 °C. All
red colonies that are not able to lose the plasmid pHDS228
are temperature-sensitive showing that both defects are closely linked.
B, growth of wild type (MSUC-3B) and dsl1-22
(YUA1-9C) cells was monitored by measuring the cell density
(A600 nm) during incubation at 25 °C. After
5 h of incubation at 25 °C an aliquot of each sample was
shifted to 37 °C for additional 5 h.
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We tried to clone the "LSD" genes from single copy or
multicopy genomic libraries containing LEU2 as a selectable
marker (27). To obtain complementing plasmids, we selected
transformants on plates lacking leucine and looked for colonies with
white sectors. The formation of white sectors indicated that the cells
had again acquired the ability to lose the SEC22-carrying
plasmid pHDS228. Those transformants, which had simply received an
additional copy of SEC22 from the library, were identified
by PCR and discarded. So far our attempts to isolate complementing
plasmids from a single copy library were successful only for the
"lsd1-1" mutant. The library plasmid that we obtained
harbored three intact open reading frames. Sequencing and subcloning
showed that the presence of YNL258c alone was sufficient to
suppress both the non-sectoring phenotype and the temperature
sensitivity of the lsd1-1 mutant. The open reading frame
YNL258c, located on chromosome XIV, encodes an essential
protein with a predicted molecular mass of 88 kDa with no similarity to
other proteins in data bases (56).
The following observations confirmed that defects in YNL258c
result in a SEC22-dependent phenotype as well as
a conditional lethal phenotype. Cloning and sequencing of the
lsd1-1 mutant allele revealed the presence of a stop codon
at position 2173 of the 2265-base pair long reading frame. This would
lead to a gene product, which is 30 residues shorter than the putative
wild type protein. By using a PCR-based method described by De Antoni and Gallwitz (47), we replaced YNL258c either by a
full-length or a shortened version, which were fused to sequences
encoding a His6 epitope followed by two copies of a c-Myc
tag. The KanMX cassette inserted downstream of the
c-Myc-tagged YNL258c sequences served as a selectable marker
that allows the transformants to grow in the presence of geneticin
(G418). Temperature-sensitive transformants were obtained only when the
C-terminally truncated version of YNL258c was introduced
into wild type cells. Western blotting analysis showed that
Ts
transformants in fact encode a shorter c-Myc-tagged
YNL258c protein than cells expressing the full-length version (data not
shown). Tetrad analysis also confirmed that these mutants need
SEC22 for growth (see below). The same results were obtained
when N-terminally tagged versions of YNL258c and its mutant
variant expressed from a centromeric vector were used to complement the
deletion of YNL258c. In summary, these data established that
the deletion of 30 C-terminal triplets from the ORF YNL258c
results in a conditional lethal phenotype. In cells carrying this
mutation the otherwise non-essential SEC22 gene is rendered essential.
While this work was in progress Waters and co-workers (43) showed that
mutations in YNL258c can make cells dependent on the
SLY1-20 mutation. The mutants identified were accordingly named dsl1-1 to dsl1-7 (dependent on
SLY1-20). SLY1-20 is a dominant mutation, which
suppresses the defects in several yeast mutants affected in ER-to-Golgi
transport (27, 37, 57-60). Accordingly, the mutant we obtained was
renamed dsl1-22. Consistent with the results obtained by
VanRheenen et al. (43), the temperature sensitivity of
dsl1-22 is suppressed by the SLY1-20 mutation on a single copy plasmid (data not shown).
Genetic Interaction of dsl1-22 with Other Genes Whose Products Act
in ER-Golgi Anterograde and Retrograde Transport--
In the process
of cloning out sequences able to complement the dsl1-22
mutation, we also obtained clones from multicopy libraries. Among these
clones were plasmids containing the YKT6 gene.
YKT6 encodes a lipid-anchored member of the synaptobrevin
family of SNARE proteins (61). This prompted us to test whether the
overexpression of other SNARE-encoding genes has similar effects.
We found that, similar to the results obtained with YKT6,
overexpression of SED5 allowed dsl1-22 mutants to
tolerate the loss of SEC22. However, the overexpression of
neither YKT6 nor SED5 was able to suppress the
Ts phenotype of ds11-22 mutants.
Overexpression of the other SNARE-encoding genes specific for ER-Golgi
transport, BET1, BOS1 or UFE1, was unable to suppress the non-sectoring phenotype of dsl1-22 mutants.
The approach, which led to the isolation of the dsl1-22, was
based on the synthetic lethality of the dsl1-22 mutation
when combined with the sec22 deletion. Therefore, we also
addressed the question whether dsl1-22 is synthetically
lethal with other defects in ER-to-Golgi transport. For this and all
subsequent assays we used dsl1-22 mutants expressing
SEC22 from its normal locus on chromosome XII: (i) a strain
obtained by backcrossing cells derived from the original mutant (Fig.
1A) twice to wild type cells (SEC22), and (ii) a
mutant in which we had introduced dsl1-22-myc
construct at the YNL258c locus (see above). The analysis of
tetrads was greatly facilitated by the presence of the KanMX cassette closely linked to the dsl1-22-myc allele which thus
allowed us to identify the dsl1-22 mutants by their
resistance to G418.
Viable double mutants were obtained when we combined the
dsl1-22 defect with sec23-1, sec22-3,
bet1-1, sed5-1, bos1 (sec31-1), and
sec27-1 mutations. The first mutation leads to a block in anterograde ER-to-Golgi transport due to a defect in COPII assembly (62); bet1-1, sec22-3, and sed5-1 are
mutations that affect genes encoding SNARE proteins involved in
ER-Golgi transport, whereas SEC27 encodes a COPI component
(63). The number of viable double mutants obtained differed to a great
extent as determined by complementation assays and analyzing their
resistance to G418 (for details see "Experimental Procedures"). The
observation that sec22-3, dsl1-22 double mutants
are viable whereas dsl1-22 mutants are inviable in the
absence of SEC22 was confirmed by plasmid shuffling
experiments using a dsl1-22 mutant and SEC22 or
sec22-3 containing plasmids (data not shown). This finding
illustrates that this assay is specific for certain alleles. Therefore,
missing or weak genetic interactions mentioned above do not rule out
that the gene products perform a related function. This may be true at
least for BOS1 and DSL1 since all the
bos1 (sec31-1), dsl1-22 double mutants
that we obtained formed very small colonies. No double mutants were
obtained when diploids heterozygous for the dsl1-22 and the
sec22, sec21-1, ret1-1,
ret1-1, sly1ts, sec20-1, or
tip20-5 mutations were subjected to tetrad analysis. The
sec21-1 (-COP), ret1-1 (-COP),
ret1-1 (-COP), sec20-1, and tip20-5
mutants primarily affect the retrograde transport from Golgi to ER, and
defects in forward transport may be secondary (20, 22, 24, 32, 33). The
strong genetic interaction between dsl1-22 and these
mutations indicates that DSL1 may be required for Golgi-ER
retrograde transport. The synthetic lethality of dsl1-22 and
sly1ts are consistent with the observation made by
VanRheenen et al. (43) who isolated dsl1 mutants
that depend on a dominant SLY1 mutation.
The dsl1-22 Mutant Shows Slight Defects in Forward
Transport--
The dsl1-22 mutant cells gave rise to
slightly smaller colonies than wild type cells even at room
temperature. Accordingly, the growth rate of dsl1-22 mutant
cells is slower when measured in liquid culture (Fig. 1B).
Growth of dsl1-22 mutants stops completely 2 h after a
shift to 37 °C. This Ts phenotype allowed us to
examine the function of Dsl1p in the secretory pathway at restrictive
temperatures. First we analyzed the secretion of periplasmic invertase
in wild type and dsl1-22 cells at different times after
shifting cells to 37 °C. Measuring total invertase activity using
intact and permeabilized cells (64) showed that the ratio of secreted
to intracellular invertase does not change significantly up to 3 h
after the shift to 37 °C (data not shown). To detect a possible
glycosylation defect due to slower ER-to-Golgi transport in
dsl1-22 mutants, intracellular and extracellular fractions
of wild type and mutant cells were separated by non-denaturing PAGE.
Invertase was visualized by an activity stain. As shown in Fig.
2A, dsl1-22 cells secrete partially underglycosylated invertase even at 25 °C. The shift to
the restrictive temperature leads to some accumulation of the ER
core-glycosylated form inside the cell. For comparison, at restrictive
temperature the sec22-3 mutation also leads to the intracellular accumulation of core-glycosylated invertase and secretion
of a small amount of underglycosylated enzyme. An incomplete block in
anterograde transport also became evident when the maturation of the
vacuolar protease CPY was analyzed in dsl1-22 cells (Fig. 2B). In pulse-chase experiments CPY appears first as a p1
precursor in the ER, is then modified to a larger form, p2, in the
Golgi, and is transported to the vacuole where it is processed to its mature form (m) by proteolysis. As expected, sec22-3 mutant
cells show a complete block in ER-to-Golgi transport 15 min after the shift to 37 °C. In this mutant only the ER form (p1) is visible consistent with a complete block in ER-to-Golgi transport. In dsl1-22 cells about half of CPY is still normally processed
even 3.5 h after the shift to 37 °C. This corresponds to the
results observed with other temperature-sensitive alleles of
DSL1 (43).
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Fig. 2.
dsl1-22 cells exhibit mild defects in
anterograde ER-to-Golgi transport. A, fate of secreted
invertase in dsl1-22 cells. Invertase synthesis was induced
for 30 min in wild type (Dsl1-myc; YUA11),
dsl1-22-myc (YUA41), and sec22-3 cells
(SHC21-12A) either at 25 or 37 °C. Preincubation at 37 °C started
30 min before induction. Glycosylation level of the enzyme in
intracellular (I) and extracellular fractions (E)
was detected by an activity stain in non-denaturing gels. The position
of highly glycosylated invertase (S) and ER
core-glycosylated invertase (ER) is indicated. B,
intracellular processing of carboxypeptidase Y in wild type (YUA11) and
mutant cells (YUA41 and SHC21-12A). Cells were shifted to 37 °C for
indicated times, pulse-labeled with
[35S]methionine/cysteine for 5 min, and chased for 0 and
30 min. The cells were lysed; CPY was immunoprecipitated, and proteins
were resolved by SDS-PAGE.
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dsl1-22 Cells Accumulate ER Membranes but Not Vesicles at
Restrictive Temperature--
The morphology of wild type and
dsl1-22 cells incubated at 25 or 37 °C was compared by
electron microscopy. As shown by Kaiser and Schekman (36), mutants with
defects in the budding reaction accumulate membranes, whereas mutants
that exhibit defects in fusion of vesicles with target membranes
accumulate vesicles. At 25 °C the morphology of dsl1-22
cells does not differ significantly from that of wild type cells grown
at 37 °C (Fig. 3, A and
B). Fig. 3C shows a representative micrograph of
a dsl1-22 mutant cell after incubation at the nonpermissive
temperature for 90 min. Compared with wild type cells (Fig.
3A) dsl1-22 cells show a strong accumulation of
membranes, which mainly emerge from the ER contiguous with the nuclear
membrane (Fig. 3, C and D, arrow). Similar structures also originate from cortical endoplasmic reticulum close to the plasma membrane (Fig. 3D,
arrowhead). No significant increase in the number of small
vesicles was observed. Thus, dsl1-22 mutants very much
resemble the coatomer mutant sec27-1 (63).
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Fig. 3.
Electron micrographs of wild type and
dsl1-22 cells. Wild type cells (MSUC-3B) grown at
37 °C for 90 min were used as a control (A). Mutant cells
(YUA1-9C) grown at 25(B) or 37 °C for 90 min
(C and D) were fixed with potassium permanganate
to highlight membrane structures. Typical cells are shown for each
condition. The arrowhead in D points to membranes
emanating from the cortical ER (E), whereas arrows in
C and D indicate sites of membrane accumulation
at the nucleus (N). V, vacuole. Bars,
1 µm in A-C; 0.1 µm in D.
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dsl1-22 Mutants Are Defective in the Retrieval of ER Proteins from
the Golgi--
The strong genetic interaction of the
dsl1-22 defect with mutations affecting retrograde
Golgi-to-ER transport and the incomplete block in anterograde transport
when growth already had ceased indicated that the primary function of
Dsl1p could be in the retrieval of proteins from the Golgi complex.
Therefore, we employed different assays to compare retrograde transport
in wild type and dsl1-22 cells.
Mutants affecting genes required in retrograde transport like
SEC20 and SEC22 secrete large amounts of the
soluble ER protein BiP/Kar2p (65). Fig.
4A shows that the same is true
for dsl1-22 and dsl1-22-myc mutants. This defect
in BiP/Kar2p localization was also observed by immunofluorescence
microscopy using an affinity-purified polyclonal anti-BiP/Kar2p
antibody. In wild type cells BiP/Kar2p antibodies stain the nuclear
periphery which is the characteristic ER staining in yeast (66). In
contrast to the typical ER staining in wild type cells, we could
observe a dot-like pattern in dsl1-22 cells even at
permissive temperature (Fig. 4B), similar to "BiP bodies" observed in several ER-to-Golgi mutants at restrictive temperature (67).
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Fig. 4.
Mislocalization of different ER proteins in
DSL1 mutant cells. A, wild type
(DSL1, MSUC-7C, and DSL1-myc, YUA11) or
dsl1-22 mutant cells (dsl1-22, YUA1-9C, and
dsl1-22-myc, YUA41) were transferred into fresh YPD medium
and grown at 25 °C. At A600 nm of 1.0 cells
were removed from the medium by centrifugation, and proteins in the
medium were precipitated by the addition of 10% trichloroacetic acid
(final concentration). Proteins were resolved by SDS-PAGE and analyzed
by Western blotting using polyclonal anti-BiP/Kar2p antibodies.
B, wild type (MSUC-3B) and dsl1-22 (YUA1-9C)
cells were grown to an early log phase at 25 °C, fixed, and stained
with affinity purified anti-BiP/Kar2p antibody (upper
panels). In addition, DAPI staining was used to localize the
nuclei (lower panels). C, Sec22- processing by
the late Golgi protease Kex2p. Immunoblot analysis was performed with
extract from wild type (DSL1, MSUC-7C, and
DSL1-myc, YUA11) or dsl1-22 mutant cells
(dsl1-22, YUA1-9C, and dsl1-22-myc, YUA41)
transformed with pWB-Acyc as indicated. Aliquots of cells were
harvested after overnight incubation at 25 °C in selective medium,
and proteins were analyzed by Western blotting. A polyclonal
anti-Sec22p serum was used to detect the Sec22p-derived hybrid protein
(Sec22-), its Kex2p cleavage product (Kex2p-c.p.), and
the endogenous Sec22 protein. D, confocal images of unfixed
cells expressing GFP-Sec22p grown at 25 °C. Images show the cellular
distribution of GFP-Sec22p in wild type (Dsl1-myc, YUA11),
dsl1-22-myc cells (YUA41), UFE1 wild type cells
(MLY-100), and ufe1-1 mutant cells (MLY101). E,
dsl1-22 cells are defective in ER retrieval of Ste2-Wbp1p.
MATa ste2 yeast cells expressing
Ste2-Wbp1p were grown on YPD plates and replica-plated to a lawn of
MAT cells. After 6 h at 30 °C to allow mating,
cells were replica-plated to SD plates selective for the growth of
diploid cells only. sec21-2 (PC82) and
dsl1-22-myc (SUA5) mutants were able to form diploids,
whereas wild type (WT) (STE2-4B) cells could not mate with
the tester strain (MSUC-2D).
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To examine the defect in retrograde transport more specifically, we
focused on the targeting of the SNARE protein Sec22p. As described
previously (18, 46) -factor fused to Sec22p through a Kex2p cleavage
site, and a c-Myc epitope is a suitable tool for analyzing targeting of
Sec22p. Several recycling mutants exhibit mislocalization of Sec22-
(18) resulting in cleavage by the late Golgi protease Kex2p. The
removal of the -factor reporter from Sec22p is easily detected by
immunoblot analysis. Fig. 4C shows the steady state
processing of Sec22- in wild type, dsl1-22 and
dsl1-22-myc strains incubated at 25 °C. About 75% of
Sec22- proteins was cleaved by Kex2p in mutant cells, whereas very
little of the reporter was cleaved by Kex2p in wild type cells.
Pre-shifting cells to 37 °C for 2 h did not result in a more
efficient cleavage (data not shown). It is unlikely that more efficient
cleavage of Sec22- in dsl1-22 is due to some Kex2p activity in the ER since mislocalization of a Sec22p-derived fusion protein was also obvious when we analyzed cells producing a GFP-tagged Sec22 protein (Fig. 4D). This fusion protein is fully
functional since it is able to suppress the growth defect of
sec22-3 mutants (data not shown). Moreover, GFP-Sec22p
behaves like C-terminally tagged Sec22 proteins when analyzed in wild
type and ufe1-1 mutant cells (Fig. 4D; see Ref.
18). In wild type cells fluorescence appeared as a ring around the
nucleus which represents ER, whereas in dsl1-22 cells a
punctated staining was detectable, very likely representing Golgi
structures (18). As with other recycling mutants, this defect already
occurs at 25 °C (20). Taken together, both the efficient Kex2p
processing of Sec22- and the localization of GFP-Sec22 indicate that
dsl1-22 mutants are defective in ER retention of Sec22p.
To examine whether the dsl1-22 mutation also interferes with
the ER retention of type I transmembrane proteins carrying the KKXX retrieval signal, we performed the
Ste2-Wbp1-dependent mating assay described by Letourneur
et al. (20). We introduced the dsl1-22-myc allele into a strain expressing a
KKXX-tagged version of the -factor receptor (Ste2-Wbp1p)
instead of the wild type STE2 gene. Wild type cells of
mating type a expressing only this receptor cannot mate with
cells of mating type since Ste2-Wbp1p is efficiently retained in
the ER due to the KKXX-tag fused to the C terminus. Mutants
that mislocalize the receptor to the plasma membrane can form diploids
with a suitable tester strain. With the Ste2-Wbp1-based assay efficient
mating occurs for instance in sec21-2 (-COP) mutants (see
Ref. 20; see also Fig. 4E). Fig. 4E shows that
dsl1-22-myc cells producing Ste2-Wbp1p can mate as
efficiently as the sec21-2 mutants indicating that targeting
of KKXX-tagged ER proteins is impaired already at a permissive temperature of 30 °C. In summary, the data show that dsl1-22 mutants are defective in the ER retention of
different types of proteins: soluble HDEL carrying proteins like
BiP/Kar2p, type II transmembrane proteins like the v-SNARE
Sec22p, as well as type I transmembrane proteins carrying a
KKXX retrieval signal.
Subcellular Distribution of Dsl1p--
According to its primary
sequence, Dsl1p contains no putative transmembrane domains. Extracts
from Dsl1-myc producing cells (YUA11) were used to examine a
possible membrane association of Dsl1p. A 500 × g
supernatant of cell lysate was treated either with buffer (B88), 5 M urea, 1% Triton X-100, or 1 M NaCl and subsequently centrifuged at 10,000 × g and
100,000 × g (Fig. 5). When incubated with buffer alone, no Dsl1-myc was detectable
in the soluble fraction, whereas both urea and detergent treatment led
to solubilization of Dsl1-myc. Less than 5% of the total
amount of Dsl1-myc became soluble upon treatment with high
salt suggesting that Dsl1p is a peripherally associated membrane
protein. In contrast, the transmembrane protein Sec22p could only be
solubilized by detergent. Experiments using a recently obtained
Dsl1-specific serum gave identical results (data not shown).
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Fig. 5.
Dsl1p is a peripheral membrane protein.
Logarithmically grown cells of YUA11 (DSL1-myc) were
disrupted using glass beads. The lysate (500 × g
supernatant) was treated as indicated (see "Experimental
Procedures") and then centrifuged at 10,000 and 100,000 × g. The resulting pellet (P10 and P100)
and supernatant (S100) fractions were resolved on a 12%
polyacrylamide gel and immunoblotted with anti-Sec22p and anti-c-Myc
antibody (9E10). In contrast to the integral membrane protein Sec22p,
Dsl1-myc became soluble after incubation with 5 M urea.
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Next we performed subcellular fractionation studies using sucrose
density gradients to compare the localization of Dsl1p with that of
known Golgi- and ER-resident proteins. Cell lysates of strain YUA11
(DSL1-myc) were prepared and loaded on top of sucrose gradients, and fractions were collected after centrifugation as described under "Experimental Procedures." Fig.
6, A and B, shows that Emp47p, a Golgi marker, the ER resident t-SNARE Ufe1p, as well as
the ER-marker BiP/Kar2p display characteristic distributions (51, 24,
66). Like Ufe1p and BiP/Kar2p Dsl1-myc protein was
detectable exclusively in the dense fractions when using the monoclonal
anti-c-Myc antibody 9E10 directed against the c-Myc epitope,
presumably reflecting ER localization (Fig. 6C).
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Fig. 6.
Dsl1p cofractionates with ER markers in
sucrose velocity gradients. Lysates of strains YUA11
(DSL1-myc) grown at 25 °C were loaded on 18-60% sucrose
density gradients. After centrifugation, fractions were collected and
subjected to Western blot analysis with antibodies directed against
Emp47p, a Golgi marker (A, see Ref. 51), Ufe1p and
BiP/Kar2p, two ER markers (B, see Refs. 24 and 66), and
c-Myc to detect tagged Dsl1 protein expressed at wild type levels
(C). The data represent average values from at least two
experiments.
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Dsl1p Interacts Physically with Coatomer--
To get additional
clues for the involvement of Dsl1p in retrograde and/or anterograde
ER-to-Golgi transport, we investigated possible interactions of Dsl1p
with proteins involved in these trafficking steps. First we tried to
address this question by expressing Dsl1p tagged with glutathione
S-transferase (GST) in yeast. The 100,000 × g supernatants from detergent-lysed yeast cells (YUA11)
expressing GST or GST-Dsl1p were loaded on glutathione-Sepharose 4B to
immobilize GST or GST-Dsl1p and associated proteins. After washing the
beads to remove unbound proteins, antibodies were used to monitor the
binding of several ER/Golgi proteins to Dsl1p. Anti-coatomer antibodies
resulted in very strong signals (data not shown), whereas only weak
signals were obtained with Emp47p-specific antibodies. These signals
were specific for the Dsl1 part of the fusion protein since no binding
was observed when lysates from GST-expressing cells were analyzed. The
SNARE proteins Bet1p, Bos1p, Sec22p, and Sed5p as well as the COPII
component Sec24p and the Rab-like GTPase Ypt1p were not retained
on the affinity matrix in significant amounts.
To verify and extend these findings, we incubated extracts of
detergent-lysed yeast cells with different GST fusion proteins purified
from E. coli. In line with the results obtained with GST
fusion proteins expressed in yeast, coatomer (COPI) showed strong
binding to GST-Dsl1p. Notably, coatomer recruitment to GST-Dsl1p from
E. coli takes place even at 4 °C (see "Experimental Procedures"), a temperature where enzymatic activities are low. As
controls, GST, GST-Sed5p, GST-Bos1p, or GST-Sec22p were not able to
recruit coatomer from cell lysates (Fig.
7). Very faint bands representing
coatomer were seen when GST-Tip20p was loaded on glutathione-Sepharose
4B (Fig. 7B, lane 5). Dsl1p may mediate this
indirect binding between GST-Tip20 and coatomer because Ito et
al. (68) recently showed that Tip20p and Dsl1p interact in two-hybrid assays. However, so far we could not observe direct binding
of Dsl1-myc to GST-Tip20p in vitro. In addition,
Dsl1-myc did not bind to GST-Bos1p, GST-Sec22p, or GST-Sed5p
(data not shown). Likewise, GST-Dsl1p was not able to bind Bet1p,
Bos1p, Sec22p, Sed5p, Ypt1p, Sec24p, or Emp47p, suggesting that the
weak binding of Emp47p mentioned above could be indirect via
coatomer.
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Fig. 7.
Specific binding of coatomer subunits to
GST-Dsl1p. Proteins from detergent-lysed yeast cells were
incubated at 4 °C for 2 h with GST alone or GST fusion proteins
purified from E. coli and immobilized on
glutathione-Sepharose 4B. Beads were washed 5 times (see
"Experimental Procedures"), and the proteins bound were analyzed by
SDS-PAGE followed by Coomassie Blue staining (A) and
immunoblot analysis using a polyclonal antibody against coatomer
(B). The positions of molecular weight markers and the
different COPI subunits are indicated.
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DISCUSSION |
Genetic Analysis Indicates That Dsl1p Is Required for Retrograde
Golgi ER Traffic--
In the present study we identified a novel
mutation that renders cells dependent on the otherwise dispensable
SNARE protein Sec22p. This mutation makes cells temperature-sensitive
for growth, allowing us to analyze the function of the affected gene.
Cloning and sequencing showed that this mutant is a new allele of the essential open reading frame YNL258c, encoding a truncated
protein that lacks its 30 C-terminal residues.
Recently, mutant alleles of YNL258c, named dsl1-1
and dsl1-2, were identified by VanRheenen et al.
(43) as mutations that make yeast cells dependent on the dominant
suppressor mutation of SLY1, SLY1-20. Thus,
screening for genetic defects that confer dependence on either Sec22p
or on the dominant SLY1-20 mutation led to the
identification of the same gene, DSL1. By comparing the
results, the Sec22p-dependent dsl1-22 mutant has
similar properties as the PCR-generated temperature-sensitive alleles
dsl1-5 and dsl1-6 obtained by VanRheenen et
al. (43). They show a slight defect in ER-to-Golgi transport of
CPY, and their growth defect at 37 °C can be suppressed by the
SLY1-20 mutation. However, the mutants obtained using the
two approaches differ in several other phenotypes. The
SEC22-dependent dsl1-22 mutant is
temperature-sensitive, and defects in vesicular transport could thus be
analyzed directly. The SLY1-20-dependent mutants
dsl1-1 and dsl1-2 are not Ts and
display secretory defects only after expression of the
SLY1-20 allele is shut off. Another difference concerns the
suppression of the non-sectoring phenotype. The dependence of the
dsl1-1 mutant on SLY1-20 could not be suppressed
by overexpression of the t-SNARE-encoding SED5 gene (43),
whereas SED5 overexpression in dsl1-22 cells could eliminate the requirement for Sec22p. This observation may imply
a direct functional link between Dsl1p and SNARE proteins like Sec22p
and Sed5p.
Both Sec22p and Sed5p show strong genetic interactions with genes
encoding proteins involved in Golgi-ER retrograde transport (37, 69,
70). Double mutant analysis revealed that the same is true for
dsl1-22. During our attempts to create double mutants harboring the dsl1-22 mutation combined with additional
mutations affecting the ER-Golgi transport cycle, we observed the
strongest genetic interactions of dsl1-22 with mutations
affecting retrograde Golgi-to-ER transport. No double mutants were
obtained when we crossed dsl1-22 strains with mutants
affected in coatomer subunit-encoding genes like RET1
(-COP), RET2 (-COP), and SEC21 (-COP) or
with mutants in SEC20 and TIP20 which are
important for fusion of Golgi-derived vesicles with the ER (32, 33). In
accordance with this, VanRheenen et al. (43) found that
overexpression of the -COP-encoding SEC21 gene partially
suppresses the Ts defect of dsl1 mutants.
Together these results strongly suggest that Dsl1p may play a role in
Golgi-ER retrograde traffic. One could speculate that the need for
Sec22p displayed by the dsl1-22 mutant may be due to the
mislocalization of SNARE proteins that can functionally replace Sec22p.
This is also indicated by the fact that the requirement for Sec22p at
least at room temperature can be alleviated either by excess of Ykt6p
or Sed5p, two other SNARE proteins. As discussed below, Sec22p as well
as Bos1p are in fact mislocalized in dsl1-22 cells.
Unexpectedly, SEC22 can be replaced in dsl1-22
mutants by the sec22-3 allele. This was surprising since the
sec22-3 point mutation has stronger effects on the growth of
certain strains than the deletion of SEC22
(sec22 cells that are not Ts can become
temperature-sensitive after introducing a sec22-3-containing plasmid).2
dsl1-22 Mutants Have a Strong ER Retention Defect--
In
dsl1-22 cells maturation of the vacuolar hydrolase CPY is
only partially inhibited, similar to what has been described for the
PCR-generated dsl1-5 and dsl1-6 mutants (43).
Invertase secretion is almost normal in dsl1-22 mutants and
a slight inhibition of anterograde transport is indicated by the
accumulation of a small amount of core-glycosylated invertase. Electron
microscopy analysis of mutant cells reveals a severe accumulation of
membranes emerging from the ER after shift to nonpermissive
temperature. Similar structures were observed in a '-COP mutant,
sec27-1 (63). Since the morphology of dsl1-22
mutant cells is almost normal at 25 °C, a temperature at which
retrograde transport is already affected (see below), this EM phenotype
at restrictive temperature is likely to be a more indirect effect due
to perturbed forward transport.
The weak inhibitory effect on forward transport appears to be a result
of a strong defect in retrograde transport back to the ER. In
dsl1-22 cells this block is already seen at permissive temperature, consistent with what has been seen with other recycling mutants (18, 20, 22). The dsl1-22 mutant allele affects the
retrieval of recycling SNARE proteins, proteins sorted by their
C-terminal KKXX motif, and the soluble ER protein BiP/Kar2p, whose recycling depends on the HDEL receptor Erd2p (65). How can
retrograde transport defects have an effect on forward transport? Obviously, one possibility is that components of the vesicle budding and fusion machineries may become limiting due to their
mislocalization. In addition, it is known that exit from the ER
requires the proper folding of cargo molecules, and this in turn
depends on chaperones like BiP/Kar2p or PDI (71, 72). These
chaperones carry a C-terminal HDEL signal that mediates their retention
in the ER. In dsl1-22 mutant cells, BiP/Kar2p and very
likely PDI are not properly retained in the ER. Insufficient amounts of
BiP/Kar2p and PDI in the ER could retard the exit of cargo
molecules (71, 72).
The following results demonstrated that dsl1-22 cells are
defective in Golgi-to-ER-retrieval of Sec22p. A GFP-tagged version of
Sec22p localizes to the ER in wild type cells, whereas in
dsl1-22 cells GFP-Sec22p displays a punctate staining
pattern typical for Golgi markers. The Sec22- fusion protein reaches
the late Golgi apparatus in dsl1-22 cells but not in wild
type cells as indicated by its Kex2p-dependent cleavage.
Fractionation studies with sucrose density gradients showed that Bos1p
exhibits a shift from ER-to-Golgi fractions in dsl1-22 cells
compared with wild type cells (data not shown). Mislocalization of the
soluble ER marker BiP/Kar2p was also demonstrated using
immunofluorescence and a secretion assay. BiP/Kar2p fluorescence in
dsl1-22 mutant cells shows a punctate pattern. Similar, more
randomly distributed structures were described previously for several
sec mutants and were named BiP bodies (67). These authors
suggested that BiP bodies could be exit sites where leaving proteins
accumulate in different mutant strains due to low efficiency of
Golgi-to-ER retrieval. Some mutants even secrete Kar2p into the medium.
Indeed, this phenomenon can be observed with dsl1-22 mutant
cells. The level of Kar2p secretion by these cells is comparable to
that of sec22-3, sec22, sec20-1
cells (65).3
Besides mislocalization of SNARE proteins and of the luminal ER protein
BiP/Kar2p, dsl1-22 cells exhibit also defects in retrieval of proteins sorted by their C-terminal KKXX motif. In this
study we used Ste2-Wbp1p as a marker protein (20). Our results
implicate Dsl1p in retrograde transport of dilysine-tagged proteins
from the Golgi compartment to the ER. We also analyzed the localization of Emp47p, a Golgi protein carrying a variant of the dilysine-motif, KXKXX (51). Unlike Ste2-Wbp1p, the localization of Emp47p is unaffected in dsl1-22 cells. This is indicated by the
results of gradient fractionation and immunofluorescence experiments
(data not shown). In this respect, dsl1-22 mutants resemble
ret1 (-COP) mutants that also mislocalize
KKXX-tagged proteins of the ER but not the
KXKXX-tagged Emp47p (23).
The Localization of Dsl1p Is Still Unclear--
Dsl1p is a
peripheral membrane protein that can be solubilized with 5 M urea and colocalizes with ER marker proteins in sucrose density gradients. Fractionation experiments were performed with a
c-Myc-tagged Dsl1 protein expressed at wild type levels. These results
were later confirmed using antibodies raised against bacterially produced Dsl1 protein. We also tried to determine the localization of
Dsl1p by immunofluorescence. Unfortunately, affinity purified polyclonal antibodies against Dsl1p still exhibited strong
cross-reactivities and were thus not helpful for immunofluorescence
analysis. Specific signals were only obtained when tagged versions of
Dsl1p were overproduced. The expression of GFP-Dsl1p led to
fluorescence pattern varying from Golgi-like staining consisting of a
few large dots in cells from early logarithmic growth phase to nuclear
staining at A600 nm >1. Overexpression of
c-Myc-tagged Dsl1p led to diffuse punctate fluorescence. A cytoplasmic
staining consisting of many small dots was also observed for Tip20p,
which is cytoplasmic when overproduced. Tip20p could be recruited to
the ER when Sec20p was overproduced as well (73). Given the tight
genetic (see above) and direct interactions (68) between Tip20p and
Dsl1p they may behave similarly. Unlike Tip20p, overproduced Dsl1p does not localize to the ER when SEC20 was overexpressed
simultaneously (data not shown).
Dsl1p Interacts Strongly with Coatomer--
As mentioned above, a
recent systematic yeast two-hybrid study revealed direct interactions
of Dsl1p with Tip20p (68). Dsl1p showed interactions with several other
proteins. However, only in the case of Dsl1p and Tip20p, this
interaction was observed with Dsl1p as bait as well as prey,
i.e. both fusion orientations. This is consistent with the
genetic data since the tip20-5 defect is synthetically
lethal in combination with dsl1-22 (this study). The genetic
as well as physical interaction between DSL1 and
TIP20 and their gene products suggest that both proteins
could be involved in the same transport step. Tip20p is able to bind to
the cytosolic region of Sec20p (73). Together they form a complex with
the SNARE proteins Ufe1p and Sec22p (32). This unconventional SNARE complex is involved in retrieval of dilysine-tagged proteins from Golgi
to ER (33). In summary, Dsl1p interacts directly with Tip20p (68), and
the dsl1-22 mutation exhibits synthetic lethality in
combination with sec22, sec20-1, and
tip20-5. Synthethic-lethal genetic interaction between
mutations in SEC22, SEC20, TIP20, as
well as UFE1 and mutations affecting coatomer subunits were established previously (69).
As expected, dsl1 mutants also exhibit genetic interactions
with coatomer mutants (see Ref. 43; this study). Final evidence for
Dsl1p playing an important role in retrograde Golgi-ER traffic is our
finding that Dsl1p interacts physically with coatomer. Coatomer could
be copurified with GST-Dsl1p from yeast cells, and it could be
recruited from yeast lysates to recombinant GST-Dsl1p purified from
E. coli. No additional factors present in the cell extracts
were required for this interaction since purified coatomer can also
bind to GST-Dsl1p (data not shown). Interestingly, the C-terminal
truncated mutant protein, Dsl1-22p, which leads to a defect in
retrograde transport, is still able to bind all coatomer subunits with
an affinity comparable to the full-length protein (data not shown).
Thus the C terminus of Dsl1p is not essential for binding of coatomer
but perhaps could represent a binding region for other proteins
involved in these transport steps.
Considering the fact that Dsl1p binds coatomer as well as Tip20p, a
component of the putative docking complex at the ER, we suggest that
Dsl1p is involved in a step between uncoating and docking. It will be
important to determine whether Dsl1p can bind both coatomer and Tip20p
at the same time or whether the interaction is sequential.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 49-551-201-1713;
Fax.: 49-551-201-1718; E-mail: hschmit@gwdg.de.
