Download Coatomer Is Essential for Retrieval of Dilysine

Ceil, Vol. 79, 1199-1207,
December
30, 1994, Copyright
0 1994 by Cell Press
Coatomer Is Essential for Retrieval
of Dilysine-Tagged Proteins
to the Endoplasmic Reticulum
Francois Letourneur,”
Erin C. Gaynor,t
Silke Hennecke,*
Corinne Demolliere,’
Rainer Duden,* Scott D. Emr,t Howard Riezman,O
and Pierre Cosson*
*Base1 Institute for Immunology
Grenzacherstrasse
487
CH-4005 Base1
Switzerland
tDivision of Cellular and Molecular Medicine
and the Howard Hughes Medical Institute
University of California, San Diego
La Jolla, California 92093-0668
*Department of Molecular and Cell Biology
University of California, Berkeley
Berkeley, California 94720
OBiozentrum der Universitat Base1
CH-4056 Base1
Switzerland
Summary
Dilysine motifs in cytoplasmic
domains of transmembrane proteins are signals for their continuous
retrieval from the Golgi back to the endoplasmic
reticulum (El?). We describe a system to assess retrieval to
the ER in yeast cells making use of a dilysine-tagged
Ste2 protein. Whereas retrieval was unaffected in most
set mutants tested (sec7, sec72, sec73, secl6, secl7,
sec78, sec79, sec22, and sec23), a defect in retrieval
was observed in previously
characterized
coatomer
mutants (sec27-7, sec27-I), as well as in newly isolated
retrieval mutants (sec27-2, retl-7). RET7 was cloned
by complementation
and found to encode the asubunit
of coatomer. While temperature-sensitive
for growth,
the newly isolated coatomer mutants exhibited a very
modest defect in secretion at the nonpermissive
temperature. Coatomer from p’-COP (sec27-7) and a-COP
(retl-7) mutants, but not from T-COP (sec27) mutants,
had lost the ability to bind dilysine motifs in vitro. Together, these results suggest that coatomer plays an
essential role in retrograde
Golgi-to-ER transport and
retrieval of dilysine-tagged
proteins back to the ER.
introduction
Signals that confer localization to the endoplasmic reticulum (ER) have been characterized in the cytoplasmic domain of many mammalian type I transmembrane proteins
that reside in the ER and in the El%Golgi intermediate
compartment. One common feature of these signals is the
presence of two lysine residues at positions -3 and -4
from the C-terminal end of the cytoplasmic domain (Nilsson et al., 1989; Jackson et al., 1990). A similar dilysine
signal was identified more recently in the cytoplasmic domain of the yeast transmembrane
protein Wbplp, a subunit of the yeast N-oligosaccharyl transferase complex in-
volved in N-linked glycosylation (te Heesen et al., 1993;
Townsley and Pelham, 1994; Gaynor et an., 1994). In both
mammalian and yeast cells, analysis of posttranslational
modifications of chimeric dilysine-tagged transmembrane
proteins indicates that they are localized to the ER by continuous retrieval from post-ER compartments back to the
ER (Jackson et al., 1993; Townsley and Pelham, 1994;
Gaynor et al., 1994).
Recently we reported that dilysine-retrieval signals interact in vitro with high affinity with the coatomer (Cosson
and Letourneur, 1994). The coatomer is a protein complex
composed of seven subunits, a-coat protein (a-COP),
P-COP, 8’-COP, y-COP, S-COP, c-COP, and <-COP (Rothman and Orci, 1992). In Saccharomyces
cerevisiae,
B-COP, 8’COP, and y-COP are the products of the SfC26,
SEC27, and SEC27 genes, respectively (Stenbeck et al.,
1992; Hosobuchi et al., 1992; Duden et al., 1994). The
coatomer complex is found free in the cytosol as well as
polymerized on the cytoplasmic side of the Gotgi compartment (Duden et al., 1991). Coatomer forms a coat around
non-clathrin-coated
vesicles that have been proposed to
be involved in transport between Golgi stacks (Rothman
and Orci, 1992) as well as between the ER and the Golgi
apparatus (Pepperkok et al., 1993). We proposed a mechanism for the ER localization of dilysine-tagged
proteins in which specific binding of the coatomer to dilysine
motifs would cause the continuous retrieval of dilysinetagged proteins back to the ER (Cosson and Letourneur,
1994) thus implicating coatomer in retrograde Golgi-to-ER
transport.
To identify the protein machinery required for retrieval of
dilysine-tagged proteins to the ER, we developed asimple
assay to monitor ER retrieval of these proteins in yeast,
making use of a fusion protein between the a..factor receptor (StePp) and a dilysine-retrieval
motif. Our results indicate that the coatomer complex plays an essential role in
retrieval of dilysine-tagged
proteins back to the ER.
Results
Retrieval of Dilysine-Tagged
Ste2p to the ER
While previous studies using dilysine-tagged
proteins
have allowed detailed analysis of retrieval to the ER in
yeast cells (Townsley and Pelham, 1994; Gaynor et al.,
1994), they have not led so far to the isolation of mutants
defective in retrieval to the ER. In an effort to isolate such
mutants, we developed an assay to monitor ER retrieval,
making use of a fusion protein between the c-factor receptor (Ste2p) and a dilysine-retrieval
motif. Stle2p is expressed on the cell surface of MATa yeast cells and is
essential for mating with MATa cells. It is composed of
seven transmembrane domains connected by loops of various lengths and transduces an activation signal via a trimerit GTP-binding
protein (Marsh et al., 1991). Sequences in its C-terminal cytoplasmic tail mediate receptor
internalization
and desensitization
but are not essential
Cell
1200
A
Figure
EX
NH2 (s?
J
1 WBPl
WBPl-SS
WBPI -RR
WBP1+4S
-KKLETFKKTN
-KKLETFSSTN
-KKLETFRRTN
-KKLETFKKTNSSSS
B
C
STES-WBPl
STES-WBPI
123
STEZ-WBPl-SS
123
STES-WBPl-SS
STE2-WBPI-RR
STE2-WBP1+4S
for pheromone binding or signaling (Konopka et al., 1988;
Rohrer et al., 1993).
To assay intracellular transport of dilysine-tagged
proteins, we replaced most of the C-terminal cytoplasmic domain of StePp with the cytoplasmic domain of Wbplp
(Ste2-Wbpl
protein), which containsafunctional
dilysineretrieval motif (Townsley and Pelham, 1994; Gaynor et al.,
1994) (Figure IA). This construct was then integrated into
the genome of MATa yeast cells deleted for STEP (dste2).
To determine the intracellular localization of the StePWbpl protein, yeast cells were labeled with [35S]methionine, lysed, and processed for immunoprecipitation
with
an antiserum to Ste2p. The immunoprecipitated
protein
was eluted, and aliquots were reprecipitated with antisera
to StePp, al ,6 mannose, or al ,3 mannose. Ste2-Wbpl
protein was reprecipitated
with antisera to StePp and to
al ,8 mannose, but notwith an antiserum to al ,3 mannose,
whereas Ste2-Wbpl-SS
protein, which bears no functional dilysine motif (Cosson and Letourneur, 1994) was
reprecipitated with all three antibodies (Figure 16). Reprecipitation with antisera to al ,3 or al ,6 mannose was relatively inefficient, probably owing to the low number of
N-linked glycosylation sites in Ste2p. Alternatively, it could
reflect a relatively slow transport of SteP fusion proteins
out of the ER. These results indicate that the Ste2-Wbpl
protein was restricted to the ER and an early Golgi compartment, where al ,6-linked mannose is added onto the
core N-linked oligosaccharides
(Graham and Emr, 1991)
but did not gain access to the medial Golgi compartment,
where al ,3 mannose is added. These observations are
very similar to those made previously with a fusion protein
constructed by fusing the transmembrane
and cytoplasmic tail of Wbpl p to invertase (lnv), which was shown to
be restricted to the ER and an early Golgi compartment
(Gaynor et al., 1994).
ER localization and the absence of surface expression
1. Retrieval of
Ste2-Wbpl
p to the ER
(A) Structure
of the STE2-WBPl
chimeras.
The WEPI-derived
sequence
added in-frame
starting at amino acid 320 of Ste2p is indicated.
Single-letter code is used for amino acids. The
extracellular
(EX), transmembrane
(TM), and
cytoplasmic
(CY) domains are indicated.
(B) lmmunoprecipitation
and mannose modificationsof Ste2-Wbplp.
Yeastcellsexpressing
either SteZ-Wbpi
or Ste2-Wbpl-SS
protein
were labeled with [%]methionine
for 30 min
and processed
for immunoprecipitation
by using an antiserum to StePp. The immunoprecipitated material was eluted and aliquots reprecip
itated with antisera
to Ste2p (lane I), al,6
mannose
(lane 2) or al,3 mannose (lane 3)
and analyzed by SDS-polyacrylamidegel
electrophoresis.
The high molecular weight band
probably
corresponds
to dimers of the Ste2
protein (Konopka et al., 1988).
(C) Mating of cells expressing
STEP-WBPI
chimeras.
MATa dste2 cells expressing
the indicated STEP-WBPI
chimera were grown on
YPD plates and replica plated to a lawn of
MATa cells. After 6 hr of mating at 24OC, they
were replica plated to SD plates selective for
the growth of diploid cells.
of the Ste2-Wbplp
would be expected to result in defective mating. Indeed, dste2 yeast cells expressing Ste2Wbpl p failed to mate, whereas when mutations were introduced into the dilysine motif that render it nonfunctional
(Ste2-Wbpl-SS,
Ste2-Wbpl-RR,
Ste2-Wbpl+4S),
efficient mating was restored (Figure 1 C). Thus, lack of mating
of cells expressing Ste2-Wbpl p reflects efficient retrieval
of the chimeric receptor to the ER. This simple assay can
be used to assess ER retrieval in previously isolated yeast
mutants and to screen for new mutants affected in ER
retrieval of dilysine-tagged
proteins.
Mutants
Defective
in Retrieval
to the
ER
Using this qualitative test, we assayed retrieval of Ste2Wbpl protein to the ER in previously characterized secretion mutants in which ER-to-Golgi transport is affected
(Novick et al., 1980; Kaiser and Schekman, 1990). In these
mutants, secretion is inhibited at 37X, making analysis
of ER retrieval impossible. To circumvent this problem,
we tested mating at various temperatures between 24%
and 37%, in search of a temperature at which inhibition
of transport would not be complete but where ER retrieval
might be affected. Mutant strains affected in budding of
ER-to-Golgi transport vesicles (secl2, sec73, sec76, and
sec23) or in their fusion with the Golgi apparatus (sec77,
secl8, and sec22) showed no mating at any temperature
tested (Figure 2; data not shown). Similarly, no mating
was observed in secl9 and sec7 mutant strains (data not
shown). These negative results suggest that the products
of these various genes are not directly involved in ER retrieval. However, we cannot rule out the possibility that
a defect in ER retrieval might be masked by a block in
secretion. In all these mutant strains, expression of the
SteP-Wbpl-SS
protein resulted in efficient mating at permissive
and semipermissive
temperatures
(data not
shown), indicating that the Ia&. of mating observed in cells
i$r$
of Coatomer
in Retrieval
to the ER
Mating Temperature
24
WT
:_
27 30
” .i
,J&:
/
.,:rI -
33
(%)
35
37
;.
.: ‘_
WT
sec22-1
sec21-1
sec21-2
sec27-1
ret1 -1
Figure
Ste2p
2. Mutants
Defective
for
ER Retrieval
of Dilysine-Tagged
MATa dsfe2 yeast cells expressing
STEZ-WBPI-SS
(A) or STE2WBPI (8) were tested for mating with MATu cells at different temperatures, as described
in Figure IC. All strains used in this study synthesized similar amounts of SteP-Wbplp
as checked
by metabolic
labeling for 10 min followed by Ste2p immunoprecipitation
and analysis on SDS-polyacrylamide
gels (data not shown).
one complementation
group, retl, that complemented all
the set mutants used in this study (data not shown). A
typical ret7 mutant allele, retl-7, exhibited efficient mating
(Figure 26), normal growth at 24OC, and a conditional
growth defect at 37OC (see Figure 4A). It was backcrossed
twice with a wild-type MATa dste2 cell. After sporulation
and dissection of tetrad& the mating abilityofdsfe2
MATa
cells was strictly linked to the thermosensitive phenotype,
thus demonstrating that both phenotypes result from the
same mutation.
To analyze genetic interactions between retl-7 and
other mutations that affect retrieval to the ER, we attempted to obtain retl, sec27 and retl, sec27double
mutants. The single mutants were crossed, tetrads from the
resulting diploid strains dissected, and spore growth
tested at 24’C. In both cases, the pattern af spore viability
suggested that the double mutants were inviable at 24OC
(20 tetrads). All complete tetrads had four thermosensitive
spores, while in tetrads with less than folur viable spores,
the missing spore or spores were inferred to be double
mutants. To determine whether this interaction was specific, we also crossed retl-7 with sec72-7. The resulting
retl-7, sec72-7 double mutants grew normally at 24OC.
Thus, the retl-7 mutation shows a synthetic lethal interaction with sec27-7 and sec27-7, suggesting that the products of these genes might interact.
Intracellular
expressing SteP-Wbplp
was only due to intracellular sequestration of Ste2-Wbplp.
In contrast, sec27-7 mutant
cells, which are mutated in the yeast y-COP, exhibited
modest mating that was particularly apparent at 30°C33Y.Z (Figure 2). More efficient mating was observed in
sec27-7 mutant cells(Figure 2), which are mutated in yeast
f3’-COP (Duden et al., 1994). This result indicated that two
characterized mutants of coatomer, sec27-7 and sec27-7,
were defective in ER retrieval, suggesting that ‘coatomer
may play a role in retrieval of dilysine-tagged
proteins to
the ER.
We also isolated new mutants with defective retrieval
of Ste-Wbplp
to the ER (ret mutants) by mutagenizing
cells expressing SteP-Wbpl p and screening for matingcompetent mutants. Three independent mutagenesis experiments yielded 30 ret mutants, of which 9 exhibited a
conditional growth phenotype with no growth at the restrictive temperature (37“C). For practical reasons, we limited
our analysis to these thermosensitive mutants.
One of the isolated thermosensitive ret mutants yielded
nonthermosensitive
diploids when crossed with wild-type
cells, but thermosensitive
diploids when crossed with
sec27-7 cells (data not shown). These thermosensitive diploids were sporulated and 24 tetrads dissected. All spores
showed a thermosensitive phenotype, demonstrating that
the isolated mutation was very tightly linked to the SEC27
locus, most likely a new allele of sec27, which we named
sec27-2 (Figure 28). The sec27-2 mutant was backcrossed twice with wild-type cells, and the thermosensitive
phenotype cosegregated strictly with the ret phenotype.
The remaining eight thermosensitive ret mutants fell into
Transport
in ER Retrieval
Mutants
To analyze quantitatively the retrieval defect in sec27,
sec27, and ret7 mutants, we made use of an Inv-Wbpl
fusion protein, consisting of the entire i~nvertase protein
fused to the transmembrane
domain and cytoplasmic tail
of Wbpl p (Gaynor et al., 1994). We have shown that the
Inv-Wbpl p is continuously retrieved from an early Golgi
compartment back to the ER. When the dilysine motif was
mutated, the fusion protein escaped the ER-early Golgi
and was transported to the vacuole, where it was processed by vacuolar proteases. The degree of processing
of Inv-Wbpl p provides a quantitative measure of the relative efficiency with which it is retrieved back to the ER
(Gaynor et al., 1994).
Cells expressing Inv-Wbpl p were pulse labeled for 10
min and chased for 0 or 60 min. The fusion protein was
then recovered by immunoprecipitation
with an antiserum
to invertase, treated with endoglycosarninidase
H (endoH), and resolved on SDS-polyacrylamide
gels (Figure
3A). The portion of the Inv-Wbplp
that escaped retrieval
and was processed in the vacuole after 60 min of chase
was quantitated by phosphorimager
analysis (Figure 3A).
As previously reported, in wild-type cells at 30°C, only
10% of the Inv-Wbplp
was processed after 60 min of
chase (Gaynor et al., 1994; Figure 3A). In1contrast, in the
retl-7 mutant at 30°C (permissive temperature), 80% of
the Inv-Wbpl p was processed after the same chase period (Figure 3A). Nearly identical results were observed
for retl-7 at 37OC (data not shown). In the sec27-7, sec272, and sec27-7 mutants, little or no retrieval defect was
observed at permissive temperature (data not shown), but
a significant fraction of Inv-Wbplp
escaped the ER at
semipermissive
temperature (Figure 3A). These results
Cell
1202
A
WT
Temp:
Chase:
retl-l
30°C
sec21-7
30°C
sec21-2
33°C
33°C
Figure 3. Intracellular
Defective for Retrieval
sec27-1
32°C
Transport
to the EA
in Mutants
Wild-type
(WT, SEY6210),
fetl-7 (EGYlOl),
sec27-7
(RSY277),
sec27-2
(EGYl03),
and
sec27-7 (CKYlOO) cells expressing
Inv-Wbpl
fusion protein were pulse labeled for 10 min
% processed:
with Tran%-label
and chased for 0 or 60 min
10%
80%
50%
50%
45%
at the indicated temperature.
(A) Inv-Wbplp
was immunoprecipitated
from
retl-1
WT
sec2l-1
sec21-2
sec27-1
Temp:
the labeled cells, treated with endoH, and ana30°C
30°C
37°C
30°C
37°C
30°C
37°C
28°C
37°C
I 0’ 60
lyzed on SDS-polyacrylamide
gel. Intact and
Chase: 10’
F-2
' '0'
60"10"0'
60"10"0'
6O"'O'
60"
processed
fusion protein migrated at 70 kDa
pZCPY,
and 56 kDa, respectively.
Percent processed
plCPYC
after 60 min was determined
by phosphormCPY imager analysis and is indicated. The asterisk
denotes a band that may represent
either an
intermediate
PEPCindependent
proteolytic
product or nonspecific
cross-reactive
material
and was not included in quantitation.
(6) CPY was recovered
by immunoprecipitation
and resolved on SDS-polyacrylamide
gels. For the experiment
at 37% with retl-7 cells, the cells
were preshifted to 37OC for 90 min prior to labeling. The position of the pl, p2, and mature (m) forms of CPY are indicated,
Inv-Wbpl
‘0’
60"
'0'
60"
'0'
60"
'0'
60"
10'
60'1
fusion -
B
are in accordance with our observations using the SteZWbplp and provide quantitative evidence that dilysinemediated retrieval is affected in sec27-7, sec27-2, and
sec27-7 mutants, especially at semipermissive temperamutant at all
tures, and is largely defective in the retl-7
temperatures tested.
Anterograde secretory transport was also assessed in
these mutants by monitoring intracellular transport of carboxypeptidase Y (CPY). During transit through the secrelory pathway, CPY is converted from the ER precursor
form (pl) to the Golgi-modified form (~2) and vacuolar form
(m). In sec27-7 and sec27-7 cells, it has previously been
reported that CPY maturation is unaffected at permissive
temperature, partially inhibited at semipermissive temperature, and at least partially inhibited at the nonpermissive
temperature (Kaiser and Schekman, 1990; Hosobuchi et
al., 1992; Duden et al., 1994). In these studies, transport
of CPY was usually tested after a short chase (15 min).
In addition, the secretion defect was less pronounced in
sec27-7 cells than in sec27-7 cells, even at 37% (Duden
et al., 1994). After a 60 min chase, we observed partial
inhibition of forward transport in sec27-7 cells at 37%,
and only a very limited inhibition in sec27-7 mutants at
37%(Figure
3B). In retl-7 cells aswell as in sec27-2cells,
only a minor defect in CPY maturation was observed at
37%, even when the cells were preshifted to 37% for 90
min prior to labeling (Figure 3B). Little or no defect in CPY
maturation was observed at lower temperatures (data not
shown). Thus, the loss of retrieval to the ER in retl-7,
sec27-2, and even sec27-7 cells is not accompanied
by
a strong block in anterograde ER-to-Golgi transport.
Retlp Is the a Subunit of Coatomer
We took advantage of the phenotype of the retl-7 mutant
to clone the RET7 gene by complementation
of the thermosensitive
defect. Three plasmids that restored growth at
37OC in retl-7 cells were isolated from two distinct genomic libraries. Two of them (pM1 and pM4) conferred only
slow growth at 37%, while one (pM5) restored normal
growth at 37OC (Figure 4A). Restriction mapping and DNA
sequencing revealed that the three plasmids contained
overlapping inserts (Figure 48). Integrative genetic mapping was used to prove that the pM5 plasmid carried the
authentic RET7 gene (see Experimental Procedures). Surface expression of Ste2-Wbplp
was abolished in retl-7
mutant cells transformed with a plasmid containing the
pM5 insert (Figure 4C), demonstrating that the pM5 insert
also restored awild-type retrieval phenotype in retl-7 cells.
Sequencing of the entire DNA insert revealed that it contained one open reading frame encoding a protein of 1201
amino acids (Figure 5A).
The recessive thermosensitive phenotype of retl-7 suggested that RET7 is an essential gene. To test this, we
disrupted the RET7 coding region. A 1.9 kb fragment encoding amino acids 282-903 of Ret1 p was replaced with
a 5 kb fragment containing the f.YS2 gene. This plasmid
was used to replace one copy of the RET7 locus of a LysPdiploid strain, and the RET7/ARET7::LYSP genotype of the
transformants was confirmed by PCR (Huxleyet al., 1990).
Upon sporulation, a pattern of two viable and two nonviable spores was observed at 24% for each tetrad analyzed
(data not shown). The viable spores were Lys-, demonstrating that Retlp is essential for germination and probably also growth at 24%.
The large size of Ret1 p as well as the synthetic lethalsec27-7,
and sec27-7 sugity observed between retl-7,
gested that RET7 might encode the as yet uncloned a
subunit of the coatomer complex. To tesr this prediction,
we purified yeast coatomer (Cosson and Letourneur,
1994; Hosobuchi et al., 1992), isolated the a subunit from
SDS-polyacrylamide
gels, and sequenced its N-terminus.
The 20 N-terminal amino acids of the yeast a-COP were
identical to the predicted N-terminus of Retlp, demonstrating that Retlp is the a subunit of the coatomer.
The most remarkable feature of the a-COP sequence
is the presence of six WD-40 repeats near its N-terminus
(Figure 58). The function of these motifs is unknown. They
were initially observed in p subunits of trimeric GTPbinding proteins (van der Voorn and Ploegh, 1992), but
they have also been found in @‘-COP (Stenbeck et al.,
7;:6
of Coatomer
in Retrieval
to the ER
Figure
37oc
24%
W3
pM4
pM5
Plasmids
Comvlementation
Jr
+I+I_.
4. Cloning
of RET7
(A) Temperature-sensitive
growth of retl-7 mutants refl-7 cells (PC70) transformed
with the
indicated plasmids (pM3, pM4, and pM5) were
plated on two SD plates supplemented
with the
necessary
amino acids and grown at 24% or
37%.
(B) Physical map of RETl. The inserts contained in various plasmids are shown, along
with their ability to complement
the thermosensitive phenotype of ret7-l cells. The thick arrow
represents
the predicted opon reading frame.
E, EcoRI; H, Hindll!; S, Sall; X, Xbal.
(C) Complementation
of the ret- phenotype
of
retl-I cells. The whole insert contained in the
pM5 plasmid was subcloned into an integrating
LYS2 vector and transformed
into retl-l cells
(PC61). Mating of the transformed
or nontransformed cells with MATn cells was tested at
24OC as described
in Figure 1.
ret1 -1
ret1 -1
+ pM5
1993; Harrison-iavoie
et al., 1993) SeclSp (Pryer et al.,
1993), and its associated 150 kDa protein (Barlowe et
al., 1994). They might represent oligomerization
motifs,
allowing interactions between the a-COP and f3’COP subunits and between Secl3p and ~150. Interestingly, in the
plasmids pM4 and pM1 the entire 5’ region of the RET7
gene is deleted, including the sequence coding for the’
WD-40 motifs (residues l-282 deleted), but these plasmids could still partially restore growth of r&7-1 cells at
37% (see Figure4A). In this case, atruncated Ret1 protein
is probably produced in the transfected cells by making
use of a cryptic promoter in the vector.
A
,F*c
34 88a.5
8&e.$
a-COP
-
PI”;
7
:
:
-
200
-
97
69
--
B
anti-o
.A
-1
Lysate
GH.................S.S.D...K.WD
T
Figure
5. Sequence
R
trlmer-C
p
c: prote1r
idh”,.ts
of Retlp
(A) The predicted
amino acid sequence
of Retlp is shown in singleletter code. The DNAsequence
isavailablefrom
the European Molecular Biology Laboratory
under the accession
number 246617.
(B) Ret1 p contains a repeated, conserved
motif. The N-terminal portion
of Retlp is displayed to align the six WD-40 motifs. The consensus
derived from a number of proteins related to the p subunit of trimeric
G proteins is also shown (van der Voorn and Ploegh, 1992).
Figure 6. Coatomer from sec27-7
Dilysine Motifs In Vitro
l[
anti-y
anti-p’
c_
_p __ -
and retl-7
Mutants
Does Not Bind
Whole-cell lysates from the indicated cells were inicubated with WBPl
peptide coupled to Sepharose
beads. The adsorbed
proteins were
separated
by SDS-PAGE
and revealed by silver staining (A) or by
immunoblot (B) using antisera to a-COP, Sec2lp (y-COP), or Sec27p
@‘COP). Whole-cell
lysates were analyzed by immunoblot
using an
antiserum to Sec27p. The position of a-COP, of its degradation
fragment (X), and of the ~100 family @COP, 6’CCP, and r-COP) are
indicated.
Cell
1204
Coatomer from sec27-1 and refl-7 Cells Does Not
Bind Dilysine Motifs In Vitro
To test the possibility that the defect in retrieval to the ER
in yeast mutants characterized above was due to a defect
in the binding of coatomer to dilysine motifs, we performed
in vitro binding experiments of coatomer to a peptide corresponding to the cytoplasmic domain of the Wbpl protein
(see Figure 1A). Whole-ceil lysates from various strains
were incubated with WBPl peptides coupled to Sepharose beads. Bound proteins were separated on SDS-polyacrylamide gels and revealed either by silver staining or
immunoblotting
with antisera to coatomer subunits. As
previously reported, proteins of 160 kDa and 100 kDa from
wild-type yeast lysates specifically interacted with dilysine
beads (Figure 6A), but not with WBPl-SS beads (Cosson
and Letourneur,
1994). These proteins correspond
to
a-COP and to the ~100 family (p-COP, p’-COP, Y-COP),
respectively, as confirmed by immunoblotting
with antisera to a-COP, 6’COP, and y-COP (Figure 6B). The protein migrating slightly faster than the ~100 family(X) corresponds to a degradation product of a-COP as analyzed
by N-terminal microsequencing.
While coatomer from
sec27-7 cells still bound efficiently to WBPl beads, coatomer from sec27-7 and retl-7 cells showed impaired binding (Figure 6). Binding of coatomer from sec27-2 cells was
indistinguishable
from binding of coatomer from sec27-7
cells (data not shown). The impaired binding of coatomer
from sec27-7 and retl-7 cells was not due to a reduction
in the total amount of coatomer present in these cells,
as shown by Western blotting of whole-cell lysates with
antisera to p’-COP (Figure 6B), a-COP, and y-COP (data
not shown), which revealed comparable amounts of the
three subunits in all cells. Thus, two classes of coatomer
mutants can be distinguished by their ability to affect binding to dilysine motifs in vitro.
Discussion
In thisstudy, we providegeneticand
biochemical evidence
that coatomer is required for retrieval of dilysine-tagged
proteins to the ER. Using a new system to assay ER retrieval of dilysine-tagged
proteins, we tested a number of
set mutants previously implicated in transport between
the ER and the Golgi apparatus (sec7, sec72, sec73,
sec76, sec77, sec78, sec79, sec27, sec22, sec23, and
sec27). Among these mutants, only sec27-7 @‘-COP) and
sec27-7 (y-COP) mutants showed a significant alteration
in ER retrieval. Moreover, we isolated new mutants deficient in retrieval to the ER. One represents a new sec27
allele (sec27-2). The others fell into one complementation
group (retl). Upon cloning and sequencing, we found that
RET7 encodes the a subunit of the coatomer (a-COP).
Mutations in [3’-COP as well as in a-COP led to the formation of a coatomer complex that had lost the ability to bind
dilysine motifs in vitro, suggesting that binding of the coatomer to dilysine motifs is essential for their retrieval to
the ER. Interestingly, a-COP and f3’COP subunits were
components, together with E-COP, of the partial coatomer
complex that bound dilysine motifs under high salt condi-
tions (Cosson and Letourneur, 1994) and thus would be
expected to comprise the dilysine-binding
site.
Despite the fact that mutations in y-COP (sec27-7 and
sec27-2) also led to adefect in ER retrieval, coatomer from
these cells remained capable of binding dilysine motifs in
vitro. This suggests that simple binding of the coatomer
to dilysine motifs is not sufficient for retrieval to the ER.
Coatomer is required for vesicle formation from isolated
Golgi fractions (Rothman and Orci, 1992) and is therefore
likely to perform other functions for which the Y-COP subunit might be essential. It is possible that coatomer in
sec27 mutant cells is incapable of binding dilysine motifs
in living cells because it is not correctly targeted to the
Golgi membrane; alternatively, binding of coatomer to
Golgi membranes may be normal in the mutant cells but
may not lead to the formation of transport vesicles.
There is still controversy about the role of coatomer in
various steps of membrane transport. In vitro, coatomercoated vesicles can bud from isolated Golgi, and they have
been implicated in transport through cisternae of the Golgi
complex (Rothman and Orci, 1992). In vivo, a block in
ER-to-Golgi transport was observed in mammalian cells
microinjected with antibodies to b-COP (Pepperkok et al.,
1993). Moreover, sec27-7 (y-COP) yeast mutant cells
(Novicketal., 1960)aswellasCHOcellsmutated
ins-COP
(Guo et al., 1994) exhibit a block of ER-to-Golgi transport
at the restrictive temperature. These results support the
idea that coatomer is also necessary for ER-to-Golgi transport. They are, however, challenged by the recent finding
that a different coat (COPII) is necessary and sufficient
for ER transport vesicle budding in vitro (Barlowe et al.,
1994).
All coatomer mutants analyzed in this study are defective for retrieval of dilysine-tagged proteins from the Golgi
to the ER. In some mutant cells (sec27-l), this block is
accompanied by an inhibition of ER-to-Golgi transport at
37%, while in others (sec27-7, sec27-2, retl-l), little or
no inhibition of ER-to-Golgi transport is observed. These
results are compatible with the widely accepted idea that
coatomer is directly involved in various steps of transport,
namely ER-to-Golgi, Golgi-to-ER, and intra-Golgi transport. In this case, the explanation for the lack of effect of
the retl-7 and sec27-2 mutations on secretion would be
that these mutant alleles do not affect secretion, and that
our selection scheme was biased in favor of such mutations. However, an alternative and highly speculative
model can be envisaged, in which coatomer is directly
involved only in retrograde Golgi-to-ER transport. Effects
seen on anterograde transport events could be indirect
effects of the inhibition of retrograde transport. For instance, a defect in the retrieval of essential V-SNARE proteins to the ER could result in a block in anterograde ER-toGolgi transport. Detailed analysis will be necessary to
determine the exact role of coatomer in various Steps Of
intracellular transport.
Experimental
Procedures
Strains,
Media, and Reagents
Yeast media have been described
(Sherman,
1991).
Yeast
strains
Role of Coatomer
1205
Table
1. Yeast
in Retrieval
to the ER
Strains
Strain
Genotype
RH31 l-3D
RH1298
RH270-2B
PC8
PC9
PC16
PC17
PC63
PC64
PC65
PCGE
PC24
PC67
PC40
PC26
PC82
PC27
PC28
PC75
PC81
PC70
PC11
PC13
PC52
PC53
SEY6210
EGYlOl
RSY277
EGYlO3
CKYI 00
MA Ta,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
MATa,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
wad
ura3,
ura3,
ura3.
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
ura3,
wag
Origin
leu2, trpl
leu2, his4, barl-1, steP::LEUZ
leu2, his4, /ysZ, barl-1, ste2::LEU2
leu2, his4, lys2, barl-1, steP::LEUZ, STEZ-WBPl::UFfA3
/euZ, his4, lys2, barl-1, ste2::LEUZ,
STEZ-WBPl-SS::lJRAS
leo2, his4, lys2, barl-1, steZ::LEU2,
STE2-WBPl-RR::URAS
leu2, his4, lys2, barl-1, steZ::LEUZ,
STE2-WBPl+4S::URA3
leo2, his4, barl-1, ste2::LEUZ,
STEZ-WBPl::lJRAS,
sec7-1
leu2, his4, lys2, barl-1, steZ::LEU2, STE2-WBPl::URA3,
secl2-1
leu2, hisd lys2, barl-1, steZ::LEUZ, STEZ-WBPl::lJRA3,
seclS1
leu2, his4, lys2, barl-1, steZ::LEUZ, STE2-WBPl::URA3,
secld1
leu2, his4, lys2, barl-1, steZ::LEU2, STE2-WBPl::URA3,
secl7-1
leu2. his4, barl-1, steZ::LEU2,
STE2-WBPl::URAS,
secl8-1
leu2, his4, lys2, barl-1, steZ::LEUZ, STEZ-WBPl::URA3,
secl9-1
leu2, his4, lys2, bad-l,
steZ::LEUZ, STEZ-WBPl::URA3,
sec21-1
leu2, his4, lys2, barl-1, steP::LEUZ, STEZ-WBPl:tlJRA3,
sec21-2
leu2, his4, lys2, barl-1, steP:tLEUZ, STEZ-WBPl::URA3,
sec22-1
leu2, his4, barl-1, steZoLEU2,
STEZ- WBPl::URA3,
sec23-1
leu2, his4, barl-1, steZ::LEUZ,
STEZ- WBPl::URAS,
sec27-1
leu2, his4, lys2, steP::LEUZ, STEZ-WBPl::URA3,
retl-1
leu2, trpl, retl-1
leu2, his4, lys2, steP::LEUL,
leu2, his4, lys2, steP::LEUZ, STEZ-WBPl::URA3
leu2, his4, barl-1, ste2::LEUZ
leu2, his4, lys2, ste2::LEU2
leu2, his3, trpl, lys2, suc2-A9
leu2, his3, trpl, sucZA9, retl-l
sec21-1
leu2, his3, lys2, sec27-2, suc2-A9
leu2, sec27-1
used in this study are listed in Table 1. All PC strains were backcrossed
at least twice with RH1298. The STE2 gene was disrupted
by use of
the plasmid pUSTE203
(Nakayama
et al., 1988). The plasmid used
to construct
the various
STEP-WBPl
chimeras
(pJR3-320Bam345Stop) was prepared by J. Rohrer as described previously
(Rohrer
et al., 1993). It has a unique BamHl site changing amino acids 319
and 320 to serine and leucine, respectively.
The STE2 gene is under
control of its own promoter, and the plasmid can be integrated in the
yeast chromosome
at the ura3 locus after linearization
with Stul.
The mutant forms of the STE2 gene were created by using standard
polymerase chain reaction (PCR)protocols(Jones
and Howard, 1990).
The entire subcloned
fragments
were sequenced
after subcloning.
Plasmid pBLYS was constructed
by inserting a 5 kb fragment containing the LYSP gene (Fleig et al., 1986) in the pBluescript
plasmid
(Stratagene).
Plasmid pEGl-KK has been described previously
(Gaynor et al., 1994).
Antibody to SecPlp (y-COP) (number 9256.9) was described
previously (Hosobuchi
et al., 1992). Rabbit antiserum
to an N-terminal
peptide of mammalian a-COP, cross-reacting
with yeast u-COP, was
a gift of C. Hatter and F. T. Wieland. For obtaining antisera to Sec27p
@‘-COP), a peptide RSDRVKGIDFHPTEPW
corresponding
to residues 12-27 (Harter et al., 1993) was synthesized
on a MAP8 matrix
(Bachem, Feinchemikalien
Aktiengesellschaft).
Rabbits were immunized with this peptide, and the polyclonal antiserum
recognizing
p’
COP was affinity purified on a column made of the same peptide used
for immunization
coupled to activated CH Sepharose 48 (Pharmacia).
Antisera to invertase (Gaynor et al., 1994) and CPY (Klionsky et al.,
1988) have been described
previously.
Cell Labeling and lmmunoprecipitation
Analysis of the intracellular
transport of the Inv-Wbplp
and CPY was
performed as previously
described (Gaynor et al., 1994). In brief, cells
expressing
Inv-Wbpl
fusion protein (pEGI-KK)
were preincubated
for
45 min at the indicated temperature,
pulse-labeled
for 10 min with
Tran”S-label,
and chased for 0 or 60 min. Equal amounts of cells were
H. Riezman
H. Riezman
H. Riezman
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
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This study
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Robinson et al., 1988
This study
Kaiser and Schekman,
This study
C. Kaiser
1991
removed, and the fusion protein was recovered
by immunoprecipitation, treated with endoH to remove N-linked oligosaccharides,
and
resolved on SDS-polyacrylamide
gels. CPY was immunoprecipitated
from the supernatant
of the invertase
immunoprecipitation
and analyzed on SDS-polyacrylamide
gels.
Labeling and immunoprecipitation
of Ste2p with an antipeptide antiserum to the N-terminus
of Ste2p were done as described
(Zanolari
et al.. 1992). For reprecipitation
Ste2p was elutecl from the beads at
50°C and subjected to a second round of immunoprecipitation
with
antisera to Ste2p, al,6 mannose,
or al,3 mannose (a gift from R.
Schekman).
Methods for SDS-PAGE
(Laemmli, 1970), silver staining (Bloom et
al., 1987; Merril et al., 1981) immunoblotting
(Towbin et al., 1979),
and immunodetection
by enhanced chemiluminescence
(Amersham,
Arlington Heights, IL) have been described.
Genetic Techniques
Standard genetic methods for mating of haploid yeast strains, complementation analysis, and tetrad analysis were employed (Sherman and
Hicks, 1991). Escherichia
coli strain DH5a was used for plasmid propagation and purification.
Plasmid DNA was purified by the alkaline lysis
method (Maniatis et al., 1982). Procedures
for transformation
of DNA
into yeast (Becker and Guarente,
1991) and E. coli (klaniatis
et al.,
1982) have been described
previously.
DNA sequencing
was carried
out using Sequenase
(United States Biochemical
Corporation,
Cleveland, OH).
Allstrainsused
in thisstudyproducedsimilaramountsof
Ste2fusion
proteins, as checked
by metabolic labeling for 10 min followed by
Ste2p immunoprecipitation
and analysison
SDS-plolyacrylamide
gels.
For mating tests, a patch of MATa cells grown on YPD plates was
preincubated
at the indicated temperature
for 2 hr, then replica plated
to a lawn of MATu cells (RH31 I-3D) and incubated at the indicated
temperature
for 8 hr. The cells were then replica plated to SD plates
where only diploid cells can grow. Sensitivity to CLfactor was tested
Cell
1206
in parallel by halo assay (Sprague, 1991), and cells that were able to
mate were also sensitive to cz factor (data not shown).
To isolate mutants,
yeast cells expressing
SteP-Wbplp
(PC13)
were mutagenized
with ethylmethane
sulfonate (50% cell death) as
described (Lawrence,
1991) and plated at high density on YPD plates.
Three days later, they were replica plated to a lawn of MATa ceils
(PC52), allowed to mate at 24OC for 6 hr, and replica plated to SD
plates supplemented
with histidine. The diploid cells that grew were
sporulated,
and following tetrad dissection,
thermosensitive
MATa
cells expressing
SteP-Wbplp
and capable of mating were isolated.
Cloning of RET1
Yeast strain PC70 was transformed
to Ura+ or Leu’ with two distinct
yeast genomic libraries constructed
in YCplac33 (pM4 and pM5) and
YCplaclll
(pMl), respectively
(Gietz and Sugino, 1988), and obtained, respectively,
from T. Wller and F. Cvrckova. The transformants
were grown at room temperature
on selective medium, then replica
plated onto selective medium and grown at 37OC. Plasmids were recovered from clones growing at 37‘C and retransformed
into PC70,
and the transformed
cells were tested for growth at 37OC as described.
The isolated plasmids were analyzed by restriction mapping and DNA
sequencing.
The whole insert in the pM5 plasmid was sequenced
using sense and antisense oligonucleotides
spaced 300 bp apart.
Integrative
genetic mapping was used to prove that the isolated
plasmids carried the authentic RET7 gene. A 2 kb EcoRI-Clal
fragment
that was excised from the plasmid and carried the 3’end of the putative
RET1 was subcloned
into p8LYS (described
above). The resulting
plasmid was linearized within the EcoRI-Clal
insert by digestion with
endonuclease
Sal1 and recombined in the genome of yeast strain PC53
via the homology presented by the insert DNA. Integration at the appropriate locus was checked by PCR as previously
described
(Huxley et
al., 1990). The strain was mated to the strain PC81 (ret+7) and the
resultant diploid subjected
to meiotic analysis (49 tetrads). For every
tetrad analyzed, the meiotic progeny exhibited the expected 2:2 segregation of thermosensitive:nonthermosensitive
and Lys2+:Lys2-.
All
thermosensitive
progeny were LysP- and, when it could be tested (in
MATa cells expressing
Ste2-Wbpl
p; 46 spores), ret-. All nonthermosensitive spores were Lys2: and showed no loss of ER retrieval. These
data demonstrate
a tight linkage of RET7 and the integrated LYSL,
thereby proving,that
the isolated plasmids carried the authentic RET7.
In Vitro Binding of Coatomer
to Dilysine Motifs
An 11-mer peptide CKKLETFKKTN
corresponding
to the cytoplasmic
domain of Wbplp with a cysteine added as the first amino acid was
synthesized
and coupled to activated thiol-Sepharose
48 (Pharmacia)
according to the recommendations
of the manufacturer
(5 mg of crude
peptide per ml of beads). The coupling reaction was quenched for 1
hr at room temperature
in 0.1 M ammonium
acetate (pH 4.0) 0.5 M
NaCI, 8.5 pM,2-mercaptoethanol,
and beads equilibrated
and stored
in PBS buffer.
Yeast cells were grown overnight
at 30°C and spheroplasts
prepared as previously
described
(Cosson and Letourneur,
1994). Spheroplasts were rapidly frozen in liquid nitrogen before lysis in Tris-Triton
buffer (Cosson and Letourneur,
1994). Binding to WBPI peptide beads
(50 ~1 of beads for a 50 ml yeast culture) and washing conditions were
as already described
(Cosson and Letourneur,
1994).
For protein sequencing,
spheroplasts
were prepared
from 1 liter
of yeast culture and processed
as described
above. Lysates were
incubatedfor
hr at4°Cwith300~lofWBPl
beads. Adsorbed proteins
were separated on a 6% SDS-polyacrylamide
gel and transferred
to
Problott membrane
(Applied Biosystems)
in 1% methanol,
100 mM
CAPS (pH 11). The Coomassie
blue-stained
band corresponding
to
c-COP (Cosson and Letourneur,
1994) was excised and sequenced
with an Applied Biosystems
sequencer
(model 475A). The sequence
obtained corresponded
to the 20 N-terminal amino acids of the predicted Fietlp. We also sequenced
the N-terminus
of the a subunit of
coatomer purified in a more classical way (Hosobuchi
et al., 1992) and
obtained a sequence
corresponding
to the eight N-terminal
amino
acids of the predicted
Retlp.
fiths, Randy Schekman,
and Jose A. Garcia-San2
for critical reading
of the manuscript,
and David Avila, Peg Scott, Fabienne Crausaz, and
Thomas Aust for technical assistance.
The laboratory
of H. Ft. was
supported
by a grant from the Swiss National Science Foundation,
the laboratory
of S. D. E. by a grant from the National Institutes of
Health and by the Howard Hughes Medical Institute, and the laboratory
of Randy Schekman
(R. D.) by the Howard Hughes Medical Institute.
R. D. was supported
by a fellowship
from the European Molecular
BiologyOrganization.The
Base1 Instituteforlmmunologywasfounded
and is supported
by F. Hoffman-LaRoche
and Company,
Limited,
CH-4002 Basel, Switzerland.
Received
September
29, 1994; revised
should
be addressed
to P. C. We thank
Gillian Grif-
31, 1994.
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GenBank
Accession
The accession
246617.
number
Number
for the sequence
reported
in this paper
is