Department of Biology, The Division of Cellular and Molecular Medicine, and The Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0668
The coatomer (COPI) complex mediates
Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of
COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to
generate new temperature-conditional mutant alleles
of one COPI gene in Saccharomyces cerevisiae, SEC21 (-COP). Unexpectedly, all of the new sec21 ts mutants
exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e.,
CPY and
-factor) were completely blocked in the ER
at nonpermissive temperature; however, other proteins
(i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical
cargo-specific ER to Golgi transport defects were also
induced by Brefeldin A. In contrast, all proteins tested
required COPII (ER to Golgi coat complex), Sec18p
(NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a
critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required
for packaging of certain cargo into ER to Golgi COPII
vesicles. Interestingly, CPY-invertase hybrid proteins,
like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII
vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid
proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the
sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not
late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although
COPI plays an important and most likely direct role both in Golgi-ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the
secretory pathway.
In the secretory pathway of eukaryotic cells, transport
between organelles is mediated by coated vesicles
that must efficiently capture and package cargo molecules, and then target, dock, and fuse with an appropriate
acceptor compartment (Rothman and Orci, 1992 Two vesicle coat complexes, COPI/coatomer and COPII,
have been identified that regulate transport between the
endoplasmic reticulum and Golgi complex in both yeast
and mammalian cells. COPII was initially identified in
yeast and is comprised of the Sec23p/Sec24p and Sec13p/
Sec31p complexes, the small GTP-binding protein Sar1p
(Barlowe et al., 1994 In contrast to the role of COPII in ER to Golgi transport, the direct role(s) of COPI/coatomer (composed of
seven subunits: More recently, COPI has been implicated as playing a
critical role in Golgi to ER retrograde transport. A dilysine (KKXX) motif directing retrieval of type I membrane
proteins (Jackson et al., 1993 To dissect the role of COPI in yeast more thoroughly, we
used random PCR mutagenesis to generate new temperature-sensitive (ts) alleles of SEC21, which encodes the yeast
Strains and Media
Yeast strains used in this study are shown in Table I. Standard genetic techniques were used throughout (Sherman et al., 1979 Table I.
Saccharomyces cerevisiae Strains Used in This Study
). The bidirectional nature of interorganelle transport is crucial for
proper functioning of the pathway: anterograde transport serves to direct the forward transport of secretory cargo
(Palade, 1975
), while retrograde transport is essential both
for retrieval of proteins which have escaped their site of
residence and for recycling of transport factors required to
mediate further rounds of anterograde traffic (Letourneur
et al., 1994
; Pelham, 1994
). Because the coat complexes
that direct these trafficking events participate directly or
indirectly in cargo selection, vesicle coats play a critical
role in maintaining the directional flow of protein transport in the secretory pathway (Aridor and Balch, 1996
;
Rothman and Wieland, 1996
; Schekman and Orci, 1996
).
), and most likely Sec16p (Espenshade et al., 1995
). The COPII complex is required for formation of cargo-containing, ER-derived transport vesicles
and thus functions directly in anterograde traffic between
the ER and the cis-Golgi (Bednarek et al., 1995
).
,
,
,
,
,
,
; Waters et al., 1991
) in
ER/Golgi transport has recently been the subject of significant debate. In vitro work in mammalian cells initially
revealed that COPI, along with the small GTP-binding protein ADP-ribosylation factor (ARF)1, binds Golgi membranes and induces formation of functional transport vesicles
(Malhotra et al., 1989
; Rothman and Orci, 1992
). Several lines of evidence support a role for COPI in anterograde
transport. In mammalian cells, both in vitro and in vivo
work has indicated that inhibition of COPI function results in a block in ER to Golgi transport (Pepperkok et al.,
1993
; Peter et al., 1993
; Dascher and Balch, 1994
; Zhang et
al., 1994
). COPI-coated vesicles have also been observed
to bud from purified yeast nuclei (ER membranes); however, unlike COPII-coated vesicles, ER-derived COPIcoated vesicles are devoid of any known cargo (Bednarek
et al., 1995
). Finally, three of the COPI subunits (
,
,
)
were initially identified as SEC gene products in yeast; sec
mutants were isolated as defective for anterograde secretory transport (Hosobuchi et al., 1992
; Duden et al., 1994
).
; Gaynor et al., 1994
; Townsley and Pelham, 1994
) was found to bind specifically to
COPI proteins in vitro (Cosson and Letourneur, 1994
).
Furthermore, a screen for yeast mutants defective for
KKXX-mediated retrieval (ret mutants) identified three
COPI subunits (
,
,
) as products of RET genes (Letourneur et al., 1994
; Cosson et al., 1996
). Interestingly, all
COPI mutants, whether sec or ret mutants, are defective
for KKXX-mediated retrieval, while only a small subset
exhibit deficiencies in anterograde ER to Golgi transport
(Letourneur et al., 1994
; Cosson et al., 1996
). These observations have led to the speculation that COPI may directly
function only in retrograde traffic, and that anterograde transport defects in these mutants are a secondary consequence of an inability to recycle transport components required for anterograde traffic to continue (Letourneur
et al., 1994
; Pelham, 1994
; Lewis and Pelham, 1996
). However, direct verification of this hypothesis has not yet been
provided.
-COP subunit (Hosobuchi et al., 1992
). In these sec21 ts
mutants, certain cargo proteins (i.e., CPY and
-factor)
were blocked in the ER, while other cargo proteins (i.e.,
HSP150, invertase, CPY-invertase hybrid proteins) were
not blocked and were secreted normally or sorted properly to the vacuole. Similar cargo-specific ER to Golgi transport
was also observed in BFA-treated cells; however, no ER
to Golgi transport was detected in COPII or NSF mutants.
Our observations support a model whereby COPI is not
directly required for anterograde transport at any step in
the secretory pathway (ER to Golgi, Golgi to plasma
membrane, or Golgi to vacuole); instead, ER to Golgi traffic of some cargo proteins may require COPI for retrieval of limiting transport factors essential for packaging these
cargo into anterograde, ER-derived COPII-coated vesicles.
This is likely to be an active process mediated by sorting
determinants contained within the cargo proteins themselves.
Materials and Methods
). Strains EGY1181-5,
EGY1231-1 and -2, EGY111-2, and EGY1221-2 were constructed by
crossing SEY5188, SF309-1C, SEY5017, and RSY454, respectively, with
either SEY6210 or SEY6211, sporulating, and isolating haploid progeny
carrying desired markers and mutations. Cells were grown in either YPD
(yeast extract, peptone, dextrose) or YNB (yeast nitrogen base) media
supplemented as necessary (Sherman et al., 1979
).
Plasmids and DNA
Plasmid p315SEC21 was provided by R. Duden and R. Schekman (University of California, Berkeley, CA) and was constructed by subcloning a
NotI-HindIII fragment (containing the entire SEC21 ORF) out of a
YEP13 complementing library plasmid (Hosobuchi et al., 1992) into the
vector pRS315 (Sikorski and Hieter, 1989
). Plasmid p416SEC21 contains
this same NotI-HindIII fragment of SEC21 in pRS416 (Sikorski and
Hieter, 1989
). To generate the sec21::HIS3 disruption construct, an AvaI-
BclI fragment of SEC21 (comprising >95% of the SEC21 ORF) was replaced with the HIS3 gene in p315SEC21. This plasmid was digested with
Spe1, and the Spe1 fragment containing the HIS3-disrupted SEC21 gene
was isolated by gel purification and used to make the strain EGY021.
Plasmids pCYI-20, -50, -156, and -433 were described previously (Johnson et
al., 1987
). Plasmid pOH containing the hemagglutinin (HA)-tagged Och1p
was provided by S. Harris and G. Waters (Princeton University, Princeton, NJ) and was described previously (Harris and Waters, 1996
).
SEC21 Mutagenesis and Double Mutant Construction
The yeast strain used in the plasmid shuffle, EGY021, was constructed by
first disrupting one chromosomal copy of SEC21 in an SEY6210/SEY6211
diploid with HIS3 (using the linearized SpeI fragment described above) by
homologous recombination. p416SEC21 (CEN, URA3, SEC21) was transformed into this disrupted diploid, which was then sporulated. His+, Ura+
haploids were isolated, one of which was designated EGY021. Mutations in SEC21 were generated by random PCR mutagenesis (Muhlrad et al.,
1992). Primers annealing 160 nucleotides 5
of the BamHI site and 150 nucleotides 3
of the BclI site in SEC21 were used to PCR-amplify a 2,200nucleotide fragment (~80% of the SEC21 ORF) using Taq polymerase
(Perkin-Elmer Corp., Norwalk, CA) under standard conditions, except
that limiting (50 µM) dATP was used. A gapped (linearized) acceptor
plasmid was made by digesting p315SEC21 (CEN, LEU2, SEC21) with
BamHI and BclI and isolating the vector by gel purification. Equimolar
amounts of gapped plasmid and mutagenized DNA were cotransformed
into EGY021. Homologous recombination in yeast resulted in mutagenized SEC21 DNA being incorporated into the p315SEC21 vector, and
transformants were selected on
Leu plates. Leu+ colonies were replica
plated onto 5-fluoroorotic acid (5-FOA, which selects against URA3-containing plasmids) plates to shuffle out p416SEC21 (CEN, URA3, SEC21;
Sikorski and Boeke, 1991
). Temperature-conditional mutants which grew
on 5-FOA at 26° but not 37°C were isolated. p315sec21 (CEN, LEU2,
sec21ts) mutant plasmids were isolated and reshuffled into EGY021 for
further mutant characterization.
Double mutant strains EGY231/213 (sec23-1/sec21-3) and EGY11/213 (sec1-1/sec21-3) were generated by crossing EGY1231-2 or EGY111-2, respectively, with EGY021, sporulating, and isolating temperature sensitive, His+, Ura+ haploid progeny exhibiting the appropriate mutant phenotype. p315sec21-3 was then shuffled into these strains as described.
Cell Labeling, Immunoprecipitation, Subcellular Fractionation, Immunoblotting, and Brefeldin A (BFA) Treatment
Cell labeling, immunoprecipitation, reimmunoprecipitation with secondary antisera, endoglycosidase H treatment, and subcellular fractionation
were performed essentially as previously described (Gaynor et al., 1994),
with the following variations. Before labeling, cells were grown to early
logarithmic phase in YNB supplemented with amino acids. Cells were labeled at 5 OD/ml in YNB media containing amino acids, 100 µg/ml
2macroglobulin, and 500 µg/ml BSA. To assay internal and external invertase, cells were converted to spheroplasts after pulse-chase by adding an
equal volume of 2× spheroplast buffer (50 mM Tris, pH 7.5, 2 M sorbitol,
40 mM NaN3, 40 mM NaF, 20 mM DTT), incubating on ice for 10 min,
then adding 25 µg zymolyase/ml cell suspension and incubating 30 min at
30°C. To separate cells or spheroplasts from media, cell suspensions were
centrifuged at 5,000 g for 5 min. To assay media proteins, media fractions
were precipitated by addition of TCA to a final concentration of 10%. After washing the pellets twice in acetone, proteins were solubilized by sonication in Laemmli sample buffer plus 5%
-mercaptoethanol, boiled, and
centrifuged at 16,000 g for 15 min, and 0.25 OD equivalents were loaded
for SDS-PAGE. For immunoprecipitation of HSP150 or invertase from
internal/external fractions, OD equivalents of cells or spheroplasts and
media were harvested and precipitated by adding TCA to a 10% final concentration. Media fractions were prepared as described. Cells were prepared for immunoprecipitation as previously described (Gaynor et al., 1994
). Spheroplasts were similarly prepared but without glass bead lysis.
Antisera to CPY (Klionsky et al., 1988
),
factor (Graham and Emr, 1991
),
HSP150 (Russo et al., 1992
), invertase (Gaynor et al., 1994
), and
1,6 and
1,3 mannose (Franzusoff and Schekman, 1989
) were described previously. Immunoblotting to detect Och1-HA was done using the 12CA5
monoclonal antibody (provided by David Levin, Johns Hopkins University, Baltimore, MD, and made by Berkeley Antibody Co., Berkeley, CA)
at a 1:5,000 dilution and visualized by ECL. BFA was from Epicentre
Technol. Corp. (Madison, WI) and was solubilized at 10 mg/ml in 95%
ethanol before use. For experiments involving BFA treatment of the erg6
mutant, cell labeling, immunoprecipitation, and subcellular fractionation
were performed as described, except that the media also contained 50 mM
Na-Hepes, pH 7.0. Either 100 µg/ml BFA or an equivalent volume of 95%
ethanol was added to the cells 2 min before labeling.
Fluorescence Microscopy
Indirect immunofluorescence was performed as previously described
(Redding et al., 1991), with the following variations. Cells were fixed for
16 h in 4% formaldehyde, spheroplasted with 45 µg/ml Zymolyase 100T
(Seikagaku America, Inc., Rockville, MD), and then treated with 1%
SDS. Fixed spheroplasts were incubated first with either the 12CA5 monoclonal antibody at a 1:400 dilution or an affinity-purified rabbit anti-Mnn1p
(Graham et al., 1994
) at a 1:10 dilution for 16 h at 4°C. This was followed
by incubations with either 0.5 µg/ml goat anti-mouse IgG or 0.5 µg/ml
goat anti-rabbit for 2 h at 4°C, and then a 1:200 dilution of Cy3-conjugated
donkey anti-goat IgG for 2 h at 4°C (antibodies from Jackson ImmunoResearch Labs., West Grove, PA).
Rationale for Generation of New sec21 Temperature-Conditional Alleles
Before this work, analysis of COPI function in yeast had
been restricted to studying a small number of COPI mutant alleles, all of which had been isolated in screens selecting specifically for either anterograde (sec mutants) or
retrograde (ret mutants) secretory transport defects. While
these mutants have yielded significant insight regarding
the role of COPI in secretory transport, the means by
which they were identified and the fact that only one or
two mutant alleles for each coatomer subunit had been
isolated both limits and biases what can be learned from
them. Additionally, the range of defects observed for different COPI mutants, coupled with the fact that it has
been impossible to determine whether these mutations
may simply alter or reduce function of the encoded proteins rather than represent true temperature-conditional loss of function alleles, has made it difficult to establish the most primary defect(s) associated with loss of COPI function.
As an alternative genetic approach, we used random PCR
mutagenesis to generate multiple new temperature-conditional mutant alleles of one COPI gene, SEC21 (-COP).
Mutants were isolated by selecting only for temperatureconditional growth and not by screening for directional
transport defects. By eliminating this bias, we hoped to reveal the primary function(s) of Sec21p/
-COP. We chose
to mutagenize SEC21 because the two existing sec21 mutant alleles exhibit different phenotypes: both are partially
defective for retrieval of dilysine-containing proteins, but
while sec21-1 also exhibits a partial block in anterograde
transport, sec21-2 does not (Hosobuchi et al., 1992
; Letourneur et al., 1994
).
SEC21, like all COPI genes, is essential. We employed
the standard techniques of PCR mutagenesis (Muhlrad
et al., 1992) and plasmid shuffle (Sikorski and Boeke,
1991
) to generate strains in which the wild type SEC21
gene was replaced with mutagenized SEC21-containing
plasmids (see Materials and Methods). Six temperatureconditional alleles were identified which were viable at 26 but not 37°C. Different growth properties at intermediate
temperatures suggested that they represented at least four
distinct alleles (Table II). Somewhat surprisingly, however, the sorting and transport phenotypes of these mutants were remarkably similar: all were partially defective for retrieval of a KKXX-containing Inv-Wbp1 fusion (data
not shown; Gaynor et al., 1994
; Letourneur et al., 1994
),
and all exhibited striking and nearly identical cargo-specific anterograde transport defects. Therefore, while the
analyses described below were performed for each new
mutant, results are only shown for one representative allele, designated sec21-3.
Table II. Growth Phenotype of sec21 ts Mutants |
The sec21 ts Mutants Exhibit a Rapid and Complete
Block in Export of CPY and -factor from the ER at
Nonpermissive Temperature
In yeast, anterograde secretory transport can be assayed
by observing the processing and maturation of a well characterized, soluble vacuolar hydrolase, carboxypeptidase Y
(CPY). CPY is synthesized initially as a proenzyme which
is first modified with core oligosaccharides in the ER, generating the p1 form, and next in the Golgi complex, where
the core sugars are elongated, generating the p2 form. After delivery to the vacuole, the pro region is cleaved to
yield the mature (m) enzyme (Stevens et al., 1982). sec21-3
cells were incubated at either permissive (26°C) or nonpermissive (37°C) temperature for 10 min and subjected to
pulse-chase analysis, and CPY was recovered by immunoprecipitation. At 26°C, processing and maturation of CPY
occurred normally (Fig. 1 A), with kinetics similar to wild
type cells (data not shown). In contrast, at 37°C, CPY was found exclusively as the p1, ER-glycosylated form (Fig.
1 A). Similar results were observed when the sec21-3 mutant was preincubated at 37°C for either 1 or 30 min before
labeling or when cells were chased for longer times (i.e., 60 min); in addition, maturation of two membrane-associated
vacuolar hydrolases, alkaline phosphatase (ALP) and carboxypeptidase S (CPS), was also completely blocked in
sec21-3 at 37°C (data not shown). This is in contrast to the partial anterograde transport defects observed for other
COPI mutants (Duden et al., 1994
; Letourneur et al., 1994
).
Even in sec21-1, for instance, we observed significant maturation of CPY at 37°C after backcrossing this mutation
into our strain background (data not shown).
To address whether the p1CPY which accumulated in
sec21-3 at 37°C had reached the Golgi complex, Golgispecific carbohydrate modifications on CPY were analyzed. It has previously been shown that the yeast Golgi
complex can be functionally divided into at least four distinct compartments. From cis to trans, these compartments
are defined by: initial 1,6 mannosyltransferase (Och1p),
elongating
1,6 mannosyltransferase,
1,3 mannosyltransferase (Mnn1p), and Kex2p activities (Franzusoff et al., 1991
;
Graham and Emr, 1991
; Nakanishi-Shindo et al., 1993
;
Gaynor et al., 1994
). p1CPY is generated in the ER by the
addition of core oligosaccharides to appropriate asparagine residues. Upon arrival in the Golgi, the core sugars
are modified first with initial
1,6 mannose in the cis-most
compartment and then with
1,2 and
1,3 mannose in a
medial compartment (Graham and Emr, 1991
). The presence of these mannose moieties indicates how far the protein has progressed in the secretory pathway. Cells were
incubated at 37°C for 10 min, pulse-labeled, and chased for
30 min. CPY was recovered by immunoprecipitation, and
then split into equal aliquots and subjected to a second immunoprecipitation with antisera specific to either CPY or
1,6 mannose linkages. In wild type cells, CPY was efficiently
1,6 mannose-modified, while in a sec18-1 (NSF)
mutant, which blocks ER to Golgi transport, CPY does not
acquire
1,6 mannose (Fig. 1 B). In sec21-3, as in sec18-1,
no CPY was recovered after immunoprecipitation with
1,6 mannose antiserum (Fig. 1 B), suggesting that ER to
Golgi transport of CPYwas blocked in sec21-3.
Finally, the subcellular location of p1CPY in sec21-3 at
nonpermissive temperature was examined by differential
centrifugation. Spheroplasts were incubated at 37°C, pulselabeled, chased for 45 min, and then osmotically lysed. After a clearing spin at 300 g to remove unbroken cells and
cell wall debris, the lysate was centrifuged sequentially to
generate 13,000 g pellet (P13), 100,000 g pellet (P100), and
100,000 g supernatant (S100) fractions. In wild type cells,
mCPY fractionated in the S100 (Fig. 1 C), consistent with
its localization to the lumen of the osmotically sensitive vacuole. In contrast, in sec21-3 at 37°C, p1CPY was found
exclusively in the P13 fraction (Fig. 1 C). It has previously
been shown that both soluble (i.e., PDI) and membraneassociated (i.e., Wbp1p) ER resident proteins fractionate
almost entirely in the P13, while Golgi proteins (i.e.,
Mnn1p and Kex2p), even cis-Golgi proteins (i.e., Och1p),
are found primarily in the P100 (data not shown; Gaynor
et al., 1994; Harris and Waters, 1996
). Indeed, immunoblot analysis indicated that ~75% of an HA-tagged Och1p (a
resident of the cis-most Golgi compartment) fractionated
in the P100 in both wild type and sec21-3 cells (Fig. 1 C).
Further support for ER localization of p1CPY in sec21-3
was provided by resolution of P13 material on a 2-step sucrose gradient, where p1CPY migrated in the ER-enriched
fraction but Och1p did not (data not shown; Gaynor et al.,
1994
). Together, these data provide strong evidence that
CPY transport was blocked at the level of the ER in sec21-3 at nonpermissive temperature.
To assay anterograde transport of a secreted protein in
the new sec21 ts mutants, pulse-chase analysis of the
mating pheromone -factor was performed.
-factor is
synthesized as a precursor which acquires up to three core
N-linked oligosaccharides in the ER. Pro-
-factor then
transits rapidly through the Golgi complex, where it is sequentially modified with
1,6 and
1,3 mannose, then proteolytically processed into the mature peptide pheromone in the trans-Golgi before being secreted (Julius et al., 1984
; Graham and Emr, 1991
). In sec21-3 at permissive temperature (23°C),
-factor was very rapidly Golgi-modified and
then converted to the mature, 13 amino acid peptide hormone (Fig. 2). This is identical to what was observed in
wild type cells; in addition, the mature
-factor peptide was
secreted to the medium in sec21-3 at 23°C (data not
shown). After a 5-min preincubation at 37°C, however, only the core-glycosylated, ER form of
-factor was recovered (Fig. 2). The protein also remained entirely intracellular, and even after long chase times, appearance of
mature
-factor was not observed (data not shown). Therefore, shifting the sec21 ts mutants to nonpermissive temperature yielded a rapid and complete ER block for several vacuolar hydrolases (CPY, ALP, and CPS), as well as
for a secreted protein,
-factor; this rapid and absolute onset also suggests that the temperature shift resulted in immediate inactivation of Sec21p.
Anterograde Transport of a Subset of Secreted Proteins (i.e., HSP150) Is Not Affected in the sec21 ts Mutants
Rather than limit our analysis of transport defects in the
sec21 ts mutants to specific known proteins, we took an alternative approach to assess general secretion defects in
these and other sec mutants. Most proteins secreted from
yeast cells remain in the periplasmic space or are associated with the cell wall; however, a few proteins are efficiently secreted into the growth medium (Robinson et al.,
1988). We took advantage of this to address (a) whether
some proteins might escape the strong ER block observed for CPY and
-factor in the sec21 ts mutants and (b),
whether COPII mutants are competent for secretion of
some cargo molecules; if so, this might suggest that COPI
and COPII can both form cargo-containing anterograde
ER to Golgi transport vesicles. Whole cells were incubated at 37°C for 30 min, pulse-labeled, and chased for 30 min. Cells and media were separated by centrifugation,
and proteins in the media were precipitated with TCA and
resolved by SDS-PAGE. At least nine protein bands were
apparent in media from wild type cells (Fig. 3, lane 1). No
media proteins were secreted from either sec18-1 (NSF) or
sec23-1 (COPII) mutants (Fig. 3, lanes 2 and 3), nor were
any media proteins secreted from a sec1-1 mutant (Fig. 3,
lane 5), which blocks docking/fusion of TGN-derived
secretory vesicles with the plasma membrane (Novick and
Schekman, 1979
; Bennett, 1995
). Interestingly, in the
sec21-3 mutant, some media proteins were efficiently secreted (Fig. 3, lane 4, arrows) while others were blocked
(
). Identical results were obtained after either a 1 or 60 min preincubation of the sec21-3 mutant at either 37 or
38°C (data not shown). This analysis thus revealed that secretion of a distinct subset of proteins was unaffected in
the COPI mutant. These proteins trafficked via the normal
secretory route, because (a) COPII, NSF, and Sec1p were
absolutely required for their secretion (which also implied
that COPII is required for all anterograde ER to Golgi
transport), and (b) all media proteins secreted from wild
type and sec21-3 cells were precipitable with Con A-sepharose (data not shown), demonstrating that they received
glycosyl modifications typical of proteins transiting the
secretory pathway.
One particularly abundant protein secreted from sec21-3
cells (Fig. 3, lane 4, top arrow) migrated at a molecular
mass similar to that observed for a previously characterized media protein, HSP150 (Russo et al., 1992); subsequent analysis with HSP150 antiserum confirmed that
HSP150 was the identity of this protein (see below). HSP150 is extensively O-glycosylated, and its synthesis increases ~6-fold upon shift to high temperature (Russo et al.,
1992
). Glycosylation in the ER causes this 414-amino acid
protein to migrate at ~80-100 kD. After carbohydrate
modification in the Golgi, HSP150 migrates at ~150 kD
(Russo et al., 1992
; Jamsa et al., 1994
). HSP150 secretion was assayed by immunoprecipitating HSP150 from intracellular (I) and extracellular (E) fractions of cells which
had been pulse-labeled and chased at 37°C as described.
CPY was also immunoprecipitated from the intracellular
fraction to ensure that each mutant assayed exhibited an
appropriate secretion block (data not shown). This analysis revealed that >95% of HSP150 was secreted from wild
type cells and from sec21-3 cells (Fig. 4, top panel) with
nearly identical kinetics (data not shown). Even under severely restrictive conditions (i.e., preincubation at 38°C for
60 min before labeling), >95% of HSP150 was secreted
from all new sec21 ts mutants (data not shown), including
sec21-5 which exhibits growth defects at <30°C (Table II)
and likely represents a nearly null allele. >95% of HSP150
was also secreted from two other COPI mutants, ret2-1
(
-COP) and sec33-1 (
-COP) (Fig. 4 top panel), each of
which exhibits defects in anterograde transport of CPY
(data not shown; Cosson et al., 1996
; Wuestehube et al.,
1996
). All other COPI mutants tested (ret1-1,
-COP;
sec27-1,
-COP; and ret3-1,
-COP) also secreted >95%
of HSP150. Thus the lack of an anterograde transport defect for HSP150 was not restricted to the new sec21 ts mutants but was observed for other COPI mutants as well.
70-80% of HSP150 was also secreted from sec20-2 and
bet1-1 mutants (Fig. 4, middle panel) under conditions where
anterograde transport of other proteins (i.e., CPY) was
blocked (data not shown; Novick et al., 1980
; Newman and
Ferro-Novick, 1987
); Sec20p has recently been implicated in retrograde Golgi-ER transport (Lewis and Pelham,
1996
), while the precise function of Bet1p, a v-SNARE-
like protein (Newman et al., 1992
), is unknown.
In sec18-1, sec23-1, and sec1-1 mutants, HSP150 export
was completely blocked (Fig. 4, middle/lower panels).
HSP150 was also retained intracellularly in sec22-1 and
sec16-1 mutants (Fig. 4, middle panel and data not shown);
Sec22p functions as an ER to Golgi v-SNARE (Lian and
Ferro-Novick, 1993; Sogaard et al., 1994
), and Sec16p has
been proposed to act as a scaffold for COPII vesicle proteins (Espenshade et al., 1995
). Importantly, in sec23-1/ sec21-3 and sec1-1/sec21-3 double mutants, HSP150 remained inside the cell and migrated at the same molecular
mass seen for the sec23-1 and sec1-1 single mutants, respectively (Fig. 4, lower panel). Based on mobility (Fig. 4,
top panel) and reimmunoprecipitation with antisera to
1,3 mannose (data not shown), HSP150 also appeared to
be appropriately Golgi-modified in the new sec21 ts and
other COPI mutants. These observations demonstrate that
HSP150 secretion from COPI mutants followed the normal secretory route and did not result from an aberrant
bypass of secretory compartments like the Golgi. Therefore, while HSP150 secretion was abolished in COPII,
Sec22p (ER-Golgi v-SNARE), Sec18p (NSF), and Sec1p
mutants, it was efficiently secreted from all COPI mutants
as well as from the putative retrograde transport mutants
sec20-2 and bet1-1.
Invertase Is Underglycosylated but Efficiently Secreted from the sec21 ts Mutants
Given the striking result that several media proteins were
efficiently secreted from sec21 ts and other COPI mutant
cells, we next decided to analyze secretion of the well characterized periplasmic enzyme invertase (encoded by the
SUC2 gene). Previous analysis of the original sec21-1 mutant
indicated that invertase secretion was partially blocked in
this mutant (Novick et al., 1980). Subsequent analysis of
sec21-1 and sec33-1 also yielded partial invertase secretion
phenotypes (Wuestehube et al., 1996
). However, these experiments were done using invertase enzyme assays following derepression of the SUC2 promoter, which necessitated long incubations in low glucose media and long
incubations at nonpermissive temperature, conditions which
could yield secondary effects that might mask the true
behavior of invertase in COPI mutants. To avoid these difficulties, we performed pulse-chase analysis using a constitutively expressed form of invertase, which eliminated
the need for SUC2 derepression. Invertase contains 9-12
utilized N-linked glycosylation sites which are heterogeneously and extensively modified in the Golgi complex. By
the time it is secreted, invertase migrates as a high molecular mass smear on SDS gels. To assay invertase secretion,
cells were incubated at 37°C, pulse-labeled, chased briefly,
converted to spheroplasts, and separated into intracellular
(I) and extracellular (E) fractions from which invertase
was immunoprecipitated. Unexpectedly, we found that invertase, like HSP150, was efficiently and rapidly secreted
from both sec21-3 and from all aforementioned COPI mutants; this was observed at all restrictive conditions described for CPY and HSP150, even 60 min preincubations
at 38°C (Fig. 5 A and data not shown). Under similar conditions, in sec18-1, sec23-1, sec22-1, and sec1-1 cells, invertase remained entirely intracellular (Fig. 5 A and data not
shown). Surprisingly, invertase secreted from both sec21-3
and ret2-1 migrated neither as the high molecular mass
smear observed in wild type cells nor as the ER-retained, core-glycosylated ladder of bands observed for sec18-1,
but instead as a distinct intermediate form (Fig. 5 A, *).
To observe postER modifications on invertase in sec21-3
and to determine if the shift in molecular mass occurred as
a result of incubation at nonpermissive temperature, cells
were incubated at 26 or 37°C, pulse-labeled, and chased.
Invertase was immunoprecipitated and split into equal aliquots for reimmunoprecipitation with antisera against either invertase, 1,6, or
1,3 mannose linkages. At 26°C, invertase from sec21-3 cells migrated as a high molecular
mass smear similar to wild type cells (Fig. 5 B), indicating
that the aberrant migration in sec21-3 was due to the temperature-conditional defect. However, unlike in sec18-1
cells at 37°C (data not shown), in sec21-3 at 37°C, invertase
was efficiently modified with both
1,6 and
1,3 mannose
(Fig. 5 B). This analysis revealed that (a) invertase secreted from sec21-3 did not bypass Golgi glycosyltransferase activities, and (b) the severe underglycosylation was
most likely due to defects in elongating
1,6 mannosyltransferase activity.
BFA Induces Cargo-selective ER to Golgi Transport Defects
As an independent test of the requirement for COPI in
ER to Golgi transport, we employed the fungal metabolite
BFA and the BFA-sensitive yeast mutant erg6 (ise1). Treatment of mammalian cells with BFA severely perturbs secretory pathway function (Pelham, 1991; Klausner et al., 1992
),
blocking ER export of several secretory proteins (Misumi
et al., 1986
) and inducing redistribution of Golgi enzymes
to the ER (Lippincott-Schwartz et al., 1989
). These events
are thought to occur because BFA inhibits a guanine nucleotide exchange factor which catalyzes the exchange of
GTP for GDP on ARF (Donaldson et al., 1992
; Helms
and Rothman, 1992
). This inhibition causes ARF and thus
coatomer to dissociate from Golgi membranes (Donaldson et al., 1990
, 1991). Not surprisingly, BFA also prevents formation of COPI-coated vesicles in vitro (Orci et al.,
1991
); however, BFA does not affect the membrane association of COPII proteins (Shaywitz et al., 1995
).
The yeast erg6 (ise1) mutant was previously demonstrated to exhibit dramatic growth and anterograde secretory transport defects upon treatment with BFA (Graham
et al., 1993; Vogel et al., 1993
). To observe the immediate
effects of BFA treatment on transport of distinct cargo
proteins from the ER to the Golgi, erg6 cells were incubated in media buffered to pH 7.0 either in the presence or
absence of 100 µg/ml BFA for 2 min, pulse-labeled for 10 min, and then chased for 30 min. Invertase and CPY were
recovered by immunoprecipitation and then subjected to
reimmunoprecipitation with antisera against either the
protein itself or
1,6 mannose linkages. Interestingly, invertase from BFA-treated cells was quantitatively
1,6
mannose modified and migrated at a mobility remarkably
similar to that observed for sec21-3 cells at nonpermissive temperature (Fig. 6 A). HSP150 was also modified with
Golgi-specific carbohydrates in BFA-treated cells (data
not shown). In contrast, BFA treatment resulted in <10%
1,6 mannose modification of CPY (Fig. 6 B). Retrograde flux of Golgi enzymes to the ER was not previously
detected in BFA-treated erg6 cells (Graham et al., 1993
).
Therefore, this analysis suggested that in the presence of
BFA, nearly all of the newly synthesized invertase and
HSP150 reached the Golgi complex, while >90% of CPY
did not.
To confirm that cargo-selective ER to Golgi transport
occurred in the presence of BFA, the subcellular distribution of invertase and CPY was determined. Invertase was
quantitatively modified with Golgi-specific carbohydrates
(Fig. 6 A); however, its secretion, like that of media proteins such as HSP150, was completely blocked after BFA
treatment (data not shown). We thus analyzed the intracellular location of invertase and CPY by differential centrifugation. erg6 cells were converted to spheroplasts, incubated in media buffered to pH 7.0 in the presence of 100 µg/ml BFA for 2 min, and then pulse-labeled and chased.
The lysed spheroplasts were then subjected to differential
centrifugation as described for Fig. 1 C, after which CPY
and invertase were recovered by immunoprecipitation. >90% of CPY was recovered in the 13,000 g pellet (Fig. 6
C), consistent with ER localization (Gaynor et al., 1994).
In contrast, 70% of invertase was found in the 100,000 g
pellet (Fig. 6 C), consistent with localization of invertase
to the Golgi (Gaynor et al., 1994
; Harris and Waters, 1996
;
Fig. 1 C). Together, these observations indicate that treatment of erg6 cells with BFA yielded nearly identical cargospecific ER to Golgi transport defects as those observed
for the new sec21 ts mutants: CPY was blocked in the ER,
while invertase was efficiently transported to the Golgi. Because secretion was also blocked in BFA treated cells,
this analysis also suggests that BFA acts at multiple sites in
the yeast secretory pathway, consistent with previous observations in mammalian cells (Pelham, 1991
; Klausner et
al., 1992
; Strous et al., 1993
; Torii et al., 1995
).
CPY-Invertase Hybrids Exit the ER and Are Sorted to the Vacuole in sec21 ts Mutants
Thus far, our analysis has shown that the sec21 ts mutations (and BFA treatment) prevented ER export of some proteins while others were entirely unaffected. We next decided to address which of these two events is dominant; for instance, does a protein like CPY contain a signal for ER retention in this mutant, or might invertase contain a signal for its active ER export even under conditions where other proteins are blocked?
We therefore examined the behavior of a series of CPYinvertase fusion proteins. These fusion proteins use the
CPY promoter and contain either 50, 156, or 433 amino
acids of CPY fused to full-length invertase (Fig. 7 A). Each
fusion protein has previously been shown to be efficiently
targeted to the vacuole and proteolytically cleaved in a
vacuolar hydrolase-dependent manner at the CPY-invertase junction (Johnson et al., 1987). Cells harboring these
fusion proteins were incubated at 37°C, pulse-labeled, and
chased. Fusion proteins were recovered by immunoprecipitation with invertase antiserum and treated with endoglycosidase H, which removes NH2-linked sugars and reduces
invertase to a single band, to reveal the vacuolar processing event. In sec18-1, the fusion proteins migrated at the
predicted molecular mass for the intact CPY-invertase hybrids (Fig. 7 B,
), indicating that they were retained in the
ER and not transported to the vacuole. In both wild type
and sec21-3 cells at nonpermissive temperature, however,
only the cleaved, vacuolar form of the hybrid proteins was
recovered (Fig. 7 B, arrow). This demonstrated that each
of these fusion proteins, even CYI-433 (which contains
~80% of CPY), escaped the sec21-3 ER block and was
subsequently targeted to the vacuole and proteolytically processed. Interestingly, this analysis also revealed that, as with Golgi to plasma membrane transport, transport and
sorting from the Golgi to the vacuole was unaffected in
sec21-3.
The cis-Golgi Is Disrupted in the sec21 ts Mutants
In mammalian cells, disruption of COPI function leads
to significant morphological changes in Golgi structure
(Donaldson et al., 1990; Guo et al., 1994
; Kreis et al., 1995
).
Our observation that invertase was significantly underglycosylated in the sec21 ts mutants suggested that Golgi
structure might also be physically disrupted upon shift of
these yeast COPI mutants to nonpermissive temperature.
We performed indirect immunofluorescence experiments to look at the distribution of two Golgi resident proteins,
Och1p (earliest cis-Golgi; Nakanishi-Shindo et al., 1993
;
Gaynor et al., 1994
) and Mnn1p (medial/trans-Golgi; Graham and Emr, 1991
; Graham et al., 1994
). For Och1p, we
used an HA epitope-tagged form of the protein which has
previously been shown to substitute functionally for and
exhibit near-identical fractionation characteristics with native Och1p (Harris and Waters, 1996
). Cells were incubated for 30 min at 26 or 37°C, and then fixed and prepared for indirect immunofluorescence with either the
12CA5 monoclonal antibody directed against the HA
epitope or an affinity-purified anti-Mnn1p antibody. In wild
type cells at 26 and 37°C (Fig. 8 A and data not shown) and
in sec21-3 cells at 26°C (Fig. 8 B), Och1p localized to punctate structures (~5-10/cell in a focal plane) characteristic
of the yeast Golgi. However, in sec21-3 at 37°C (Fig. 8 C),
Och1p exhibited a much more diffuse staining pattern,
with some faint punctate spots visible but with most of the
signal dispersed through the cell, perhaps staining either
small vesicular or tubulo-vesicular structures. This staining
was specific to Och1p, since cells not harboring the HAOch1p plasmid did not stain with the 12CA5 antibody (data not shown). The diffuse staining in sec21-3 at 37°C
did not result from decreased protein expression, transport to and/or degradation in the vacuole, or significant accumulation of the protein in the ER, since immunoblot
analysis yielded a similar signal and subcellular fractionation pattern for HA-Och1p in both wild type and sec21-3
cells at 26 and 37°C (Fig. 1 C and data not shown). After
longer preincubations at 37°C (
1 h), ~10% of sec21-3
cells also exhibited modest ER staining (data not shown), suggesting that appearance of Och1p in the ER may represent a secondary, more terminal phenotype in this mutant.
Interestingly, in contrast to Och1p, Mnn1p exhibited a
punctate Golgi staining pattern in both wild type cells at
26 or 37°C (Fig. 8 D and data not shown) and in sec21-3
cells at either 26 (Fig. 8 E) or 37°C (Fig. 8 F), with ~8-15
distinct punctate structures observed per cell in a focal
plane. Therefore, while the sec21-3 mutation caused a dramatic change in distribution of a cis-Golgi protein, a medial/trans-Golgi protein appeared to be unaffected. This
suggests that COPI plays a role in maintenance of early
but not late Golgi structure.
Recent controversy over the precise and primary role of
the COPI/coatomer complex has yielded several speculative models to explain how COPI may function both in retrograde and anterograde ER/Golgi transport (Pelham, 1995;
Aridor and Balch, 1996
; Lewis and Pelham, 1996
; Schekman and Orci, 1996
). These models have remained speculative in part because of uncertainty over whether existing
yeast COPI mutants were simply weak alleles which may partially allow both retrograde and anterograde transport
to continue. Thus it has been difficult to establish a primary phenotype associated with loss of COPI function in
yeast. We have isolated and anlyzed multiple new temperature-conditional sec21 (
-COP) mutants (represented by
sec21-3 in this study). Surprisingly, even though these mutants were not identifed by screening for directional transport defects, each new mutant rapidly and completely
blocked ER export of some cargo proteins. However, further analysis of these mutants revealed that (a) other
cargo proteins were unaffected in these and other COPI
mutants, (b) these cargo proteins may contain active recognition determinants directing their packaging into COPII
vesicles, (c) anterograde transport to the cell surface and
to the vacuole is not affected in the sec21 ts mutants, and
(d) these COPI mutations cause significant perturbation
of early but not late Golgi structure and function. We also
found that BFA induced nearly identical cargo-specific
ER to Golgi transport defects as observed in the sec21 ts
mutants. Together, our results are consistent with a model
whereby COPI does not act directly in anterograde transport but instead plays a critical role in regulated retrograde transport between the Golgi and the ER, and that
this role indirectly influences ER to Golgi transport of selected cargo proteins.
Model for an Indirect Requirement for COPI in ER to Golgi Transport of Some but Not All Cargo
In the transport model we propose (Fig. 9), cargo destined
for export from the ER would first be concentrated and
recruited into budding COPII-coated vesicles (COPIICVs) by cargo-specific "receptors" or transport factors.
All cargo would then travel to the earliest cis-Golgi (Och1p)
compartment in COPII-CVs. Although our model implies
that this step is mediated by a single type of COPII-CV, our
data do not rule out the possibility that multiple types of
COPII-CVs may carry distinct cargo to the cis-Golgi. After
COPII vesicles fuse with the cis-Golgi, transport factors (i.e., receptors) for cargo such as CPY and -factor would
cycle back to the ER in COPI-CVs to be reutilized for further rounds of traffic. The absence of Sec21p/COPI function would result in rapid depletion of receptor pools in
the ER, causing anterograde transport of cargo dependent
on these receptors to cease. Without COPI, for instance,
these receptors may not be recognized in the cis-Golgi
and, like other proteins dependent on COPI for retrieval to the ER (i.e., invertase-Wbp1 fusion proteins; Letourneur et al., 1994
), be directed to the vacuole and degraded.
Anterograde transport of cargo insensitive to the COPI
mutant block (i.e., HSP150 and invertase) could be maintained by one of three putative mechanisms. One possibility is that these cargo do not require sorting receptors and
thus are not sensitive to a block in retrieval of these proteins. Second, HSP150 and invertase may use receptor(s) with sufficiently high rates of expression such that their de novo synthesis allows efficient packaging of these cargo
into COPII vesicles. Third, HSP150 and invertase may interact with receptor(s) which do recycle but which do not
absolutely require Sec21p/COPI for recognition in the
cis-Golgi and retrieval to the ER. The latter two possibilities seem most plausible (Fig. 9), since evidence for cargoselective transport in other yeast mutants already exists
(Schimmoller et al., 1995
), and proteins like invertase and
HSP150 are likely to be actively packaged into COPIICVs. If these receptors do recycle, how might this continue in the sec21 ts mutants and BFA treated cells? Disruption of COPI function in mammalian cells has been
shown to induce formation of Golgi-ER tubules that lead
to unregulated transport of Golgi proteins to the ER (Lippincott-Schwartz et al., 1990
; Kreis et al., 1995
). One speculative possibility is that normally the invertase/HSP150 "receptors" may or may not recycle in COPI vesicles; however, under mutant/COPI-deficient conditions, tubule formation
could allow return of these but not other "receptor" proteins.
Despite the cargo-selective anterograde transport defects observed in the sec21 ts mutants, models supporting a
direct role for COPI in anterograde ER to Golgi transport
seem unlikely given the following contradictory observations. First, there is no evidence in favor of COPII-independent ER to Golgi transport (Fig. 3; Barlowe et al., 1994;
Aridor et al., 1995
; Bednarek et al., 1995
; Doering and
Schekman, 1996
), and COPI and COPII are unlikely to
coat the same transport vesicle (Barlowe et al., 1994
; Aridor et al., 1995
; Bednarek et al., 1995
). Second, while our
results are most consistent with an ER block for both CPY
and
-factor in the sec21 ts mutants (Figs. 1 and 2),
-factor is packaged exclusively into COPII-CVs in in vitro ER
budding assays (Bednarek et al., 1995
), and these vesicles
continue to bud even when COPI is washed off the membrane (Barlowe et al., 1994
). Finally, no soluble cargo proteins have thus far been found within ER-budded COPI
vesicles (Bednarek et al., 1995
).
Finally, if our model is correct, then other mutants defective for the retrograde transport process might also be
expected to exhibit cargo-specific anterograde transport
defects. Recently, Lewis and Pelham showed that Sec20p,
an ER membrane protein, and Ufe1p, a candidate retrograde ER t-SNARE, are likely to participate in retrieval of
dilysine-containing proteins (Lewis and Pelham, 1996). In
the SNARE hypothesis (Rothman, 1994
), targeting of
transport vesicles to an appropriate acceptor compartment
is mediated by the interaction of a v-SNARE protein on
the vesicle with its cognate t-SNARE protein on the target
membrane. Interestingly, the sec20-2 mutant as well as the
bet1-1 v-SNARE mutant also exhibited cargo-specific ER
to Golgi transport defects (Fig. 4). These results suggest that Bet1p might act as a retrograde v-SNARE involved in
targeting of recycling Golgi to ER vesicles with Ufe1p on
the ER. Recently, rat homologs of Sec22p and Bet1p
(rsec22 and rbet1) were identified (Hay et al., 1996
). Surprisingly, these two proteins localized to distinct subcellular
compartments: rsec22 localized to the ER, consistent with
its role as an anterograde COPII-CV v-SNARE, while rbet1
localized to the Golgi. Together, these data support a role for
Bet1p as a candidate v-SNARE for retrograde COPI-CVs.
Active Cargo Concentration into COPII Vesicles via cis-acting Sorting Determinants
Essential to our model is the existence of cargo "receptors" and whether such receptors might actively recruit
and concentrate cargo into budding COPII-CVs. If this is
the case, then the ER would provide the first point of discrimination between distinct cargo proteins. Two candidate cargo receptor families have already been identified.
One, the p24 family of membrane proteins, has been detected in both COPI- and COPII-CVs (Schimmoller et al., 1995; Stamnes et al., 1995
), and the cytoplasmic domains
of these proteins have recently been shown to bind COPI
proteins in vitro (Fiedler et al., 1996
). Interestingly, yeast
strains deleted for the first member of this family to be
cloned, EMP24, exhibit a delay in anterograde transport
of some but not all cargo proteins (Schimmoller et al., 1995
).
However, unlike the sec21-3 mutant,
emp24 strains affect
transport of invertase but not CPY; this also supports the
idea that packaging of proteins like invertase into COPII
vesicles requires "receptors" or transport factors (Fig. 9).
It has also been suggested that lectin-like membrane proteins may comprise another candidate cargo "receptor"
family (Fiedler and Simons, 1995
; Schekman and Orci,
1996
). In yeast, a KKXX-containing protein exhibiting lectin homology (Emp47p) has been identified and shown to
cycle between the Golgi and ER in a COPI-dependent
manner (Schroder et al., 1995
; Lewis and Pelham, 1996
).
emp47 strains do not exhibit transport defects for any proteins tested (Schroder et al., 1995
). However, BLAST
(Basic Local Alignment Search Tool) analysis of the recently completed yeast genome sequence revealed a protein with significant homology to EMP47, suggesting that
the two proteins may function similarly or even substitute
for each other. Given that HSP150 and invertase (two of
the proteins observed to be unaffected by the sec21-3 transport block) are both extensively glycosylated, it
seems plausible that a lectin-like protein may play a role in
their selective transport out of the ER.
Concentration of cargo into COPII-CVs has also been
observed in both yeast and mammalian cells; however, the
mechanism for this event is unknown, and whether this is
an active process has been postulated but not yet demonstrated (Aridor and Balch, 1996; Schekman and Orci,
1996
). In support of this being an active process, it is interesting to note that in the sec21 ts mutants, newly synthesized CPY and
-factor did not escape the ER, even after long incubations at nonpermissive temperature. Thus these
proteins were not simply swept along (in a "bulk flow"
manner) into anterograde vesicles along with invertase and
HSP150, both of which were efficiently transported out of
the ER in a COPII-dependent manner (Figs. 4 and 5 and
our unpublished observations). In contrast, CPY-invertase hybrid proteins, like invertase, were efficiently transported out of the ER under conditions where CPY transport was blocked (Fig. 7). Thus CPY is not likely to be "actively retained" in the ER in the COPI mutant. Previous
work demonstrated that HSP150 can act as a "carrier,"
which, when fused to nonyeast proteins, directs their
transport through the media (Simonen et al., 1994
). Together, these observations suggest that both invertase and
HSP150 may contain active sorting determinants mediating their packaging into anterograde COPII-CVs. Interestingly, a candidate motif for such a determinant may already exist. Previous work demonstrated that a single
point mutation in the NH2-terminal region of invertase resulted in ER accumulation of this "S2" mutant invertase in
wild type cells (Schauer et al., 1985
). We are currently investigating whether this region of invertase is also responsible for its COPI-independent ER to Golgi transport.
COPI Is Required for Early but Not Late Golgi Structure and Function
Our data also indicate that COPI plays a critical role in
maintenance of early Golgi function and structure in yeast.
In the sec21-3 mutant at nonpermissive temperature, immunofluorescence revealed that the cis-Golgi was noticeably perturbed, while the medial/trans Golgi appeared
normal (Fig. 8). We also observed striking underglycosylation of invertase in all COPI mutants, most notably sec21-3
and ret2-1 (Fig. 5). Because initial 1,6 and
1,3 mannosyltransferases were still functional, this was likely due to a
defect in elongating
1,6 mannosyltransferase activity.
One interpretation of these data is that early Golgi compartments no longer retain functional integrity because
they are either "mixed," dissociated, or vesiculated in this
mutant, thereby causing inactivation of enzymes whose activities are dependent on a specific environment. "Mixing"
might occur if COPI-mediated recycling of mannosyltransferases is required for maintenance of functionally distinct
early Golgi compartments. For instance, loss of COPI
function might cause a more random distribution of the
mannosyltransferases, yielding the diffuse Och1p staining pattern and perhaps accounting for inactivation of the
elongating
1,6 mannosyltransferase. Alternatively, COPI
may act structurally to stabilize the cis-Golgi; in this case,
loss of COPI function could result in either mixing or
rapid dissociation/vesiculation of early Golgi membranes.
The yeast Golgi is difficult to detect at the ultrastructural
level, and EM analysis of sec21-3 shifted for 1 h to nonpermissive temperature did not reveal significant accumulation of vesiculated or tubulated membranes (our unpublished observations). However, consistent with this interpretation of our data, rapid and dramatic vesiculation of Golgi
membranes was observed by EM in a mammalian
-COP
mutant after incubation at nonpermissive temperature (Guo
et al., 1994
).
Despite early Golgi aberrancies in the sec21-3 mutant,
this organelle remained remarkably competent for trafficking. Neither HSP150 nor invertase bypassed this organelle en route to the plasma membrane: both proteins
were Golgi modified in the sec21-3 mutant, and in a sec213/sec23-1 double mutant, HSP150 transport was blocked (Figs. 4 and 5). Later Golgi compartments also seemed to
be unaffected in the sec21-3 mutant, both structurally and
functionally; indeed, both TGN to plasma membrane and
TGN to vacuole transport steps occurred normally and involved factors that suggested that the trans-Golgi retained
wild type characteristics (i.e., secretion required Sec1p,
and transport to the vacuole required a vacuolar sorting
determinant). If COPI is required for regulated anterograde vesicular transport through the Golgi, it is possible
that fusion of Golgi cisternae in the sec21 ts mutants could
mask this requirement by creating a syncitium allowing
(unregulated) anterograde transport to continue. However, given the distinct localization patterns of Och1p and
Mnn1p and the fact that, with the exception of the elongating 1,6 mannosyltransferase, Golgi function appears
nearly normal in the sec21 ts mutants, this seems unlikely.
In summary, we have provided evidence that COPI is not likely to act directly in anterograde secretory transport but instead, along with other proteins (i.e., Sec20p and Bet1p), is more likely to be required for recycling of transport factors required to package selected cargo into COPII vesicles in the ER. Future identification and characterization of both these transport factors as well as sorting determinants in cargo proteins directing their packaging into COPII vesicles will provide additional mechanistic insight into this essential sorting process.
.
Please address all correspondence to Scott Emr, Department of Biology, the Division of Cellular and Molecular Medicine, and the Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0668. Tel.: (619) 534-6462; Fax.: (619) 534-6414.We thank members of the Emr lab for many helpful discussions during the course of this work, especially Beverly Wendland, Chris Burd, and Matthew Seaman for also critically reading the manuscript, and Eden Estepa for excellent technical assistance. We thank Randy Schekman, Rainer Duden, Pierre Cosson, Francois Letourneur, Marja Makarow, Sandy Harris, Gerry Waters, Todd Graham, David Levin, Chris Kaiser, and Susan Ferro-Novick for providing strains, plasmids, and/or antisera.
This work was supported by grants from the National Institutes of Health (GM32703 and CA58689) to S.D. Emr. S.D. Emr is supported as an Investigator of the Howard Hughes Medical Institute.
ALP, alkaline phosphatase; ARF, ADP-ribosylation factor; BFA, Brefeldin A; CPS, carboxypeptidase S; CYP, carboxypeptidase Y; CV, coated vesicle; HA, hemagglutinin; ts, temperature sensitive.