Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637
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Abstract |
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Golgi stacks are often located near sites of "transitional ER" (tER), where COPII transport vesicles are produced. This juxtaposition may indicate that Golgi cisternae form at tER sites. To explore this idea, we examined two budding yeasts: Pichia pastoris, which has coherent Golgi stacks, and Saccharomyces cerevisiae, which has a dispersed Golgi. tER structures in the two yeasts were visualized using fusions between green fluorescent protein and COPII coat proteins. We also determined the localization of Sec12p, an ER membrane protein that initiates the COPII vesicle assembly pathway. In P. pastoris, Golgi stacks are adjacent to discrete tER sites that contain COPII coat proteins as well as Sec12p. This arrangement of the tER-Golgi system is independent of microtubules. In S. cerevisiae, COPII vesicles appear to be present throughout the cytoplasm and Sec12p is distributed throughout the ER, indicating that COPII vesicles bud from the entire ER network. We propose that P. pastoris has discrete tER sites and therefore generates coherent Golgi stacks, whereas S. cerevisiae has a delocalized tER and therefore generates a dispersed Golgi. These findings open the way for a molecular genetic analysis of tER sites.
Key words: apparatus, Golgi; endoplasmic reticulum; Pichia; Saccharomyces; microtubules ![]() |
Introduction |
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THE transitional ER (tER)1 is a specialized ER subdomain at which proteins destined for the Golgi
apparatus are packaged into transport vesicles (Palade, 1975). tER sites are defined by the presence of
COPII vesicles, which carry secretory cargo out of the ER
(Kuehn and Schekman, 1997
). Vertebrate cells contain
multiple tER sites, and the COPII components Sec23p,
Sec13p, and Sar1p have been localized to these sites (Orci
et al., 1991
; Kuge et al., 1994
; Shaywitz et al., 1995
; Paccaud et al., 1996
; Tang et al., 1997
). Although tER sites often display an elaborate architecture (Bannykh and Balch,
1997
), the mechanisms that generate these structures are
still mysterious.
In many cell types, ranging from algae to pancreatic acinar cells, some or all of the tER sites are directly apposed
to the cis-face of Golgi stacks (Whaley, 1975; Farquhar
and Palade, 1981
). Such morphological data led early investigators to propose the cisternal progression model for
Golgi function (Beams and Kessel, 1968
; Morré, 1987
). In
this view, a new cis-Golgi cisterna forms by the fusion of
membranes derived from the tER. The cisterna then
progresses through the stack to the trans face, where it fragments into secretory vesicles. However, this model
failed to explain how resident Golgi proteins maintain a
polarized distribution across the stack, or why vesicles appear to bud from all Golgi cisternae. As an alternative, it
was proposed that the Golgi consists of a series of stable
compartments, and that transport vesicles carry the secretory cargo from one compartment to the next (Dunphy and Rothman, 1985
; Farquhar, 1985
; Rothman and
Wieland, 1996
). According to the stable compartments
model, the juxtaposition of tER and Golgi structures
might facilitate vesicular trafficking, but this juxtaposition
would not reflect a continual formation of new Golgi cisternae.
Recently, cisternal progression has been the subject of
renewed interest (Bonfanti et al., 1998). An updated
model postulates that cisternal progression is coupled to
retrograde vesicular transport of resident Golgi proteins
(Schnepf, 1993
; Mironov et al., 1997
; Bannykh and Balch,
1997
; Love et al., 1998
). This "cisternal maturation"
model can account for many experimental findings, including the polarity of the Golgi stack and the existence
of Golgi-derived transport vesicles (Glick and Malhotra,
1998
; Pelham, 1998
).
Can the cisternal maturation model help explain why
Golgi structure varies so dramatically between cell types
(Mollenhauer and Morré, 1991)? If the Golgi is indeed an
outgrowth of the tER, then some of the variation in Golgi
structure may be due to differences in tER organization.
To test this idea, we examined two budding yeasts that display different Golgi morphologies. Pichia pastoris has
stacked Golgi organelles (Gould et al., 1992
; Glick, 1996
).
By contrast, the Saccharomyces cerevisiae Golgi rarely
shows a stacked structure, but exists primarily as individual cisternae dispersed throughout the cytoplasm (Preuss
et al., 1992
). Here we demonstrate that tER organization
is also fundamentally different in the two yeasts. P. pastoris contains discrete tER sites. In S. cerevisiae, the entire
ER network apparently functions as tER. We suggest that
fixed tER sites in P. pastoris produce coherent Golgi
stacks, whereas the delocalized tER in S. cerevisiae produces a dispersed Golgi. Thus, tER organization may be a
key determinant of Golgi structure in budding yeasts.
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Materials and Methods |
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Strains and Plasmids
Yeast strains and plasmids are listed in Table I. Experiments with S. cerevisiae were carried out using strain DBY1034 and derivatives thereof.
DBY1034 was transformed with plasmid pOH to obtain expression of
Och1p-HA. Strain DBY1034-S13G, in which the endogenous SEC13 gene
has been replaced with SEC13-GFP, was constructed as follows. The
URA3 cassette from pUC1318-URA3 (Bénédetti et al., 1994) was excised
with HindIII, blunted, and inserted into the SspI site of pUC19 (Yanisch-Perron et al., 1985
) to create pUC19-URA3. An 1,166-bp HincII-HindIII
fragment spanning the 3' portion of SEC13 was then amplified by PCR
from S. cerevisiae genomic DNA and inserted into the corresponding sites
in pUC19-URA3. The resulting plasmid was mutagenized using the
QuikChange kit (Stratagene Inc.) to replace the SEC13 stop codon with a
SnaBI site. The EGFP gene was excised from pEGFP-1 (Clontech) with
BamHI and NotI, blunted, and inserted into this SnaBI site. The resulting construct was linearized at the unique BstEII site and integrated into the
chromosomal SEC13 gene. This "pop-in" strain was then plated on 5-fluoroorotic acid (Rothstein, 1991
) to select for the "pop-out" recombinant
strain DBY1034-S13G. Strain DBY1034-S23G, in which the endogenous
SEC23 gene has been replaced with SEC23-GFP, was constructed in the
same manner, beginning with the insertion into pUC19-URA3 of a
1,120-bp AflII-HindIII fragment spanning the 3' portion of SEC23. Similar strategies were used to replace the endogenous SEC24 and SEC31
genes with EGFP fusion genes. To construct strain DBY1034-S12m, in
which the endogenous SEC12 gene has been replaced with SEC12-myc, a
1,685-bp HincII-XbaI fragment spanning the 3' portion of SEC12 was inserted into pUC19-URA3; a c-myc epitope sequence was then inserted
before the stop codon using the QuikChange kit, and the resulting
construct was linearized with SspI for "pop-in" integration into the
SEC12 locus.
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Experiments with P. pastoris were carried out using the prototrophic
wild-type strain PPY1 or the isogenic his4 arg4 auxotroph PPY12 and derivatives thereof (Table I). General methods for growth and transformation of P. pastoris have been described elsewhere (Sears et al., 1998).
Strain PPY12-OH, which expresses Och1p-HA, was constructed as follows. A modified gene encoding tagged Och1p (Chapman and Munro,
1994
) was excised from plasmid pOCHFT (a gift of Sean Munro, Medical
Research Council, Cambridge, UK) by digesting at an upstream HindIII
site (sequence including the start codon: AAGCTTAGAGATCATG),
blunting, and digesting at an XbaI site immediately after the stop codon.
This fragment was subcloned into pIB2 (Sears et al., 1998
) that had been
digested with SmaI and SpeI. The resulting plasmid was digested with
BstEII and PstI, and the corresponding BstEII-PstI fragment from pOH
was inserted to create pIB2-OH, in which a gene encoding triple-hemagglutinin epitope (HA)-tagged Och1p is downstream of the strong constitutive GAP promoter. pIB2-OH was linearized with SalI and integrated
into the his4 locus of PPY12. Strain PPY12-S13G, in which the endogenous SEC13 gene has been replaced with SEC13-GFP, was constructed
as follows. pSG464 (Gould et al., 1992
) was digested with SnaBI and NdeI,
blunted, and religated to yield pUC19-ARG4. This plasmid lacks the
PARS2 sequence present in pSG464, and therefore can only transform P. pastoris by integration. A 461-bp HindIII-NsiI fragment spanning the 3'
end of SEC13 was then amplified by PCR from P. pastoris genomic
DNA and inserted into pUC19-ARG4 that had been cut with HindIII and
PstI. This plasmid was mutagenized to replace the SEC13 stop codon
with a SnaBI site, and the EGFP gene was inserted into this site as described above. The resulting construct was linearized at the unique MscI
site and integrated into the chromosomal SEC13 gene, yielding an intact
SEC13-GFP fusion gene plus a 3' fragment of authentic SEC13. A similar strategy was used to create strain PPY12-S12m: a 1,328-bp BamHI
fragment spanning the 3' end of SEC12 was inserted into pUC19-ARG4;
a c-myc epitope sequence was then inserted just upstream of the HDEL
sequence, and the resulting construct was linearized with XhoI for integration into the SEC12 locus. To express myc-tagged S. cerevisiae Mnt1p
(Chapman and Munro, 1994
) in P. pastoris, the gene encoding tagged
Mnt1p was excised from pYMT1BS (a gift of Sean Munro) and subcloned into pOW3, a P. pastoris episomal vector that contains the GAP promoter (Gould et al., 1992
; Waterham et al., 1997
; O.W. Rossanese, unpublished observations).
Plasmid pAFB584, which encodes glutathione S-transferase fused to the NH2-terminal acidic domain of S. cerevisiae Sec7p (up to the NaeI site in the coding sequence), was provided by Alex Franzusoff (University of Colorado, Denver, CO).
Nocodazole Treatment
P. pastoris cultures were grown overnight at 30°C to an OD600 of ~0.15 in 1% yeast extract, 2% peptone, 2% glucose, 20 mg/liter adenine sulfate, 20 mg/liter uracil, 50 mM sodium maleate, pH 5.5. Half of a culture then received nocodazole at a final concentration of 15 µg/ml, diluted from a 10 mg/ml stock solution in water-free dimethyl sulfoxide. As a control, the other half of the culture received only dimethyl sulfoxide. Incubation was continued for up to 2.5 h, and the cells were fixed for microscopy.
Antibodies for Immunofluorescence
Anti-HA monoclonal antibody (16B12; Berkeley Antibody Co.), anti-
myc monoclonal antibody (9E10; Boehringer Mannheim Biochemicals) and anti-green fluorescent protein (GFP) monoclonal antibody (a mixture from clones 7.1 and 13.1; Boehringer Mannheim Biochemicals) were
used at 5 µg/ml; in cells not expressing a tagged protein, each antibody
gave only a faint background signal. The anti-GFP antibody was used to
supplement the endogenous GFP signal, which is diminished by the immunofluorescence procedure. Anti--tubulin monoclonal antibody (KMX-1;
Boehringer Mannheim Biochemicals) was used at 1 µg/ml. The following
rabbit polyclonal antibodies were used. Anti-Pdi1p serum (a gift of Peter
Walter, University of California, San Francisco, CA; originally produced
by Victoria Hines, Chiron Corp., Emeryville, CA), raised against SDS-PAGE-purified S. cerevisiae Pdi1p, was used at a dilution of 1:350. Alex
Franzusoff generously provided an antiserum raised against a fusion between
-galactosidase and the NH2-terminal portion of Sec7p (Franzusoff et al., 1991
); this antibody was used at a dilution of 1:500. Oregon green
488-conjugated goat anti-mouse IgG and Texas red-X-conjugated goat
anti-rabbit IgG (Molecular Probes, Inc.) were used at 20 µg/ml.
Controls were performed (not shown) to check the specificity of the polyclonal antibodies. In the case of anti-Pdi1p, the immunofluorescence signal could be quenched by preincubating with a protein fragment (a gift of Robert Freedman, University of Kent at Canterbury, UK) comprising the COOH-terminal 308 residues of S. cerevisiae Pdi1p. The anti-Sec7p antibody specifically recognized a glutathione S-transferase-Sec7p fusion protein on an immunoblot of an extract from Escherichia coli cells expressing this protein.
Many rabbit antisera contain traces of reactivity against -1,6-mannose
linkages, and because S. cerevisiae and P. pastoris both add
-1,6-mannose
residues to glycoproteins transiting through the Golgi (Orlean, 1997
;
Trimble et al., 1991
), these contaminating antibodies give a spurious labeling of Golgi structures (not shown). With S. cerevisiae, anti-
-1,6-mannose antibodies label Golgi cisternae in immunoelectron microscopy experiments (Preuss et al., 1992
); and when used at very high dilutions for
immunofluorescence, such antibodies primarily label early Golgi elements. Similarly, with P. pastoris, anti-
-1,6-mannose antibodies give a
strong and relatively specific Golgi labeling. In several cases, we observed punctate staining with antisera raised against various COPII proteins, but
this staining was actually due to contaminating anti-
-1,6-mannose antibodies. This problem can be avoided by preincubating a diluted antibody
either with fixed S. cerevisiae cells or with 0.5 mg/ml purified yeast mannan (M-7504; Sigma Chemical Co.).
Electron Microscopy
Thin-section electron microscopy was performed essentially as described
(Kaiser and Schekman, 1990; Gould et al., 1992
). In brief, a 50-ml culture
of yeast cells in rich glucose medium was grown to an OD600 of ~0.5. The
culture was concentrated to a volume of <5 ml with a bottle-top vacuum
filter, and 40 ml of ice-cold 50 mM KPi, pH 6.8, 1 mM MgCl2, 2% glutaraldehyde was added rapidly with swirling. After fixation for 1 h on ice, the
cells were washed repeatedly, and then resuspended in 0.75 ml 4%
KMnO4 and mixed for 1 h at room temperature. The cells were washed,
and then resuspended in 0.75 ml 2% uranyl acetate and mixed for 1 h at
room temperature. Finally, the cells were embedded in Spurrs resin; 50 ml
of yeast culture yielded enough cells for three BEEM capsules. The resin was polymerized for 2 d at 68°C. Sections were stained with uranyl acetate
and lead citrate, and viewed on an electron microscope (100 CXII; JEOL
U.S.A. Inc.).
For immunoelectron microscopy, we used a modification of previously
published methods (Kärgel et al., 1996; Rieder et al., 1996
). A 50-ml log-phase culture was concentrated to a volume of <5 ml (see above) and
fixed by adding 40 ml of 50 mM KPi, pH 6.8, 1 mM MgCl2, 4% formaldehyde, 0.1% glutaraldehyde (aldehydes were EM grade from Ted Pella,
Redding, CA). After 1 h at room temperature, the cells were washed twice
with 20 ml PBS, pH 7.4, containing 0.5% 2-mercaptoethanol, 0.5 mM
o-phenanthroline, 0.25 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, and then once more with 1 ml of the same solution. Treatment with
periodate was omitted because this compound may destroy certain antigens by reacting with amino acid side chains (Clamp and Hough, 1965
). The washed cells were embedded in 100 µl of low-melting-temperature agarose, which was cut into cubic-millimeter blocks and infiltrated for 2 h
at room temperature in 1.7 M sucrose, 25% polyvinylpyrrolidone K15
(Fluka Chemical Corp.) in PBS, prepared according to Tokuyasu (1989)
.
Individual agarose blocks were then mounted on copper pins, frozen in
liquid nitrogen, and cryosectioned at
80°C. Thawed cryosections were
prepared and labeled as described (Griffiths, 1993
; Kärgel et al., 1996
), using a blocking buffer consisting of PBS, 20 mM glycine, 1% fish gelatin
(Sigma Chemical Co.), and 10% fetal calf serum; the serum had been
heated at 60°C for 1 h, and then centrifuged to remove complement. Dilutions were 1:10 for an affinity-purified polyclonal anti-HA antibody (a gift
of Jan Burkhardt, University of Chicago, Chicago, IL), 1:25 for an affinity-purified polyclonal anti-GFP antibody (a gift of Charles Zuker, University of California, San Diego, San Diego, CA), and 1:50 for protein A-gold
(10 nm; Goldmark Biologicals). After the protein A-gold step, the sections were postfixed for 30 min in 1% glutaraldehyde in PBS, and then
washed with distilled water and stained with 1.5% silicotungstic acid in
2.5% polyvinyl alcohol (MW 15,000; ICN Biomedicals Inc.). The best results were obtained using relatively thick sections and a thin layer of stain.
Electron micrographs were digitized and imported into Photoshop (Adobe Systems Inc.) to adjust brightness and contrast and to create composite images. For Figs. 3 and 7, the gold particles were darkened to improve visualization. Images were printed on an NP-1600 dye-sublimation printer (Codonics).
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Immunofluorescence Microscopy
We modified previously described methods (Pringle et al., 1991) to enhance both the preservation and the visualization of intracellular structures. Improved preservation was achieved by rapidly digesting the cell
wall with protease-free lyticase in sorbitol-free wash buffer, and by postfixing the cells in acetone. Improved visualization was achieved by adhering the cells directly to coverslips (Weiss et al., 1989
; Hell and Stelzer,
1995
) rather than to wells on a slide. A detailed protocol follows.
Fixation and Spheroplasting. A yeast culture is grown overnight in rich or selective medium with good aeration to an OD600 of 0.25-1.0. 8 ml of this culture is pipetted onto a 150-ml bottle-top filter, and the liquid is removed by vacuum. The cells are immediately resuspended in 5 ml of freshly prepared 50 mM KPi, pH 6.5, 1 mM MgCl2, 4% formaldehyde (EM grade; Ted Pella). After fixation for 2 h at room temperature in a 15-ml tube (Falcon Labware), the cells were centrifuged for 3 min at 1,000 g (2,000 rpm) in a tabletop centrifuge, and the supernatant is aspirated completely with a Pasteur pipet. The cells are resuspended in 5 ml of freshly prepared wash buffer (100 mM KPi, pH 7.5, 1 mM MgCl2, 0.25 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin), and then centrifuged again as above. Finally, the cells are resuspended in wash buffer to an OD600 of 10. To 100 µl of concentrated cell suspension is added 0.6 µl of 2-mercaptoethanol followed by 20 µl of recombinant yeast lytic enzyme (20,000 U/ml; ICN Biomedicals Inc.). After mixing end-over-end for 15-30 min at room temperature, the spheroplasts are centrifuged for 2 min at 400 g (2,000 rpm) in a microfuge, gently resuspended in 100 µl wash buffer, and then centrifuged and resuspended once again.
Adhesion and Permeabilization.
To create "wells" on a coverslip, a suitable piece is cut from the adhesive backing of an express courier document pouch, holes are made with a rotating-head hole punch, and the perforated plastic is attached to the coverslip. 10 µl of 0.1% polylysine (P-1524; Sigma Chemical Co.) is added to each well, and then removed by
vacuum aspiration. Each well is washed three times with water, and 10 µl
of spheroplast suspension is added. After 3 min, excess liquid is blotted off
with a cotton swab, and the dried coverslip is immersed in 40 ml of acetone precooled to 20°C in a 50-ml tube (Falcon Labware). (This organic
solvent fixation step is important for preserving tER and Golgi structure.
For some antigens methanol works better, but in most cases acetone is superior.) After 5 min, the coverslip is removed, inverted onto a paper towel to blot off excess solvent, and allowed to dry.
Antibody Incubations.
Each well is precoated for 30 min with a drop of
PBS-Block (PBS, pH 7.4, 1% dried milk, 0.1% bovine serum albumin,
0.1% octyl glucoside). 10 µl of primary antibody mixture in PBS block is then added, and the coverslip is incubated in a humid chamber for 1 h.
Each well is washed eight times with PBS block, and 10 µl of secondary
antibody mixture in PBS block is added. 2 µg/ml 33258 (Hoechst AG) is
included in the secondary antibody solution to stain DNA. After a 30-min
incubation in the dark, each well is washed eight times with PBS block.
The liquid is aspirated completely after the final wash. Each well then receives 5 µl of phenylenediamine-containing mounting medium (Pringle et
al., 1991). The coverslip is inverted onto a slide and sealed with nail polish.
Microscopy. Samples were viewed on an Axioplan microscope equipped with a 100× Plan-Apo 1.4 NA objective (Carl Zeiss, Inc.) and with band-pass filters for visualizing Hoechst dye, fluorescein/Oregon green, and Texas red. Images were captured with a Photometrics CCD camera using the Openlab software package (Improvision). For each field of cells, multiple images were recorded from focal planes spaced 0.2 µM apart; three to four of these images were deblurred using a simple deconvolution algorithm, and then combined to form a single projected image. Where indicated, the same cells were also photographed in differential interference contrast (DIC) mode at a single focal plane. Photoshop was used for colorization, adjustments of brightness and contrast, and creation of composite and merged images. Manipulations with the Openlab and Photoshop programs improved the quality of the images, but did not significantly alter their information content. Fluorescence and DIC images were printed on a Stylus Photo color inkjet printer (Epson Electronics America Inc.).
To quantify the overlap between Och1p-HA and Sec7p, digital images were examined with Photoshop. Cells that showed clear staining with both antibodies were used for quantitation. A spot of one color was scored as overlapping with a spot of the second color if the center of the first spot was contained within the second spot. To estimate the percentage of Golgi structures that are perinuclear in P. pastoris, the perinuclear and peripheral Och1p-HA spots were counted in 50 cells that showed faint nuclear envelope staining (see Fig. 2 C). To estimate the average percentage of the cell area occupied by fluorescent spots of a given marker protein, 25 cells stained to reveal Och1p-HA and Sec7p were analyzed using NIH Image (http://rsb.info.nih.gov/nih-image/): the total areas of the Och1p-HA and Sec7p spots were summed and divided by twice the total areas of the cells.
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Visualization of GFP Fusion Proteins in Intact Cells
Yeast cells expressing a GFP-labeled COPII protein were grown to log phase with good aeration. A 400-µl aliquot of the culture was then mixed rapidly with 400 µl of 100 mM KPi, pH 6.5, 2 mM MgCl2, 8% formaldehyde, 0.5% glutaraldehyde. After fixation for 1 h in the dark, the cells were washed twice with 1 ml PBS, and then resuspended in 50 µl PBS. For viewing by fluorescence microscopy, 1-2 µl of the resuspended cells were spotted on a slide and spread with a coverslip.
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Results |
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Golgi Stacks in P. pastoris Are Closely Associated with tER Sites
Stacks of Golgi cisternae can be readily visualized in P.
pastoris cells, with each stack containing about four cisternae (Fig. 1) (Gould et al., 1992; Glick, 1996
). Previously
published images of P. pastoris showed Golgi stacks near
the nucleus (Gould et al., 1992
; Rambourg et al., 1995
;
Glick, 1996
). Golgi structures might form by growing out
of the nuclear envelope, which constitutes a large fraction
of the ER in budding yeasts (Preuss et al., 1991
). Alternatively, P. pastoris Golgi stacks might be positioned near the nucleus by microtubule-dependent transport toward
the centrosome, as occurs in vertebrate cells (Thyberg
and Moskalewski, 1985
; Kreis, 1990
; Lippincott-Schwartz,
1998
). Such a role for microtubules is excluded by the following two observations.
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First, thin-section electron microscopy indicates that a
typical P. pastoris cell contains several distinct Golgi
stacks, only some of which are located near the nucleus
(Fig. 1 A). Other Golgi stacks are found next to the peripheral ER elements that underlie the plasma membrane.
Second, treatment of P. pastoris cells with nocodazole
does not visibly alter the structure or positioning of Golgi
stacks (Fig. 1 B). As with S. cerevisiae (Jacobs et al., 1988),
nocodazole treatment of P. pastoris disrupts microtubules
and inhibits nuclear division (see Fig. 4), but Golgi stacks
are still observed next to the nuclear envelope and peripheral ER. In both untreated and nocodazole-treated cells,
vesicular profiles are frequently seen in ER regions adjacent to the Golgi cisternae (Fig. 1) (Gould et al., 1992
;
Glick, 1996
). These vesiculating ER regions resemble the
tER sites seen in vertebrate cells (Palade, 1975
; Bannykh
and Balch, 1997
). Hence, the morphological data suggest
that Golgi stacks in P. pastoris are associated not with the
centrosome, but rather with tER sites.
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Comparison of ER and Golgi Structures in the Two Yeasts by Immunostaining
Electron microscopy indicates that general ER structure is
similar in P. pastoris and S. cerevisiae, but that Golgi
organelles in P. pastoris are more coherent (see above).
We confirmed this interpretation by visualizing various
marker proteins (Table I) using immunofluorescence microscopy. For these experiments, we developed a modified immunofluorescence protocol that consistently yields
high-quality images (see Materials and Methods). Because P. pastoris is closely related to S. cerevisiae (Higgins and
Cregg, 1998), polyclonal antibodies raised against S. cerevisiae antigens often cross-react with the P. pastoris homologues. Alternatively, marker proteins were modified with
epitope tags and visualized using specific monoclonal antibodies.
To visualize the general ER in P. pastoris, fixed cells
were labeled with an antibody against protein disulfide
isomerase (Pdi1p), a marker for the general ER (Fig. 2 D,
red) (Sitia and Meldolesi, 1992; Nishikawa et al., 1994
).
The same cells were also incubated with Hoechst dye (Fig.
2 D, blue) to label DNA. The anti-Pdi1p antibody highlights the nuclear envelope as well as peripheral ER elements. This pattern resembles the ER distribution seen in
S. cerevisiae (Rose et al., 1989
; Preuss et al., 1991
).
Fig. 2 A shows the S. cerevisiae Golgi. This organelle appears in immunofluorescence images as a set of punctate
spots, with different Golgi markers often displaying only a
partially overlapping localization (Segev et al., 1988; Franzusoff et al., 1991
; Antebi and Fink, 1992
; Lussier et al.,
1995
). In S. cerevisiae cells expressing an HA-tagged version of the early Golgi marker Och1p (Nakayama et
al., 1992
; Gaynor et al., 1994
; Harris and Waters, 1996
;
Gaynor and Emr, 1997
), an anti-HA monoclonal antibody
(Fig. 2 A, green) reveals multiple spots of Och1p-HA labeling per cell. The same cells were also labeled with a
polyclonal antibody (red) against Sec7p, a protein that is
concentrated in late Golgi elements (Franzusoff et al.,
1991
). The two markers show no significant overlap (Fig. 2
A, Merged; Table II).
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Do early and late Golgi elements ever colocalize in
S. cerevisiae? In temperature-sensitive sec14 mutants of S.
cerevisiae, multilamellar Golgi structures accumulate at
the nonpermissive temperature (Novick et al., 1980; Rambourg et al., 1996
); these structures are reminiscent of the
Golgi stacks seen in higher eukaryotes. Therefore, we
tested whether Och1p-HA and Sec7p colocalize in a sec14
mutant after incubation at the nonpermissive temperature of 37°C. The 37°C treatment causes redistribution of Sec7p
into large clusters (Fig. 2 B, red) (Franzusoff et al., 1991
).
However, the staining pattern of Och1p-HA is largely unchanged in the sec14 mutant (Fig. 2 B, green), and there is
still no significant overlap between Och1p-HA and Sec7p.
It seems that S. cerevisiae is incapable of generating coherent Golgi stacks that contain both early and late Golgi proteins.
Fig. 2 C shows the P. pastoris Golgi. Using an integrating expression vector (Sears et al., 1998), we generated a
P. pastoris strain that stably expresses HA-tagged S. cerevisiae Och1p. P. pastoris contains an
-1,6-mannosyltransferase activity like that ascribed to Och1p (Trimble et al.,
1991
; Nakanishi-Shindo et al., 1993
), and expression of
Och1p-HA in P. pastoris has no effect on growth or Golgi
morphology (not shown). Och1p-HA localizes to two to
six spots per P. pastoris cell (Fig. 2 C, green). As predicted
from electron microscopy, a subset (~45%) of the Och1p-HA spots clearly adjoin the nucleus (see Fig. 2 C, legend).
When P. pastoris cells are labeled with the antibody
against S. cerevisiae Sec7p, two to six spots are again detected (Fig. 2 C, red), presumably because this antibody
reacts with the P. pastoris homologue of Sec7p. By contrast to the situation in S. cerevisiae, Och1p-HA and Sec7p
exhibit nearly quantitative overlap in P. pastoris (Fig. 2 C,
Merged; Table II). Similarly, when a tagged version of the S. cerevisiae medial-Golgi protein Kre2p/Mnt1p (Chapman and Munro, 1994
; Lussier et al., 1995
) is expressed in
P. pastoris, it colocalizes with Sec7p (not shown). Colocalization at the light microscopy level indicates that two
markers are either in the same compartment or in closely
apposed structures. These data support the conclusion that
Golgi organelles are more coherent in P. pastoris than in S. cerevisiae.
To confirm that the structures visualized in P. pastoris by immunofluorescence are indeed Golgi membranes, we performed immunoelectron microscopy on thawed cryosections. Using a polyclonal anti-HA antibody to detect Och1p-HA, we see a specific labeling of Golgi stacks (Fig. 3; Table III).
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Golgi Organization in P. pastoris Is Unaffected by Microtubule Disruption or Cell Cycle Progression
P. pastoris resembles vertebrate cells in having stacked
Golgi cisternae. We tested whether the two cell types are
also similar with regard to Golgi dynamics. Pre-Golgi elements in vertebrate cells are transported along microtubules (Lippincott-Schwartz, 1998), but our electron microscopy data indicate that in P. pastoris, microtubules do
not influence Golgi structure (see Fig. 1). This conclusion
was confirmed at the immunofluorescence level. In untreated P. pastoris cells, microtubules are present and nuclei partition into daughter cells during mitosis (Fig. 4 A).
Microtubule distribution shows no detectable relationship
to Golgi distribution (Fig. 4 A). Nocodazole treatment depolymerizes microtubules and blocks nuclear migration,
but the Golgi staining pattern is unaffected (Fig. 4 B).
Moreover, the Golgi markers Sec7p and Och1p-HA still colocalize in nocodazole-treated P. pastoris cells (not shown).
To determine whether cell cycle progression alters
Golgi organization in P. pastoris, as it does in vertebrate
cells (Rabouille and Warren, 1997), we analyzed P. pastoris in the G1, S/G2, and M phases of the cell cycle. Immunofluorescence was used to quantify the average number of Golgi structures per cell. This number is similar in
each phase of the cell cycle (Table IV). Thus, when observing P. pastoris under a variety of conditions, we consistently find that each cell contains a small number of distinct Golgi stacks.
|
COPII Coat Proteins Show Different Distributions in S. cerevisiae and P. pastoris
The electron microscopy data suggest that P. pastoris contains discrete tER sites, whereas S. cerevisiae lacks such
sites. To explore this idea further, we compared the localizations of COPII proteins in the two yeasts. GFP was
fused to the COOH terminus of S. cerevisiae Sec13p, a
coat protein that is incorporated at a late stage of COPII
vesicle assembly (Pryer et al., 1993; Kuehn and Schekman, 1997
). Replacement of the endogenous SEC13 gene with the SEC13-GFP gene has no detectable effect on
cell growth, indicating that the Sec13p-GFP fusion can
perform the essential function of Sec13p (Pryer et al.,
1993
). When S. cerevisiae cells expressing Sec13p-GFP are
viewed by fluorescence microscopy, each cell contains
~30-50 tiny spots (shown in Fig. 5 A, although this pattern
is difficult to photograph accurately). The spots are distributed almost evenly throughout the cytoplasm, with
some cells showing an apparent concentration of spots on
the nuclear envelope. We surmise that these spots represent individual COPII vesicles. A vesicle is smaller than
the resolution limit of light microscopy (Lacey, 1989
), so
the apparent size of the spots is probably misleading, but a
single COPII vesicle contains many copies of Sec13p-GFP
and hence should produce a detectable fluorescence signal. GFP was also fused to three other S. cerevisiae COPII coat proteins (Kuehn and Schekman, 1997
): Sec23p,
Sec24p, and Sec31p. All of these fusions are functional,
and they all give the same fluorescence pattern as Sec13p-GFP (Fig. 5 B and data not shown), indicating that we
have visualized the normal distribution of COPII vesicles.
These results strongly support the notion that COPII vesicles bud from the entire ER in S. cerevisiae.
|
In parallel, the P. pastoris SEC13 gene was replaced with a SEC13-GFP fusion gene. The resulting fluorescence pattern is strikingly different from that seen in S. cerevisiae. Sec13p-GFP in P. pastoris is concentrated in only two to six large spots per cell (Fig. 6 A). When examined by immunofluorescence microscopy, the Sec13p-GFP spots are adjacent to, but not quite overlapping, the Golgi spots marked by the anti-Sec7p antibody (Fig. 6 B). This result suggests that Sec13p-GFP is localized to tER sites. Indeed, immunoelectron microscopy with an anti-GFP antibody revealed that Sec13p-GFP is present on tubulovesicular structures at the interface between ER membranes and Golgi stacks (Fig. 7; Table III). We conclude that COPII vesicle budding is restricted to discrete tER sites in P. pastoris.
|
Sec12p Localizes to tER Sites in P. pastoris
The earliest known player in the COPII assembly pathway
is Sec12p, a membrane-bound guanine nucleotide exchange factor that recruits the small GTPase Sar1p to the
ER membrane (Nakano et al., 1988; Nakano and Muramatsu, 1989
; Barlowe and Schekman, 1993
; Kuehn and
Schekman, 1997
). In previous studies of S. cerevisiae, Sec12p exhibited general ER staining (Nishikawa and Nakano, 1991
, 1993
). Our results confirm those earlier findings. S. cerevisiae Sec12p has traditionally been visualized
in strains overexpressing this protein (Nishikawa and Nakano, 1993
; Nishikawa et al., 1994
). To eliminate possible
ambiguities resulting from overexpression, we replaced
the chromosomal SEC12 gene with a myc-tagged version. The resulting strain grows like the wild-type (not shown),
implying that Sec12p-myc can functionally replace the essential wild-type protein (Nakano et al., 1988
). Sec12p-myc (Fig. 8 A, green) colocalizes with Pdi1p (Fig. 8 A, red)
in the nuclear envelope and in peripheral ER membranes.
Although Sec12p-myc sometimes shows a discontinuous staining pattern, the fluorescence signal is relatively weak, and we have observed that the ER network often appears
discontinuous when it is weakly stained (not shown).
Hence, the combined data suggest that Sec12p-myc is
present throughout the ER in S. cerevisiae.
|
We also replaced P. pastoris SEC12 with a myc-tagged version. Once again the fluorescence signal is weak, but the pattern is clearly visible. In this case, the anti-myc antibody does not give a general ER staining, but instead labels several spots per cell (Fig. 8 B, green). Like Sec13p-GFP (see above), Sec12p-myc localizes to sites that are immediately adjacent to Sec7p-containing Golgi structures (Fig. 8 B). Thus, in P. pastoris, components at both early and late stages of the COPII assembly pathway are concentrated at tER sites.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Why is the Golgi apparatus more photogenic in P. pastoris than in S. cerevisiae? These two yeasts are morphologically very similar; yet Golgi cisternae in P. pastoris
are organized into stacks, whereas Golgi cisternae in S.
cerevisiae are scattered throughout the cytoplasm. We propose the following hypothesis. In P. pastoris, COPII vesicles bud from fixed tER sites, and then fuse with one another to create new Golgi cisternae, which mature to
yield polarized stacks (Glick and Malhotra, 1998). In
S. cerevisiae, COPII vesicles bud throughout the ER, and
therefore each Golgi cisterna forms at a different location (Fig. 9).
|
Our immunofluorescence data confirm that S. cerevisiae and P. pastoris have fundamentally different Golgi
structures. A hallmark of the Golgi in S. cerevisiae is
that various marker proteins often show distinct punctate distributions (Antebi and Fink, 1992; Chapman and
Munro, 1994
; Lussier et al., 1995
). For example, the early
Golgi protein Och1p-HA exhibits virtually no overlap
with the late Golgi protein Sec7p. Although multilamellar Golgi structures are seen in temperature-sensitive
sec7 and sec14 mutants of S. cerevisiae (Novick et al.,
1980
; Svodoba and Necas, 1987
; Rambourg et al., 1993
,
1996
), we find that early and late Golgi markers still do
not colocalize in sec14 mutant cells (Fig. 2), indicating that S. cerevisiae cannot make coherent Golgi stacks.
With P. pastoris, on the other hand, Och1p-HA and
Sec7p overlap almost completely (Fig. 2), as expected if
each Golgi stack represents an ordered set of early, middle, and late cisternae.
Does this difference in Golgi structure correlate with a
difference in ER organization? The ER of both yeasts
comprises the nuclear envelope plus peripheral elements.
However, in P. pastoris, vesicles can often be seen budding
specifically from regions of the ER adjacent to Golgi
stacks (Fig. 1) (Gould et al., 1992; Glick, 1996
). Such vesiculating ER regions have not been seen in S. cerevisiae
(Kuehn and Schekman, 1997
). These observations suggested to us that P. pastoris contains discrete tER sites, whereas S. cerevisiae does not. To test this interpretation,
we used COPII coat proteins as markers for the tER (Orci
et al., 1991
; Kuge et al., 1994
; Shaywitz et al., 1995
; Paccaud et al., 1996
; Tang et al., 1997
). With S. cerevisiae, fusing GFP to Sec13p or other COPII coat proteins revealed
many small fluorescent spots that probably represent individual COPII vesicles (Fig. 5). These spots are found
throughout the cytoplasm, consistent with the notion that
COPII vesicles bud at random from the entire ER. By contrast, a Sec13p-GFP fusion in P. pastoris localizes to a
small number of discrete regions (Fig. 6). These regions
are immediately adjacent to Golgi stacks, and they
contain tubulovesicular membranes (Fig. 7). Hence, we
propose that Sec13p-GFP marks a compartment in P.
pastoris that is analogous to the tER sites described previously in vertebrate cells (Palade, 1975
; Bannykh and
Balch, 1997
).
How is Sec13p recruited to tER sites in P. pastoris? The
assembly of COPII vesicles is an ordered process that begins with the action of Sec12p (Barlowe and Schekman,
1993; Kuehn and Schekman, 1997
). We compared the localization of epitope-tagged Sec12p in the two yeasts. Consistent with previous reports (Nishikawa and Nakano,
1993
; Nishikawa et al., 1994
), Sec12p-myc in S. cerevisiae is
distributed throughout the ER. In P. pastoris, on the other hand, Sec12p-myc is concentrated at tER sites (Fig. 8).
This observation suggests a working model for the organization of tER and Golgi compartments in the two yeasts.
We postulate that in P. pastoris, Sec12p is anchored at tER
sites by unknown partner proteins that comprise a "tER
scaffold." Because Sec12p initiates the assembly of COPII
vesicles, these vesicles bud exclusively from tER sites, and
successive Golgi cisternae form at fixed locations to generate polarized stacks. This model assumes that tER sites
are relatively stable entities, and, indeed, our studies of
Sec13p-GFP dynamics in P. pastoris confirm that tER sites
are long lived and slow moving (B.J. Bevis, unpublished
observations). The situation is quite different in S. cerevisiae
this yeast apparently lacks a tER scaffold, so Sec12p
is free to diffuse throughout the ER. As a result, COPII
vesicles bud from the entire ER, and successive Golgi cisternae form at different locations, yielding a dispersed organelle (Fig. 9). In this view, Golgi structure and positioning are strongly influenced by tER organization.
An important test of our model will be to alter tER organization in P. pastoris and ask whether Golgi structure is
correspondingly affected. Such an experiment will reveal
whether the existence of Golgi stacks in P. pastoris is due
solely to the presence of fixed tER sites. The simplest view
is that Golgi stacking is a kinetic phenomenon, with cisternal formation and maturation occurring too quickly for
successive cisternae to diffuse away from one another. Alternatively, Golgi cisternae in P. pastoris might be held together by a cytosolic matrix (Mollenhauer and Morré,
1978; Cluett and Brown, 1992
; Staehelin and Moore, 1995
;
Barr et al., 1997
). These possibilities can be distinguished
by generating P. pastoris mutants in which COPII proteins
are delocalized, and then asking whether the loss of tER
sites leads to Golgi dispersal. One promising approach focuses on determining whether P. pastoris Sec12p contains
a tER localization signal that is recognized by specific
partner proteins.
Although S. cerevisiae lacks discrete tER sites, this
yeast can be used to explore the more general question of
whether the Golgi is an outgrowth of the ER. Early in S-phase of the S. cerevisiae cell cycle, the small buds invariably contain both ER and Golgi structures (Segev et al.,
1988; Redding et al., 1991
; Preuss et al., 1991
, 1992
; O.W.
Rossanese, unpublished observations). It is likely that the
ER elements present in the emerging bud give rise to new
Golgi cisternae. If so, mutants that fail to transport ER
membranes into the bud should also lack Golgi cisternae
in the bud. We are currently testing this prediction by
characterizing S. cerevisiae mutants defective in Golgi inheritance.
A comparison of budding yeasts with other eukaryotes
can indicate which aspects of the tER-Golgi system are
cell type-specific. First, unlike many eukaryotes, S. cerevisiae contains neither stacked Golgi organelles nor discrete
tER sites. It is unclear whether the absence of these structures in S. cerevisiae is adaptive, or whether it reflects a
loss-of-function mutation during the evolution of this
yeast. Second, tubular connections between Golgi stacks
are present in vertebrate cells (Mironov et al., 1997), but
have not been detected in P. pastoris, the fission yeast
Schizosaccharomyces pombe (Chappell and Warren, 1989
)
or certain insect cells (Stanley et al., 1997
). Third, the
Golgi breaks down during mitosis in vertebrate cells
(Rabouille and Warren, 1997
; Acharya et al., 1998
), but
not in higher plants (Driouich and Staehelin, 1997
) or budding yeasts (Table IV) (Makarow, 1988
).
Which aspects of the tER-Golgi system are universal?
We propose that Golgi cisternae always form by the coalescence of tER-derived membranes. This relationship is
particularly evident in cells that contain Golgi stacks immediately adjacent to tER sites (Whaley, 1975; Farquhar
and Palade, 1981
; Bracker et al., 1996
). We have now documented such an association in P. pastoris. A close apposition between tER sites and Golgi stacks is probably a
general feature of cells that do not transport Golgi elements along cytoskeletal tracks. Consistent with this idea,
microtubules have no influence on Golgi structure or positioning in P. pastoris (Figs. 1 and 4). However, in some
cell types, nascent Golgi elements are transported away
from tER sites, thereby obscuring the tER-Golgi connection. For example, in S. pombe, microtubules play a role
in cisternal stacking and possibly in Golgi movement
(Ayscough et al., 1993
). In higher plants, Golgi structures
are transported along actin filaments (Driouich and Staehelin, 1997
; Boevink et al., 1998
). The best-studied example of cytoskeleton-mediated Golgi movement is provided by vertebrate cells, which employ microtubules and associated motor proteins to generate a juxtanuclear
Golgi ribbon (Lippincott-Schwartz, 1998
; Burkhardt, 1998
).
As a consequence, tER sites and Golgi elements normally do not colocalize in vertebrate cells. Yet recent evidence suggests that nascent Golgi structures in vertebrate
cells initially coalesce at tER sites (Presley et al., 1997
;
Scales et al., 1997
; Rowe et al., 1998
); in the presence of
microtubule-depolymerizing agents, entire Golgi stacks
are found next to tER sites (Cole et al., 1996
; Storrie et
al., 1998
). Thus, when microtubules have been disrupted,
the tER-Golgi system in a vertebrate cell resembles the
tER-Golgi system in P. pastoris. It seems that in all eukaryotes, the Golgi can be viewed as a dynamic outgrowth of the tER.
![]() |
Footnotes |
---|
Address correspondence to Benjamin S. Glick, Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637. Tel.: (773) 702-5315. Fax: (773) 702-3172. E-mail: bsglick{at}midway.uchicago.edu
Received for publication 4 June 1998 and in revised form 23 February 1999.
Thanks for reagents and advice to Randy Schekman, Alex Franzusoff,
Chris Kaiser, Akihiko Nakano, Vytas Bankatis, Suresh Subramani, Jim
Cregg, Steve Gould, Sean Munro, Peter Walter, Gerry Waters, Tom
Stevens, Jan Burkhardt, Charles Zuker, and Robert Freedman, and for
help with microscopy to Bob Josephs, Yimei Chen, Sharon Parmet, Judith
Austin, and Jim McIlvain. We are grateful to Adam Linstedt, Lelio Orci,
and Hewson Swift for help with initial explorations of the P. pastoris system, and to Nava Segev, Vivek Malhotra, and Adam Hammond for critical reading of the manuscript.
O.W. Rossanese, J. Soderholm, and B.J. Bevis were supported by National Institutes of Health training grant 5-20942. E.K. Williamson operates the EM core facility funded by the University of Chicago Cancer Research Center. B.S. Glick was supported by National Science Foundation
grant MCB-9604342, a Pilot and Feasibility Study Award from the Diabetes Research Foundation, a Basil O'Connor Starter Scholar Research
Award (5-FY96-1138) from the March of Dimes Birth Defects Foundation, a Young Investigator Award from the Cancer Research Foundation,
an Institutional Research Grant from the American Cancer Society, and a
grant from the Pew Charitable Trusts.
![]() |
Abbreviations used in this paper |
---|
DIC, differential interference contrast; GFP, green fluorescent protein; HA, hemagglutinin epitope; tER, transitional ER.
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