Download I m munoisolation of Kex2p-containing organelles from yeast

Journal of Cell Science 106, 815-822 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
815
Immunoisolation of Kex2p-containing organelles from yeast demonstrates
colocalisation of three processing proteinases to a single Golgi compartment
Nia J. Bryant and Alan Boyd*
Department of Biochemistry, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK
*Author for correspondence
SUMMARY
One of the Golgi compartments of Saccharomyces cere visiae is defined by the presence of a specific endoproteinase, Kex2p, which cleaves precursor polypeptides at
pairs of basic residues. We have used antibodies directed
against the cytoplasmically disposed C-terminal domain
of Kex2p to develop an immuno-affinity procedure for
the isolation of Kex2p-containing organelles. The
method gives a high yield of sealed organelles that are
essentially free of contamination from other secretory
pathway organelles while being significantly enriched
for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings
provide clear evidence for a single yeast Golgi compartment containing all three late-processing enzymes,
which is likely to be the functional equivalent in yeast
of the mammalian trans-Golgi network.
INTRODUCTION
organelle based upon its content of a well-characterised
yeast Golgi protein, the Kex2 protease, which is responsible for the endoproteolytic processing of certain precursors
late in the secretory pathway (Julius et al., 1983; Fuller et
al., 1989a,b). Recent immunofluorescence studies have
revealed the presence of 1-5 Kex2p-containing organelles
in a single yeast cell (Franzusoff et al., 1991; Redding et
al., 1991). We now show that Kex2p-containing organelles
can be isolated by use of a simple immuno-affinity procedure, and that they also contain two other late-processing
proteinases, dipeptidyl aminopeptidase A and the Kex1 carboxypeptidase. Our data help to place the Kex2p compartment at the extreme trans side of the yeast Golgi and, taken
in conjunction with the finding that it lies on the pathway
to the vacuole (Graham and Emr, 1991), suggest that the
organelle we have isolated is the functional equivalent in
yeast of the mammalian trans-Golgi network.
The Golgi apparatus plays a central role in the endomembrane system of eukaryotic cells (Farquhar, 1985; Mellman
and Simons, 1992). In mammalian cells the organelle
usually seems to be divided into three functionally defined
areas: the cis-Golgi network, which receives vesicular traffic from the endoplasmic reticulum; the stacked cis, medial
and trans cisternae, where sequential oligosaccharide modifications are carried out; and the trans-Golgi network,
where late post-translational modifications occur and where
sorting of proteins to various post-Golgi destinations is
thought to be achieved (Griffiths and Simons, 1986; Sossin
et al., 1990).
The exact nature of the Golgi in the yeast Saccharomyces
cerevisiae is uncertain. It is clear that yeast cells possess a
functional equivalent of the mammalian Golgi, where glycoproteins are modified, proproteins are proteolytically
processed and proteins are sorted to either the cell surface
or the vacuole; and that these various post-translational
modifications occur in separate cellular compartments
(Cunningham and Wickner, 1989; Franzusoff and Schekman, 1989; Graham and Emr, 1991). However, it has
proved difficult to visualise any morphological equivalent
of the mammalian stack of cisternae in budding yeast (but
see Preuss et al., 1992). In order to exploit fully the progress
that has been made in the identification and characterisation of specific proteins involved in the operation of the
yeast endomembrane system it is essential to characterise
the yeast Golgi: to this end we have developed a procedure
for the isolation of one clearly defined compartment of this
Key words: Golgi, immunoisolation, Kex2p, sec7, yeast
MATERIALS AND METHODS
Strains and growth media
Escherichia coli strain NM522 (Gough and Murray, 1983) was
used as the host for plasmid constructions and the production of
Protein A fusion proteins; the strain pop2136 (Kusters et al., 1989)
was used for the production of βGal fusion proteins. Bacteria were
grown in LB medium supplemented with appropriate antibiotics.
For most of the experiments involving Saccharomyces cere visiae the strains used were the haploid strain JRY188 (MATα
leu2-3, 112 ura3-52 trp1 his4 sir3 rme; Brake et al., 1984) or its
homozygous diploid derivative NBY10. To produce this diploid
strain JRY188 was first transformed with the plasmid pGAL-HO
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N. J. Bryant and A. Boyd
(Herskowitz and Jensen, 1991). Transformed cells were transferred to galactose-containing medium to allow mating type interconversion. A number of single colony isolates were transferred
back on to glucose-containing medium and selection for pGALHO was removed to allow the isolation of plasmid-free segregants.
NBY10 was chosen for further study as a diploid (it did not produce α-factor, it failed to mate with tester strains of either mating
type, and it sporulated at low frequency to produce four-spored
asci). In the experiment in which the distribution of epitope-tagged
Mnt1p was analysed we used strain 6210-mntmyc (obtained from
Dr S. Munro); and in experiments in which the effects of a sec7
mutation were analysed we used SF294-2B (MATa sec7-1; Novick
et al., 1980). For experiments in which β-lactamase activity was
measured the strain used was JRY188 transformed with the plasmid pYJS50. This is a 2 µm-LEU2-based plasmid encoding a
prepro-α-factor to β-lactamase fusion protein. Yeast strains harbouring this plasmid produce and secrete active β-lactamase: in a
mid-log culture 95% of detectable β-lactamase protein and
enzyme activity are in the culture supernatant (A. J. Mileham and
A. Boyd, unpublished data). Yeast cultures were grown in SD
medium (0.67% nitrogen base without amino acids (Difco); 2%
glucose) supplemented with appropriate nutrients.
Immunological procedures
Antibodies that specifically recognise either the luminal N-terminal domain or the cytoplasmically disposed C-terminal domain of
Kex2p were obtained using bacterially produced fusion proteins.
To obtain the C-terminal domain antibody, a spa-KEX2 gene
fusion encoding a protein consisting of the 100 C-terminal amino
acids of Kex2p fused to a portion of staphylococcal Protein A was
constructed by insertion of a 1.25 kb EcoRI/BamHI fragment from
the KEX2-containing plasmid pGA714 (obtained from Dr G.
Ammerer) into similarly digested pKpra (Zueco and Boyd, 1992).
E. coli cells harbouring this plasmid produced a fusion protein
with a relative molecular mass of 29,000, which was purified from
bacterial lysates by chromatography on IgG-Sepharose (Lowenadler et al., 1986). For the N-terminal domain antibody a spaKEX2 gene fusion encoding a protein containing amino acids 119614 of Kex2p was constructed by inserting a DNA fragment
derived from the HindIII-PvuII region of the KEX2 gene into
pKpra. E. coli cells harbouring this plasmid produced a fusion
protein with a relative molecular mass of 75,000, which was purified from bacterial lysates by isolation of insoluble inclusion
bodies (Marston and Hartley, 1990) followed by preparative SDSPAGE.
The purified fusion proteins were injected into rabbits. Antibodies that recognise the Kex2p-derived portions of the SpaKex2p fusion proteins were affinity-purified from immune sera
using columns of Affi-gel (Bio-Rad) to which had been conjugated appropriate βGal-Kex2p fusion proteins (encoded by lacZKEX2 gene fusions created in the pEX series of vectors (Kusters
et al., 1989) and containing the same Kex2p amino acids as the
corresponding Spa-Kex2p fusion protein).
Immunoisolation of membranes
An immunoadsorbent (Kex2-ImAd) was formed by binding 500
µg of anti-Kex2p antibody (C-terminal domain) to 100 µl Pansorbin (10% (w/v) fixed Staphylococcus aureus cells; Calbiochem)
that had previously been incubated with Solution A (20 mM
Hepes, pH 7.4, 2 mM MgCl2, 150 mM KCl) containing 10 mg/ml
BSA. Binding of antibodies to the Pansorbin was achieved by
incubation with the affinity-purified antibody on a rotating wheel
at 4°C overnight. Antibody-coated Pansorbin was then washed
twice using Solution A and once in Solution B (0.8 M sorbitol,
10 mM triethanolamine, 1 mM EDTA, brought to pH 7.4 with
acetic acid).
Glass bead lysates were prepared as follows: cells were harvested (by centrifugation for 5 minutes at 3,000 g) from a culture
that had been grown to an A600 of 0.7, washed once in distilled
water and resuspended to 100 A600 units/ml in Solution B containing protease inhibitors (4 mM phenylmethylsulphonyl fluoride
(PMSF), 4 mM EDTA, 4 mM EGTA, 2 µg/ml each of pepstatin,
leupeptin, chymostatin and antipain). Glass beads (40 mesh) were
added to the level of the meniscus and the tubes were vortexed
4×, for 30 seconds, with 2 minute cooling intervals on ice. The
lysate was collected by centrifugation through the glass beads at
3,000 g for 10 minutes, resulting in a pellet of cell debris and a
supernatant of cell lysate containing all detectable Kex2p. Spheroplast lysate was prepared as follows: cells were harvested and
washed as before and then resuspended in spheroplast buffer (1.4
M sorbitol, 50 mM potassium phosphate, pH 7.4, 10 mM NaN3,
40 mM β-mercaptoethanol) containing zymolyase (5 mg/ml). Cell
wall digestion was achieved at 25°C, and this incubation was carried out until at least 80% of the cells present had been converted
to spheroplasts (30 minutes). Spheroplasts were harvested (2 minutes at 4,000 g) and washed once in spheroplast buffer before
being resuspended in 1 ml Solution B containing protease
inhibitors (as above) per 100 A600 units of the original culture,
and then homogenised using a Dounce homogeniser (7 strokes)
on ice. The homogenate was cleared of cell debris by centrifugation (2 minutes at 4,000 g) before use, resulting in a homogenate
containing all detectable cellular Kex2p.
ImAd was resuspended in yeast cell lysate that was prepared
from 30 A600 units of cells.
For the recovery of Kex2p-containing membranes the ImAd
was incubated with cell lysate for 3 hours at 4°C on a rotating
wheel to allow gentle mixing, recovered by centrifugation for 2
minutes at 4,000 g and then washed three times in Solution B.
Immunoblotting
Samples were subjected to 10% SDS-PAGE (Laemmli, 1970)
prior to transfer to nitrocellulose. Material bound to ImAd was
analysed by resuspension in SDS sample buffer, followed by incubation in a boiling water bath for 5 minutes after which the S.
aureus cells were removed by centrifugation prior to SDS-PAGE.
Immunoblots were developed using the ECL detection system
(Amersham). Primary antibodies were as described, secondary
antibodies were goat anti-rabbit IgG/horseradish peroxidase conjugate and goat anti-mouse IgG/horseradish peroxidase conjugate
as appropriate.
Enzyme assays
NADPH cytochrome c reductase, a marker for the presence of ER
membranes, was assayed by following the sample’s ability to
reduce NADPH, as described by Yasukochi and Masters (1976).
Carboxypeptidase Y (CPY) and dipeptidyl aminopeptidase
(DPAP) B activities were used to detect the presence of vacuolar
contents and membranes, respectively. To determine CPY activity
the sample’s activity against the peptide substrate N-carbobenzoxyphenylleucine was assayed as described by Wolf and Weiser
(1977). Total DPAP activity present in a sample was determined
by its activity against alanylprolyl-p-nitroanilide as described by
Roberts et al. (1991). The activity of DPAP A (which is heat
stable) was assayed by heating the sample to 65°C for 15 minutes prior to its addition to the reaction mixture, and the activity
of DPAP B was estimated by subtracting the amount of heat-stable
DPAP activity in a sample from the total amount of DPAP activity
in that same sample. GDPase activity was assayed as described
by Abeijon et al. (1989) with the amount of free phosphate being
determined as described by LeBel et al. (1978). Kex2 protease
activity was determined as described by Fuller et al. (1989a). For
enzymes other than Kex2, the amount of enzyme activity detected
Immunoisolation of the yeast Golgi
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in ImAd-bound material was expressed as a percentage of the total
present in the cell lysate from which the material was bound.
Material recovered from the same lysate using Pansorbin that had
not been coated by antibody was also assayed for the presence of
the various enzymes and it was found that this technique led to
less than 1% of cellular activity of each of the enzymes being
recovered (data not shown). The amount of Kex2 protease activity
bound by ImAd was estimated by measuring the depletion of
activity from the cell lysate following removal of ImAd (this
approach was shown to be valid by immunoblotting material
bound by ImAd, and material remaining in the cell lysate following treatment with ImAd to detect Kex2p; data not shown).
This was necessary, since it was found that the presence of Pansorbin inhibited the Kex2 protease activity assay.
Sucrose gradient fractionation
Total cell extract was prepared from 250 A600 units of cells by
vortexing harvested cells in the presence of glass beads in 2 ml
55% sucrose (w/w) containing 10 mM MES, 2-(Nmorpholino)ethane sulphonic acid, pH 6.5, for 1 minute. The
extract was transferred to a centrifuge tube, and then overlaid with
the following sucrose solutions (as described by Bowser and
Novick, 1991): 1 ml 50%, 1 ml 47.5%, 1.5 ml 45%, 1.5 ml 42%,
1.5 ml 40%, 1 ml 37.5%, 1 ml 35%, 1 ml 30%, all containing 10
mM MES, pH 6.5. The gradients were spun at 170,000 g in a
SW41 rotor (Beckman Instruments Inc.) for 16 hours and 1 ml
fractions were collected from the top of the gradient for further
analysis.
For determination of the latency of a soluble secretory cargo
enzyme two lysates were prepared from yeast cells secreting the
bacterial enzyme β-lactamase. One lysate was prepared using glass
bead lysis and the other by homogenisation of spheroplasts. The
activity of β-lactamase in 5 µl of each lysate (equivalent to 0.5
A600 unit of original culture) was assayed using the substrate nitrocefin (O’Callaghan et al., 1972) in both the presence and absence
of 0.1% Triton X-100 in order to determine what proportion of
the enzyme was contained within membrane structures (5 mg
nitrocefin was dissolved in 0.5 ml DMSO before being diluted to
0.5 mg/ml with 0.1 M sodium phosphate, pH 7.0; 50 µl of this
stock nitrocefin solution was added to 900 µl 0.1 M sodium phosphate, pH 7.0, 0.8 M sorbitol ; the sample was then diluted into
50 µl of Solution B, added to the assay, and the increase in A490
was followed). The activity measured in the presence of detergent
was taken as the total lysate activity (lysate prepared by glass bead
lysis contained 0.12 arbitary unit of activity (∆A490/min) and that
prepared by homogenisation contained 0.09 unit) and the activity
measured in the absence of detergent was expressed as a percentage of this.
Digestion with Proteinase K
Proteinase K was added to samples to a final concentration of 50
µg/ml as indicated (Triton X-100 was added to a final concentration of 0.1% prior to this, where appropriate). The samples were
incubated on ice for 60 minutes after which PMSF was added to
a final concentration of 3 mM.
RESULTS AND DISCUSSION
Immunoisolation of Kex2p-containing membrane
vesicles
The Kex2 protein is a type I glycoprotein with a cytoplasmically disposed domain consisting of the C-terminal 115
residues of the protein (Fuller et al., 1989a). As the basis
for immunoisolation of Kex2p-containing organelles we
Fig. 1. Immunoisolation of Kex2p-containing membrane vesicles.
(a) The immunoisolated fraction contains Kex2p. Shown is an
immunoblot developed using antibodies directed against the Cterminal domain of Kex2p. Lane 1 contains a sample of total yeast
cell lysate; lane 2 contains material bound to a Kex2-specific
immunoadsorbent (ImAd) prepared using fixed S. aureus cells and
affinity-purified anti-Kex2p antibodies; and lane 3 contains
material bound to control fixed S. aureus cells.
(b) Immunoisolated Kex2p is in sealed membrane vesicles.
Samples of ImAd-bound material were exposed to proteinase K
digestion with and without the addition of detergent. Shown is an
immunoblot developed using antibodies directed against the Nterminal (luminal) domain of Kex2p.
prepared affinity-purified antibodies from a rabbit antiserum raised against a bacterially produced fusion protein
incorporating the C-terminal 100 residues of Kex2p. These
antibodies recognised a single polypeptide of relative molecular mass 135,000 in yeast protein extracts (Fig. 1(a), lane
1), which was identified as Kex2p: it was absent from a
lysate prepared from a strain carrying a disrupted copy of
the KEX2 gene and was present in elevated amounts in a
lysate prepared from a strain harbouring a multicopy plasmid carrying the KEX2 gene (data not shown).
Affinity-purified anti-Kex2p antibodies were bound to
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N. J. Bryant and A. Boyd
Fig. 2. Recovery of Golgi dipeptidyl aminopeptidase in the
immunoisolated membranes is dependent upon the method of cell
lysis. (a,c) Marker enzyme content of ImAd-bound material
isolated from a glass bead lysate (a) and from homogenised
spheroplasts (c). (b) Latency of a soluble secretory cargo enzyme
in cell lysates prepared either by glass bead lysis or by gentle
homogenisation of spheroplasts. The activity of β-lactamase in a
sample of each lysate was assayed in both the presence and
absence of Triton X-100 in order to determine what proportion of
the enzyme was contained within membrane structures. The
activity measured in the absence of detergent is expressed as a
percentage of total activity.
fixed S. aureus cells (Pansorbin) forming an immunoadsorbent (ImAd) that was used to isolate Kex2p-containing
membranes from yeast cell lysates prepared using glass
bead breakage. As shown in Fig. 1(a), the ImAd-bound
material was found to contain Kex2p, which was not
recovered when Pansorbin lacking bound antibodies was
used in place of ImAd (note that the heterogeneous smear
of immunoreactive material present in the bound fractions
is due to the presence of protein A-derived polypeptides
in the samples analysed). This recovery of Kex2p by ImAd
was completely blocked by preincubation of the ImAd
with the βGal-Kex2 fusion protein against which the
Kex2p-specific antibodies had been affinity-purified, but
not by preincubation with an unrelated βGal fusion protein (data not shown). The membrane association of the
ImAd-bound Kex2p was confirmed in a protease accessibility experiment (Fig. 1(b)). For this experiment it was
necessary to use an antibody directed against the luminal
domain of Kex2p. This antibody revealed the presence in
cell lysates of an additional, truncated form of the protein
(relative molecular mass 100,000) as has been previously
reported (Fuller et al., 1989a). Addition of proteinase K
to ImAd-bound material caused limited digestion of
Kex2p, resulting in its conversion to a polypeptide of relative molecular mass 100,000, consistent with the removal
of the C-terminal domain from the protein, whereas in the
presence of detergent the proteinase K completely digested
the protein. From these results, we conclude that the
Kex2p recovered using ImAd is contained within sealed
membrane vesicles, orientated with their N-terminal
domains inside, protected from externally added protease
and with their C-terminal domains exposed, as found
within the cell.
Under optimised binding conditions 80% of total lysate
Kex2 protease activity was removed from cell lysate by
ImAd (Fig. 2), whereas essentially none was bound by control Pansorbin (data not shown). Although it was not possible to assay Kex2 protease activity accurately in the bound
fraction due to interference by Pansorbin, the presence of
this proportion of lysate Kex2p in ImAd-bound material
was confirmed by immunoblot analysis (not shown). As
well as being recovered in high yield, the Kex2p-containing membranes were substantially free of activities associated with marker enzymes of other organelles (Fig. 2(a)).
Thus, ImAd-bound membranes containing 80% of cellular
Kex2p contained less than 1% of the endoplasmic reticulum (ER) membrane marker enzyme NADPH-cytochrome
c oxidoreductase (Roberts et al., 1991) and less than 0.5%
of the activities associated with two vacuolar enzymes
(Roberts et al., 1991): the integral vacuolar membrane protein dipeptidyl aminopeptidase B (DPAP B) and the soluble vacuolar enzyme CPY.
It is generally thought that Kex2p resides in a compart-
Immunoisolation of the yeast Golgi
ment of the yeast Golgi that lies distal to those active in
the addition of mannose residues to glycoprotein oligosaccharides. In agreement with this is the finding that membranes containing GDPase activity (a marker for all subcompartments active in mannosyltransfer reactions) are
separable on density gradients from Kex2p-containing
membranes (Bowser and Novick, 1991; and see Fig. 4a).
Immunoisolated membranes were found to lack GDPase
activity (Fig. 2a), consistent with the notion that our
immunoisolation procedure leads to the recovery of a single
Golgi compartment.
Co-recovery of Kex1p and DPAP A with Kex2p
The peptides released from the pro-α-factor precursor following Kex2 cleavage are further processed by two other
proteases (Julius et al., 1984; Fuller et al., 1988): the STE13
gene product, dipeptidyl aminopeptidase A (DPAP A)
(Julius et al., 1983), and a carboxypeptidase encoded by the
KEX1 gene (Kex1p) (Dmochowska et al., 1987). We anticipated that membranes isolated by virtue of their Kex2p
content might also contain these two proteins, but in fact
found that an ImAd-bound fraction containing 80% of cellular Kex2p contained less than 5% of lysate DPAP A
activity. Since glass bead breakage of yeast cells had been
used to generate the lysate for this experiment we tested
the idea that extensive fragmentation of the Kex2p-containing organelle might be taking place, resulting in physical separation of the two proteins into separate vesicles.
Accordingly, we turned to the use of a potentially more
gentle lysis method, homogenisation of spheroplasts. The
integrity of secretory pathway organelles in lysates prepared
by the two methods was assessed by determining the degree
of latency of a soluble secretory cargo protein. As shown
in Fig. 2b, the proportion of a soluble cargo protein remaining latent following cell lysis was greatly increased by the
use of spheroplast homogenisation, suggesting that disruption of organelles was much reduced. Using these lysis conditions it was still possible to recover 80% of the Kex2pcontaining membranes from a cell lysate without
compromising the purity of the fraction as determined by
assays of the ER and vacuolar membrane marker enzymes,
but the ImAd-bound material now contained 25% of lysate
DPAP A activity (Fig. 2c). Furthermore, when prepared
using this method, the immunoisolated material was found
to contain a large proportion of the cellular content of
Kex1p as revealed by immunoblotting (Fig. 3). In contrast
to its content of DPAP A and Kex1p, the material
immunoisolated from a homogenate did not contain
GDPase activity (Fig. 2c), indicating no co-recovery of
other Golgi membranes. This conclusion gains support from
an experiment in which immunoisolation was performed
from a lysate of yeast cells producing an epitope-tagged
version of the MNT1-encoded Golgi α(1-2) mannosyltransferase (Fig. 3b). This protein is thought to reside in a
medial compartment of the Golgi (Graham and Emr, 1991;
Nakajima and Ballou, 1975), and was found to be absent
from immunoisolated Kex2p-containing membranes (Fig.
3b). From these results we conclude that we have isolated
a single Golgi compartment on the basis of its content of
Kex2p and that this also contains the other two Golgi lateprocessing proteases, DPAP A and Kex1p.
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(a)
(b)
Fig. 3. (a) Immunoisolated organelles contain Kex1p. Immunoblot
analysis was performed on immunoisolated material using an antiKex1p antibody(supplied by Dr H. Bussey). Lane 1, membranes
prepared by centrifugation of the cell lysate at 100,000 g for 1
hour; lane 2, ImAd-bound material; lane 3, material bound by
control Pansorbin. The membrane fraction and the ImAd-bound
fraction (lanes 1 and 2) were prepared from the same amount of
cell lysate. (b) Immunoisolated organelles do not contain epitopetagged Mnt1p. Kex2p-containing organelles were isolated from a
homogenate prepared from the yeast strain 6210-mntmyc
(supplied by Dr S. Munro). These were shown, by immunoblot
analysis using an anti-c-myc monoclonal antibody (Oncogene
Science), not to contain any epitope-tagged protein. Lane 1, a
sample (equivalent to 10%) of the homogenate from which the
organelles were isolated; lane 2, ImAd bound material; lane 3,
material bound by control Pansorbin.
We have been unable to use our immunoisolation procedure to recover any more than 25% of cellular DPAP A
in Kex2p-containing organelles. It is of course possible that
Kex2p and DPAP A are based in the same organelle but
spend a proportion of their time in different retrieval or
recycling pathways, the existence of which have been
suggested by various workers (Franzusoff et al., 1991;
Seeger and Payne, 1992): if Kex2p and DPAP A were recycled from secretory vesicles to the Golgi in different populations of retrieval vesicles, then this would go some way
towards explaining our results. A second possible explanation arises from the idea that these proteins might be organised within the plane of the organellar membrane to form
homogeneous domains or patches of proteins. If this were
the case then our results would be explained by assuming
that Kex2p and DPAP A reside in different domains that
are completely separated by glass bead lysis but not by
gentle homogenisation of spheroplasts. Whilst we cannot
rule out the explanation that DPAP A resides, not in the
same compartment as Kex2p, but in a physically associated
compartment that is specifically recovered along with that
containing Kex2p, we currently favour some combination
of the above explanations in which the Kex2p-compartment
is the last stable compartment of the Golgi.
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N. J. Bryant and A. Boyd
Fig. 4. Separation of Kex2p- and GDPase-containing membranes
by sucrose density centrifugation. (a) The separation of Kex2
protease activity from that associated with GDPase obtained using
the yeast strain NBY10. (b) and (c) The profiles obtained using
cells bearing the temperature-sensitive sec7-1 mutation grown
under permissive (b) and restrictive (c) conditions. (d)
Demonstration of the recovery of GDPase activity from fractions
of the gradients described in (b) and (c). In this experiment all of
the Kex2 activity was recovered by ImAd from each of the
fractions.
Perturbation of Golgi organisation in a sec7
mutant
We were interested to see whether physiological conditions
could be found under which the nature of the Kex2p-containing compartment was altered significantly. Morphological evidence suggests that the organisation of the Golgi is
altered in sec7 mutant cells at the restrictive temperature
(Novick et al., 1980). In order to see whether these changes
result in a loss of the ability to separate membranes containing Kex2p from those containing GDPase, we attempted
immunoisolation from such cells. Fig. 4 shows the results
of sucrose density gradient analysis of membranes isolated
from a sec7 mutant at the permissive (25°C) and restrictive
(37°C) temperatures. Following growth at the permissive
temperature, Kex2 protease and GDPase activities were
clearly separated on the gradient (Fig. 4(b)), whereas when
cells had been exposed to the restrictive temperature,
GDPase activity shifted down the gradient to overlap extensively with the Kex2p distribution (Fig. 4(c)). We found
that it was not possible to use ImAd to isolate Kex2p-containing membranes directly from lysates prepared from sec7
cells grown at 37°C, since most of the cellular Kex2p was
now present in the pellet of the clearing spin performed
prior to immunoisolation. We therefore immunoisolated
Kex2p-containing membranes from gradient fractions spanning the peak of Kex2 activity. As shown in Fig. 4(d), the
membranes immunoisolated from cells grown at the permissive temperature did not contain any significant GDPase
activity, whereas up to 30% of the GDPase activity present
in the relevant gradient fractions of membranes prepared
from cells grown at the restrictive temperature was recovered along with Kex2p. The ability of ImAd to co-recover
GDPase and Kex2p from sec7 mutant cells exposed to the
restrictive temperature is consistent with the idea that Golgi
organisation changes under these conditions: it is possible
that a partial coalescence or mixing of the two compartments occurs, perhaps due to a breakdown in retention or
retrieval systems; or, alternatively, the two compartments
may remain intact but become more tightly associated with
one another. Further analysis of the nature of the organelles
immunoisolated from sec7 cells may help to clarify this
point.
CONCLUSIONS
Our results extend and strengthen current views of Golgi
organisation in yeast cells. By investigating the post-translational modifications carried by secretory cargo proteins
accumulated during arrest of a sec18 mutant, Graham and
Immunoisolation of the yeast Golgi
Emr (1991) were led to conclude that the yeast Golgi is
divided into a minimum of three subcompartments: a cis
compartment containing α(1-6) mannosyltransferase; a
medial compartment containing Mnn1p-dependent α(1-3)
mannosyltransferase; and a trans compartment containing
Kex2p. This placement of Kex2p in a separate post-ER
compartment was consistent with earlier work, which had
demonstrated physical separation of organelles containing
either α(1-3) mannosyltransferase or GDPase activity from
those containing Kex2p (Cunningham and Wickner, 1989;
Bowser and Novick, 1991). Thus, all of the evidence available hitherto has placed the Kex2p-containing compartment
near the ER-distal limit of the yeast Golgi. Our data
strengthen this view, since they remove any need to postulate the existence of a subsequent compartment: all known
late-processing enzymes (Kex2p, DPAP A and Kex1p)
seem to be present in this single organelle. Presumably, this
organelle is that which has been detected by immunofluorescence studies of the localisation of both Kex2p (Redding
et al., 1991) and Kex1p (Cooper and Bussey, 1992).
Analogous processing proteases in mammalian cells are
associated with the trans-Golgi network, which is also
thought to be the site at which various classes of proteins
are sorted (Griffiths and Simons, 1986; Sossin et al., 1990).
Since it is clear that in yeast cells both secretory and vacuolar cargo traverse the Kex2p compartment (Graham and
Emr, 1991), we are led to the conclusion that the organelle
we have isolated may be the functional equivalent of the
trans-Golgi network in that it is the site at which sorting
occurs of proteins destined for the cell surface and the vacuole.
Immunoisolation has proved to be an important technique
in cell biology, contributing to the understanding of both
structure and function of organelles (Franzusoff et al., 1992;
Salamero et al., 1990). We envisage that the availability of
our immunoisolation procedure will pave the way for further characterisation of the molecular composition of the
Kex2p compartment and contribute to the development of
cell-free assays of membrane traffic events in this area of
the yeast secretory pathway.
We thank Paul Whitley for his contribution to this work, Gustav
Ammerer for providing a plasmid carrying KEX2, Howard Bussey
and Sean Munro for their generous gifts of anti-Kex1p antiserum
and the 6210-mntmyc strain, respectively, the Scottish Antibody
Production Unit for supplying antibody conjugates and Gwyn
Gould for suggesting the use of Pansorbin. Jeff Haywood is
thanked for his help as is Stella Hurtley for her useful discussions.
This work was supported by grants from the UK Science and Engineering Research Council and the Wellcome Trust.
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(Received 21 June 1993 - Accepted 28 July 1993)