Article |
Address correspondence to Randy Schekman, Dept. of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720. Tel.: (510) 642-5686. Fax: (510) 642-7846. E-mail: schekman{at}UCLink4.berkeley.edu
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Abstract |
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Key Words: protein sorting; TGN; endosome; VPS; yeast secretion
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Introduction |
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Secretory proteins are sorted and packaged into various types of vesicles at the trans-Golgi network (TGN),*and cargo bound for lysosomes is sorted at this point from most proteins destined for the cell surface (Keller and Simons, 1997; Traub and Kornfeld, 1997). Many lysosomal proteins probably first reach early endosomes where they are again sorted into carrier vesicles that transport them to late endosomes (Ludwig et al., 1991; Press et al., 1998), whereas other lysosomal proteins bypass early endosomes and are transported to lysosomes either directly or via late endosomes or some other intermediate compartment (Cowles et al., 1997b; Piper et al., 1997; Stepp et al., 1997; Press et al., 1998). Although it is not known whether the latter route transits late endosomes or is direct to lysosomes, this distinction may not be significant because late endosomes fuse with lysosomes (Luzio et al., 2000), and the two compartments are thought to be in a dynamic equilibrium (Mellman, 1996).
Like the TGN, early endosomes have a highly tubular morphology and are specialized for protein sorting (Mellman, 1996; Lemmon and Traub, 2000; Woodman, 2000). Endocytosed proteins destined for degradation are sorted in early endosomes for transport to late endosomes along with newly synthesized lysosomal proteins, whereas most plasma membrane proteins recycle back to the cell surface. Transport from early endosomes to the plasma membrane has been best characterized for the trafficking of transferrin receptor (Mellman, 1996; Brown et al., 2000) and of synaptic vesicle components (Hannah et al., 1999). However, recycling and transcytotic proteins and membranes are not the only cargo that reach the cell surface from early endosomes; two illustrations of newly synthesized surface proteins passing through early endosomes are the biosynthetic transport of transferrin receptor (Futter et al., 1995) and asialoglycoprotein receptor H1 (Leitinger et al., 1995; Laird and Spiess, 2000). Major histocompatibility complex class II molecules are also transported directly from the TGN to various endosomal compartments where they bind partially degraded endocytosed antigens to present them on the cell surface (Wolf and Ploegh, 1995). Major histocompatibility complex class IIcontaining compartments were once believed to be specialized organelles with late endosome and lysosome-like properties unique to antigen-presenting cells, but it now appears that they include conventional compartments of perhaps all stages of the endocytic pathway (Kleijmeer et al., 1997). Although biosynthetic transport through endosomal compartments may exist in all cell types, these are most likely relatively minor routes, since time-lapse imaging of green fluorescent proteintagged cargo molecules in polarized cells revealed only a direct TGN-to-plasma membrane route (Keller et al., 2001).
The complexity of the late secretory pathways has made it difficult to characterize both the many transport routes and the molecular machinery involved in cargo sorting and vesicle formation. Genetic screens in the yeast Saccharomyces cerevisiae have identified a large number of mutants that are blocked at various points along the exocytic, endocytic, and vacuolar/lysosomal pathways, and these mutants have greatly facilitated the identification of components required for membrane and protein trafficking in all eukaryotic cells (for reviews see Stack et al., 1995; Kaiser et al., 1997; Conibear and Stevens, 1998). Secretory cargo proteins appear to share similar requirements in ER-to-Golgi transport and in vesicle targeting and fusion with the cell surface, and the many proteins that mediate these transport steps have been particularly well characterized (Kuehn and Schekman, 1997; Guo et al., 2000). However, relatively few mutants have been found that block transport of exocytic cargo from the Golgi, and in both yeast and mammalian cells much less is known about the machinery responsible for sorting and packaging cargo into the vesicles that transport them to the cell surface.
The purification and analysis of secretory vesicles that accumulate in late (post-Golgiblocked) exocytic yeast mutants has identified two vesicle populations with different densities and distinct enriched cargo, indicating that exocytic cargo can be transported by at least two routes (Harsay and Bretscher, 1995; David et al., 1998). In the past, defects in yeast exocytosis may have gone undetected because only a single secretory enzyme (usually invertase) was followed. Furthermore, cargo in a blocked pathway may be rerouted and secreted by an alternative unblocked route. Therefore, assaying for the transport of cargo in both exocytic pathways should allow identification of new exocytic mutants and reassessment of mutants judged previously as normal for exocytosis. In the present study, we show that some vacuolar protein sorting (VPS) proteins play an important role in cargo transport in at least one branch of the exocytic pathway, and we present evidence suggesting that this pathway may transit an endosomal compartment before reaching the cell surface.
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Results |
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Like vps4,
vps27 blocks exit from an endosomal compartment and traps
vacuolar and endocytosed proteins and recycling Golgi proteins in an
exaggerated endosome (Piper et al.,
1995), a characteristic of defects in 13 VPS genes
grouped together as class E (Raymond
et al., 1992). However, unlike vps4
sec6-4, the vps27
sec6-4 mutant did not
missort invertase into light-density vesicles; instead, invertase
accumulated at an intermediate density peak (Fig. 1, F and G). Because the density of
sec6-4 invertase vesicles was consistent between fractionation
experiments, this density shift is significant and indicates either
altered properties of the invertase exocytic vesicles or the missorting
or accumulation of invertase in some other compartment. We favor the
latter possibility, as a vps27 mutant but not a vps4
mutant (without a sec6-4 background) has a mild secretory
defect (unpublished data).
A vps mutant that lacks the
sorting receptor for CPY, vps10 (Marcusson et al., 1994), was capable of sorting
invertase properly (Fig.
1 E). However, unlike for all other mutants fractionated
invertase sorting in the vps10
sec6-4 mutant
varied between experiments. Lowering the pH of the growth medium to pH
4.5 (from pH 6.5 in standard rich medium reduced missorting, suggesting
that a lower pH may be more optimal for proper invertase sorting.
However, for all mutants except vps10
sec6,
lowering the pH of the medium made no difference other than resulting in
slightly lower levels of invertase activity. All results shown in
Figs. 1 and 2
are from experiments in which cells were shifted into pH 4.5
medium.
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Although a
vps4 mutation does not cause a kinetic lag in invertase
transport (unpublished data), it is possible that when combined with a
complete secretory block caused by the sec6-4 mutation,
invertase is backed up into an upstream compartment. We wished to
explore this possibility by shifting a vps4
sec6-4 strain to a semipermissive temperature at which there is
only a small block in invertase transport. As shown in Fig. 2, all invertase was still in
light-density vesicles under these conditions. Therefore, it is likely
that invertase is transported in light-density vesicles in the
vps4
mutant without a secretory block, and the
invertase-containing membranes that accumulate in vps4 sec6-4
cells represent exocytic vesicles rather than an upstream
compartment.
CPY and invertase cofractionate in
vps sec6 mutants
Exocytic vesicles that accumulate in
sec6-4 cells did not cofractionate (copeak) with other
organelles (Fig. 3 A;
Harsay and Bretscher,
1995), although Sec4p on exocytic vesicles was only slightly
denser than the early endosome/Golgi syntaxin Tlg1p, so we cannot
exclude the possibility that Tlg1p is present on light-density vesicles.
The fractionation of the TGN/endosomal marker Kex2p was difficult to
assess, since Kex2p is known to become unstable in cells shifted to
37°C (Wilcox et al.,
1992), and this instability was exacerbated in
sec6-4 cells (Fig.
3 A). In wild-type cells, Kex2p peaks at the top of the
gradient and in intermediate-density fractions (Fig. 3 B) that correspond to the density of
accumulated invertase in vps27 sec6-4 cells (Figs. 1 and 2). Kex2p is believed to cycle between a late
Golgi compartment and endosomes (Cereghino et al., 1995; Bryant and Stevens, 1997), but the
identities and relative densities of Golgi and endosomal compartments in
density gradients are unclear (Singer-Krüger et al., 1993; Holthuis et al., 1998a). The late
endosomal syntaxin, Pep12p, the ER membrane protein, Sec61p, and the
vacuolar membrane protein, alkaline phosphatase (ALP), clearly do not
copeak with exocytic vesicles (Fig.
3 A). Similar results were obtained for vps sec6
cells (unpublished data; we did not examine Kex2p and Tlg1p in
double mutants).
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Immunoisolated
Pma1p-transporting vesicles contain invertase in vps sec6
mutants
The cofractionation of Pma1p and missorted invertase in
vps sec6-4 mutants suggests that the proteins may be packaged
into a common carrier. However, it is also possible that invertase is
missorted into a different class of vesicles with fractionation
properties very similar to that of Pma1p-transporting vesicles. To
distinguish between these two possibilities, we immunoisolated
Pma1p-transporting vesicles from vps sec6-4 mutants and
assessed whether these vesicles contain invertase (Figs. 4 and 5).
Immunoisolations were performed with membranes fractionated on Percoll
step gradients. The purpose of gradient fractionation was to separate
light and dense secretory vesicles and to remove soluble invertase
released from organelles and float vesicles away from proteases that
reduce immunoisolation efficiency. A Percoll gradient was used rather
than Nycodenz in order to maintain osmotic conditions during the
gradient fractionation and immunoisolation procedures. We found that the
invertase vesicles are particularly sensitive to osmotic changes. The
low viscosity of Percoll also enabled organelles to reach equilibrium
density after a 1-h centrifugation so that immunoisolation of fragile
vesicles could be performed more quickly. Fig. 4 A shows the Percoll gradient fractionation
profile of invertase from sec6-4 and vps1
sec6-4 cells. Using two different anti-Pma1p monoclonal
antibodies bound to magnetic beads (see Materials and methods), we could
isolate close to 60% of the invertase present in vps1 sec6
invertase peak fractions (Fig.
4 B). Very similar results were obtained for the light
vesicle cargo protein Bgl2p (Fig.
4 C). The fraction of invertase and Bgl2p not isolated may
correspond to leakage from the vesicles or their presence in
cofractionating organelles with lower amounts of Pma1p. To demonstrate
the specificity of the immunoisolation procedure, we used peptides
corresponding to the mapped epitopes of each of the antibodies
(Serrano et al., 1993)
in competition experiments (Fig. 4, B
and C). In each case, the corresponding peptide competed
specifically with the monoclonal antibody, with the peptide for antibody
#17 being a more effective competitor (most likely due to a closer
resemblance to the corresponding sequence in the folded native
protein).
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Thin section
electron microscopic examination of immunoisolated membranes from
vps4 sec6-4 cells indicated 100-nm vesicles and
some tubular membranes bound to the beads (Fig. 6).
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Discussion |
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A likely explanation for the requirement of these genes in one branch of the late exocytic pathway is that a subset of exocytic cargo transits through an endosomal compartment before reaching the cell surface. Invertase and vacuolar proteins in the CPY pathway may be transported together from the late Golgi to endosomes where they are sorted to the cell surface and to the vacuole, respectively. When the common pathway to endosomes is blocked, both invertase and CPY are rerouted to the cell surface via light secretory vesicles. Consistent with this model, we find that two mutants that are blocked in a vacuolar pathway that bypasses endosomes, apl6 and vps41, have little or no effect on the sorting of invertase. A class E vps mutant, vps4-ts, which blocks the exit of vacuolar, endocytosed, and recycling Golgi proteins from endosomes, rapidly missorts invertase into light vesicles, presumably because components required for normal invertase sorting or transport are not recycled from endosomes. Two other newly synthesized yeast proteins, a mutant form of the plasma membrane ATPase Pma1p (Luo and Chang, 2000) and the iron oxidase Fet3p (Radisky et al., 1997; Yuan et al., 1997), have been proposed to reach the cell surface from a post-Golgi compartment.
An alternative explanation for the missorting of exocytic cargo in vps mutants is that traffic between the Golgi and endosomes is somehow required for the maintenance of the normal sorting competence of the Golgi. Although we cannot rule out this possibility, the rapid onset of invertase missorting in conditional vps mutants suggests a more direct role of VPS proteins in invertase transport. The characterization of organelles that accumulate in vps mutants may provide further evidence for such a direct role, but our preliminary attempts to do so were hindered by the relatively small amounts of accumulated proteins (consistent with efficient missorting in many vps mutants). However, we have immunoisolated clathrin-coated vesicles from wild-type cells and showed that they contain invertase. Clathrin has been shown to play a role in Golgi-to-endosome transport in both yeast and mammalian cells (Kornfeld and Mellman, 1989; Seeger and Payne, 1992) but also mediates other transport routes by associating with different adaptin complexes (Robinson and Bonifacino, 2001).
Invertase and CPY transport in the
vps10 sec6 mutant
A vps10 sec6 mutant, which
lacks the sorting receptor for CPY (Marcusson et al., 1994), accumulates both
invertase and CPY in dense vesicles, although this result was variable
and influenced by the pH of the growth medium (unpublished data). An
initial expectation for this mutant was the missorting of CPY into both
exocytic pathways, or primarily into light vesicles, which are the most
abundant membranes in our gradients (Harsay and Bretscher, 1995). However, CPY can be
sorted into dense vesicles even in the absence of Vps10p, indicating a
Vps10p-independent sorting step. Therefore, there may be two sorting
steps for CPY, first at the Golgi and then at the early endosome, and
only the second sorting step may be directly dependent on Vps10p. The
effect of growth conditions on sorting may reflect secondary effects due
to defective transport of Vps10p-dependent cargoes. Another possibility
is that Vps10p functions at both the Golgi and endosome, but an
alternative pH-sensitive sorting mechanism also functions at the Golgi.
A pH-sensitive sorting step has been indicated for another soluble
vacuolar hydrolase, proteinase A, which is sorted to the vacuole in the
absence of Vps10p in low pH media, but its efficient sorting requires
Vps10p when the pH of the growth medium is >5.0 (Seaman et al.,
1997).
Although Vps10p has been proposed to function in sorting from the Golgi (Marcusson et al., 1994; Cooper and Stevens, 1996), it is not surprising that it may also sort cargo at early endosomes. A recycling pathway from endosomes to the plasma membrane has been demonstrated for yeast (Holthuis et al., 1998b; Chen and Davis, 2000; Wiederkehr et al., 2000); therefore, proteins en route to the vacuole need to be sorted from recycling proteins at the endosome. The mannose 6-phosphate receptor, which sorts soluble lysosomal enzymes from the Golgi to early endosomes (Press et al., 1998), is found predominantly in late rather than early endosomes (Bleekemolen et al., 1988; Griffiths et al., 1988) consistent with a role in early endosome to late endosome sorting before recycling back to the Golgi.
The role of class E VPS
proteins in exocytic and vacuolar cargo transport
A better
understanding of the functions of class E VPS proteins may help to
clarify the roles of early and late endosomes in yeast. The different
effects of two class E mutants, vps4 and vps27, on
invertase transport suggests that Vps4p and Vps27p may regulate
different transport steps either from the same compartment or from
different compartments. Although vps4 and vps27 are
the most well-characterized class E mutants and have very similar
phenotypes (Piper et al.,
1995; Babst et al.,
1997), the same prominent phenotype (accumulation of the
"class E compartment") may be a more direct effect of one
mutation than of the other, or the mutations may have different effects
on additional organelles. Perhaps one mutation blocks exit primarily
from early endosomes, whereas the other blocks exit from late endosomes
or from a different type of early endosome. Several types of early
endosomes have been recognized in mammalian cells based on their
distinct morphologies and functions (Brown et al., 2000; Lemmon and Traub, 2000). Yeast endosomes are much
less clearly characterized; however, the existence of three
different Rab proteins that function in the early endocytic pathway
(Singer-Krüger et al.,
1994) suggests a similar complexity. The vps27
mutant appears to have a more severe effect on invertase transport than
other vps mutants, and its mammalian homologue, Hrs, has been
localized to early rather than late endosomes (Hayakawa and Kitamura, 2000; Raiborg et al., 2001). Cargo in the
invertase-transporting pathway may, therefore, transit early endosomes
rather than (or in addition to) late endosomes. Direct traffic from the
TGN to early endosomes is also consistent with other examples in which
newly synthesized proteins transit an endosome before exocytosis
(Futter et al.,
1995; Leitinger et
al., 1995; Sariola
et al., 1995). However, we found a strong effect on
invertase sorting by pep12 mutations (thought to block vesicle
fusion with late endosomes), so it is possible that invertase is
transported from early to late endosomes from which it then reaches the
cell surface. CPY and other vacuolar proteins may likewise traffic
through both early and late endosomes as has been shown for lysosomal
hydrolases in mammalian cells (Ludwig et al., 1991; Press et al., 1998). If such is the
case, then some VPS proteins are likely to function primarily in early
endosome-to-late endosome transport, and missorting takes place in early
endosomes rather than at the TGN.
Why transport
exocytic cargo through endosomes?
All previous examples of newly
synthesized proteins transiting through endosomes before reaching the
cell surface are of membrane proteins that have special functions in, or
recycle through, endosomal compartments, so it is not entirely
unexpected that they are targeted to endosomes directly from the Golgi.
However, no prior evidence exists for newly synthesized soluble exocytic
proteins transiting endosomes en route to the cell surface, and this is
thought to be a specialized rather than a general route. Why might
soluble cargo transit through an early endosomal compartment? The answer
to this question may also provide an explanation for why yeast exocytic
cargo is sorted into separate pathways.
One reason for divergent exocytic pathways in yeast may be that different types of cargo may have different processing requirements and are therefore routed through different compartments. Alternatively, sorting into divergent routes may reflect the need to regulate differentially a pathway mediating surface expansion and a pathway exporting soluble proteins destined for release from the cell. This possibility is consistent with the nature of the cargo thus far identified in the two pathways. Another feature of markers in dense exocytic vesicles is that they are required only under certain physiological conditions (invertase in low glucose and acid phosphatase in low phosphate). Although both secreted invertase and acid phosphatase are regulated at the transcriptional level (Johnston and Carlson, 1992), a second level of regulation in the form of differential trafficking may exist also, and this form of regulation may be important for other cargo that share this pathway. One such cargo may be the general amino acid permease Gap1p, which is regulated both transcriptionally and posttranslationally by differential sorting in the late secretory pathway (Roberg et al., 1997). When cells are grown on urea, Gap1p is transported to the plasma membrane, whereas in cells grown on glutamate Gap1p is sorted to the vacuole. Perhaps proteins that are required only under certain growth conditions are first transported to early endosomes, which receive traffic from and may have sorting properties affected by the external environment, and from this point the various cargoes are sorted either to the plasma membrane or to late endosomes for vacuolar degradation. This type of regulation may be useful especially for proteins whose external levels need to be rapidly adjusted to environmental conditions or for soluble proteins, which unlike membrane proteins cannot be efficiently retrieved from the cell surface if they are no longer needed.
The involvement of VPS proteins in one branch of the yeast exocytic pathway shows promise for the isolation of additional mutants that block transport of only a subset of exocytic cargo from the Golgi or from endosomes. We have yet to identify mutants that specifically block the major, Pma1p-transporting, pathway, and screening for a complete secretory block in a vps mutant background may facilitate the isolation of such mutants. The characterization of more mutants blocked uniquely in one exocytic pathway should contribute to our knowledge of exocytic cargo sorting and vesicle formation at the late Golgi.
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Materials and methods |
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Yeast
strains and plasmids
Yeast strains used in this study are listed
in Table I. Standard yeast genetic
techniques were used to perform crosses and tetrad analysis (Guthrie and Fink, 1991). Yeast
transformations were by the lithium acetate method (Schiestl and Gietz, 1989). For
plasmid construction, DNA fragments were isolated using a QIAEX II kit
from QIAGEN, and plasmid DNA was isolated using a QIAPrep Spin kit
(QIAGEN). Escherichia coli transformations were performed using
INVF' competent cells from Invitrogen. All other recombinant
DNA techniques were performed as described by Ausubel et al. (1987).
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To construct a temperature-sensitive clathrin mutant in
our strain background, we used the integrating plasmid
YIpchc521-ClaI (Tan et al.,
1993) to replace the wild-type copy of CHC1 in the
strain NY10, congenic with NY17 (Salminen and Novick, 1987); the resulting
strain was then crossed with EHY226 (sec6-4) to generate the
chc1-521 sec6-4 mutant (confirmed by complementation
analysis).
Subcellular fractionation
Overnight
primary yeast cultures (OD600 0.52) were
inoculated into YPD or SD with required amino acids (500 ml culture per
gradient) and grown for 1216 h at 24°C to OD600
0.50.7. For SD cultures, growth was continued for an additional 2
h at 24°C in 250 ml YPD to allow cells to adjust to rich medium.
Cells were then shifted to 37°C for 60 min (unless otherwise
indicated) in 250 ml prewarmed YPD to induce the sec6-4
secretory block. All cells constitutively expressed invertase (Suc2p),
so it was not necessary to derepress SUC2 by lowering glucose
concentrations. Conversion of cells to spheroplasts and lysis was
performed as described by Harsay and
Bretscher (1995) except spheroplasts were washed only once
and lysis was performed with 15 strokes in a Dounce homogenizer,
sufficient to result in
6090% cell lysis. Fractionation of
secretory vesicles was performed as described (Harsay and Bretscher, 1995) with the following
changes: for differential centrifugation, a 700-g spin was not
performed routinely so that the first spin was at 13,000 g for
20 min. The supernatant from this spin was divided into two 17-ml tubes
per gradient (SW28.1; Beckman Coulter), and a 70 µl cushion of
40% Nycodenz in lysis buffer was placed through the sample onto the
bottom of each tube with a Pasteur pipette. After centrifugation at
100,000 gav for 90 min, the supernatants were
removed by pipetting, leaving
400 µl at the bottom of each
tube. The small cushion, which was mixed with the membranes before
loading gradients, allowed easy resuspension so that incubation on ice
to loosen pellets was not necessary. Linear 1530% Nycodenz/0.8 M
sorbitol gradients were formed in 12.5-ml tubes (SW41; Beckman
Coulter) using a two-chambered gradient mixer attached to an Auto
Densi-Flow II C fractionator (Labconco). Membranes were mixed with 80%
Nycodenz in lysis buffer so that the load volume for each gradient was
1.5 ml in
32% Nycodenz. The membranes were loaded through the
gradients and centrifuged at 100,000 gav for
1516 h (to equilibrium); 0.4-ml fractions were collected
from the top using an Isco Model 640 density gradient fractionator.
Fraction densities were determined by reading refractive indices on a
Bausch and Lomb refractometer and converting these values to g/ml based
on a standard curve generated by five weighed standards. The equation
for the standard curve is
= 3.384
-3.536,
where
is density in g/ml and
is the refractive
index.
For Percoll density gradient fractionation, we shifted
vps1 sec6-4 and sec6-4 cells for 40 min
and vps4-ts sec6-4 for 30 min in YPD at 37°C. A high speed
spin (100,000 gav) membrane fraction was prepared as
above except rather than a Nycodenz cushion a 200 µl 67% Percoll
solution in lysis buffer was placed through the samples into the bottoms
of the SW28.1 tubes. When preparing the 67% Percoll solution, we
adjusted the sorbitol buffer concentration to account for the
significant volume taken up by Percoll particles. A 25 ml 67% Percoll
solution contained 16.75 ml Percoll, 7.5 ml 3x
sorbitol-triethanolamine lysis buffer (3x is 2.4 M sorbitol, 30 mM
triethanolamine, 3 mM EDTA, pH 7.2), and 0.75 ml H2O;
final pH was adjusted to pH 7.2 with acetic acid. The membranes were
adjusted to 58% Percoll in 3.5 ml (475 µl membranes, 3.025 ml 67%
Percoll solution), placed into the bottom of an SW41 tube, and the
following gradient steps were added: 2 ml 55%, 1.5 ml 50%, 1.5 ml 40%, 2
ml 30%, and 1.5 ml 20% Percoll. The steps were prepared by mixing lysis
buffer containing 2 mg/ml BSA with 67% Percoll solution that contained 2
mg/ml BSA. The gradients were spun for 1 h at 100,000
gav, and 0.5-ml fractions were collected from the
top. Percoll gradient fractionation of wild-type cells for the isolation
of clathrin-coated vesicles was performed in a similar manner with the
following changes. Cells were grown at 30°C in YPD. Lysis was
performed in a buffer optimized for clathrin coat stability (as in
Payne and Schekman,
1985; except with higher sorbitol concentration): 100
mM MES-NaOH, pH 6.5, 0.8 M sorbitol, 0.5 mM MgCl2, 1 mM EGTA,
2 mM DTT, and protease inhibitor cocktail (Compete Mini, EDTA free;
Roche). The 67% Percoll solution was made up with this lysis buffer in
the manner described above for sorbitol-triethanolamine lysis buffer.
The high speed spin membranes were adjusted to 58% Percoll in 2.6 ml,
loaded into an SW41 tube, and 1.5 ml of each of the following steps were
added: 55, 50, 40, 35, 30, and 25% Percoll. The gradients were spun for
1 h at 100,000 gav, and 0.5 ml fractions were
collected from the
top.
Immunoisolations
Anti-Pma1p monoclonal
antibodies (#15 and #17) (Serrano et
al., 1993) were purified from hybridoma tissue culture
supernatants by ammonium sulfate precipitation (60% saturation) followed
by protein A chromatography using binding and elution buffers optimized
for mouse IgG1 (Pierce Chemical Co.). Peak fractions from the protein A
column were exchanged into 50 mM sodium phosphate, pH 6.9, using
Excellulose desalting columns (Pierce Chemical Co.) and stored in this
buffer with 5 mg/ml BSA, 10 mM NaN3. Affinity purified
polyclonal anti-Clc1p antibodies were prepared as follows: antiserum
against GST-Clc1p (Deloche et al.,
2001) was preadsorbed against fresh cells with a deletion of
CLC1, and immunoglobulins were purified on T-Gel columns
(Pierce Chemical Co.). GST and GST-Clc1p were purified according to a
protocol from Amersham Pharmacia Biotech and crosslinked to Amino-Link
Plus coupling gel (Pierce Chemical Co.) for preparation of affinity
columns. Anti-GST antibodies were purified from the immunoglobulin
preparation, and the remaining IgG was used for affinity-purifying
anti-Clc1p according to Harlow and
Lane (1988)(. Antibodies were concentrated on protein A
columns, desalted, and stored as described above for
anti-Pma1p.
Immunoisolations were performed with magnetic Dynabeads (Dynal). Dynabeads protein G were incubated overnight with purified antibodies, and bound antibodies were quantified by SDS-PAGE. Anti-Pma1p beads contained 0.1 µg antibody/µl beads; anti-Clc1p and anti-GST beads contained 0.2 µg antibody/µl beads. In one experiment (Fig. 6), Dynabeads M-500 subcellular were used, which were prepared according to the manufacturer's instructions and contained 40 ng primary antibody/µl beads. For immunoisolation of Pma1p-containing vesicles, we prepared a 1-ml reaction containing Dynabeads (amounts as specified), lysis buffer, 5 mg/ml BSA, and 30 µl membranes from Percoll gradient peak fractions obtained by fractionating 1 g of cells. The reactions were rotated gently at 4°C for 2 h and washed twice over 2 h. The beads were resuspended in 200 µl lysis buffer (5x original concentration). For immunoisolation of clathrin-coated vesicles, we mixed Dynabeads with 200 µl membranes from clathrin-enriched Percoll gradient fractions obtained by fractionating 1 g of cells in a final reaction volume of 1 ml. Incubations and washes were the same as described for anti-Pma1p, and washed beads were resuspended in 250 µl lysis buffer (4x original concentration).
Peptide competition experiments to confirm the specificity of the anti-Pma1p immunoisolations were as follows: peptides corresponding to the mapped epitopes of each antibody (VSAHQPTQEKPAKTYDDAAS for antibody #15 and IEELQSNHGVDDEDSDNDG for antibody #17 [New England Peptide]) were dissolved in water at 2 mg/ml. Beads bound with antibodies were preincubated with peptide (40 µg peptide per 1 µg bound antibody, 400 µl volume), and 100 µg peptide (50 µl stock) was included in a 1-ml immunoisolation reaction containing 2.5 µg bound antibody.
EM of
immunoisolated membranes was performed as described (Harsay and Bretscher, 1995) except
the beads were mixed 1:1 with 4% intermediate melting temperature
agarose after the primary fixation and were processed as 1
mm3 cut agarose chunks.
Protein and enzyme
assays
For invertase reactions, Nycodenz gradient fractions were
assayed as described (Goldstein and
Lampen, 1975; Harsay and Bretscher, 1995). Units are expressed
as nmol glucose produced per min, per µl fraction, normalized to 1 g
wet cells. Samples from Percoll gradient fractions and immunoisolations
were assayed in a similar manner except longer reaction times were used
(2050 min), and the reactions were spun after the boiling stop
bath to remove precipitated Percoll or beads before the glucose assay.
Exoglucanase activity was determined as described (Harsay and Bretscher, 1995) except
that rather than 6-h incubations, 1020 µl of undiluted
fraction in 250 µl reaction buffer was incubated for 912 h at
30°C; activities are expressed as the percent total of the
activity measured in the fractions. ATPase and GDPase activities were
assayed as described (Harsay and
Bretscher, 1995); units are expressed as pmol inorganic
phosphate produced per min per µl fraction, normalized to 1 g wet
cells. The bottom three fractions of the gradients (load volume)
contained high levels of soluble enzymes and were not routinely
assayed.
Immunoblotting was performed as described (Harsay and Bretscher, 1995) except a tank transfer apparatus (Amersham-Pharmacia Biotech) was used to transfer proteins. Rabbit polyclonal antisera were used to detect Bgl2p, Sec61p, and CPY (this laboratory) and Tlg1p (Holthuis et al., 1998a). Monoclonal antibodies from Molecular Probes, Inc. were used to detect ALP, Vps10p, and Pep12p. A monoclonal antibody (Brennwald and Novick, 1993) was used to detect Sec4p. Anti-HA monoclonal antibody HA.11 from Covance was used to detect HA-Kex2p and HA-Pma1p. HRP-conjugated secondary antibodies were from Amersham Pharmacia Biotech. Blots were developed using the standard ECL or ECL-Plus kits (Amersham Pharmacia Biotech).
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Footnotes |
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (GM26755) and the Human Frontier Science Program (G-501-95) to R. Schekman. E. Harsay was supported in part by a National Research Service award. R. Scheckman is an investigator of the Howard Hughes Medical Institute.
Submitted: 12 November 2001
Revised: 7 December 2001
Accepted: 7 December 2001
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References |
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