Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK
* Author for correspondence (e-mail: k.ayscough{at}bio.gla.ac.uk)
Accepted 4 March 2003
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Summary |
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Key words: Sla1p, Sla2p, Actin, Yeast, Endocytosis
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Introduction |
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Characterisation of Sla1p has revealed that it is a multivalent adaptor
protein required for cortical actin patch structure and organisation in
budding yeast (Ayscough et al.,
1999). Sla1p has three Src homology-3 (SH3) domains in its
N-terminal third and a C-terminal domain composed of multiple repeats rich in
proline, glutamine, glycine and threonine
(Fig. 1A). These repeats also
contain motifs for phosphorylation by the actin-regulating kinase Prk1p
(Zeng et al., 2001
). We have
recently demonstrated that Sla1p can act as an adaptor protein linking the
Arp2/3-activating proteins Las17p/Bee1p (the yeast WASP homologue) and Abp1p
to the endocytic machinery (Warren et al.,
2002
).
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Sla2p is the yeast homologue of mammalian HIP1 (Huntingtin interacting
protein-1), a protein that localises to clathrin-coated pits
(Engqvist-Goldstein et al.,
1999; Engqvist-Goldstein et
al., 2001
) (Fig.
1A). Sla2p has an ENTH domain, which, in other proteins, has been
shown to interact with membrane inositol phospholipids
(De Camilli et al., 2002
), and
its C-terminal 200 amino acids have homology to talin, another mammalian
protein that binds to actin and is found at focal contact sites
(Hemmings et al., 1996
;
Priddle et al., 1998
). In
vitro, this C-terminal domain of Sla2p is able to bind to actin, and in vivo
expression of the C-terminal domain of Sla2p alone localises it to actin
structures (McCann and Craig,
1997
; Yang et al.,
1999
). However, the behaviour of other Sla2p mutant proteins in
cells suggests, firstly, that Sla2p has a cortical localisation domain in its
N-terminus. This is potentially due to the ENTH domain acting to localise the
protein to regions of clathrin coats. Secondly, a region upstream of the talin
homology region may mask its ability to bind to actin
(McCann and Craig, 1997
;
Wesp et al., 1997
;
Yang et al., 1999
). Thus Sla2p
may only bind to actin via its talin-like domain when it is modified (e.g.
phosphorylated) or when associated with other proteins that allow appropriate
unmasking of the site.
Here we show that Sla1p and Sla2p associate both in vivo using a two-hybrid approach and in vitro using purified proteins. We also define the interacting domain as a stretch of 243 amino acids in Sla1p and of 458 amino acids in the coiled-coil region of Sla2p. The role of this interaction is investigated further using genetic and cell biological approaches. In addition, deletion of either or both genes is detrimental to the ability of cells to undergo endocytosis. Furthermore, in cells lacking both Sla1p and Sla2p, the cortical actin cytoskeleton is unable to polarise, and the majority of actin is localised to the distal end of the cell. On the basis of the experimental data shown here and of results from other published reports we propose a model in which Sla1p acts as a link between Sla2p, clathrin and other endocytic machinery and the cortical actin cytoskeleton. Within this complex Sla1p destabilises and facilitates depolymerisation of F-actin. Sla2p and other proteins may then promote formation of new F-actin structures, which are important for driving endocytosis.
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Materials and Methods |
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Yeast strains and growth conditions
Yeast strains and plasmids used in this study are listed in
Table 1 and
Table 2, respectively. Unless
stated otherwise, cells were grown in a rotary shaker at 30°C in liquid
YPD medium (1% yeast extract, 2% Bacto-peptone, 2% glucose supplemented with
40 µg/ml adenine), except for KAY419, KAY439, KAY508 and KAY657 cells,
which were grown under selection in synthetic media lacking uracil. Halo
assays were performed as previously described
(Ayscough et al., 1997). The
mutant Sla1
118-511 was myc-tagged using oligonucleotides and methods
previously described (Warren et al.,
2002
).
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Protein purification and interactions
GST alone and GST-Sla1(118-511SH3#3) were expressed from DH5
bacteria by following pGEX vector protocols (Amersham Pharmacia). GST-Sla1 was
expressed and purified from 4 l of KAY419 cells as previously described
(Warren et al., 2002
).
SixHis-tagged Sla2p was purified on a Ni-NTA resin column from KAY445 cells as
described previously (Yang et al.,
1999
).
Pulldown assays were carried out with purified GST-Sla1p bound to
glutathione Sepharose beads as described previously
(Warren et al., 2002). Whole
cell lysates were spun at 2000 g for 4 minutes to remove
unbroken cells, and the supernatant spun at 300,000 g for 20
minutes at 4°C. The final supernatant was removed and 100 µl incubated
with either 20 µl of GST-Sla1p coated glutathione beads or with 20 µl of
glutathione sepharose for 3 hours at 4°C with rotation. The beads were
spun down at 500 g for 4 minutes and washed three times in 10
bed volumes of binding buffer. Bound proteins were eluted in sample buffer and
separated on a 10% SDS polyacrylamide gel before transfer to PVDF for
analysis.
Purified 6xHis-tagged Sla2p was eluted from Ni-NTA beads in binding buffer (50 mM K-HEPES pH 7.5, 100 mM KCl, 1 mM EDTA, 1 mM EGTA + protease inhibitors as above) + 0.01% Triton X-100. Proteins were eluted from the beads in sample buffer and separated on a 10% SDS polyacrylamide gel before being transferred to PVDF for analysis. Western blotting of purified 6xHis-Sla2p revealed that no Sla1p was co-purified (data not shown).
For investigating binding of GST-Sla1(118-511SH3#3) to His-tagged
Sla2p, the Sla2p beads were firstly washed in high stringency buffer (2 M
NaCl, 2% Triton-X100, 20 mM imidazole, 50 mM NaH2PO4
pH8). 25 µl of Sla2p on beads were incubated with GST or GST-Sla1 fragment
(final concentration 0.1 mg/ml) in binding buffer (50 mM
NaH2PO4 pH8, 20 mM imidazole, 0.3 M NaCl) for 3 hours at
4°C. The beads were washed three times in binding buffer and bound
proteins were eluted in sample buffer.
Yeast whole cell lysates were prepared as previously described
(Warren et al., 2002) and
samples were run on 10% SDS polyacrylamide gels. For immunoblots,
affinity-purified rabbit anti-yeast Sla2 antibodies (a gift from D. Drubin, UC
Berkeley) were diluted 1:500. GST and GST-Sla1p were detected using
affinity-purified polyclonal rabbit antiserum against the GST epitope at a
1:1000 dilution. Anti mouse and rabbit HRP-conjugated secondary antibodies
(Sigma) were used at 1:5000 and detected using ECL chemiluminescence.
Yeast two-hybrid screen
The yeast two-hybrid screen used bait and activation plasmids and a yeast
strain pJ69-2A designed and constructed by Philip James
(James et al., 1996). The
region of Sla1p to be tested in the two-hybrid assay was subcloned from a
plasmid carrying the entire SLA1 gene [pKA51
(Ayscough et al., 1999
)].
Eco R1 digestion was used to cut out the fragment corresponding to amino
acids 118 -511 from the gene, and this fragment was then cloned into the
pGBDU-C1 bait vector. The bait plasmid was checked for self-activation before
the library screen was carried out. For checking the requirement of the third
SH3 domain, EcoR1 was used to digest plasmid pKA116, which had been
constructed previously (Ayscough et al.,
1999
). This released the fragment as above but without the SH3
domain.
For the screen, 2 µg of bait plasmid (pKA237) was transformed into the
two-hybrid yeast strain pJ69-2A to give a strain KAY500.
2x109 KAY500 cells, grown in liquid media lacking uracil,
were transformed with 20 µg library DNA from each reading frame (library
was a gift from Francis Barr, Munich, Germany). In total, the 202 colonies
that grew within 7 days were picked from plates lacking histidine, uracil and
leucine. Following re-streaking and selection for the inability to grow on
media lacking adenine and containing 5-fluororotic-acid (5-FOA), a total of 57
yeast strains remained and plasmids were extracted from these. The plasmids
were then re-transformed into pJ69-2A with the bait plasmid to ensure that
activation of reporter genes still occurred. Twenty-six of the 57 yeast
strains could not grow on activation plates. The remaining 31 plasmids were
sequenced to identify the region responsible for the two-hybrid interaction,
and the resulting sequence was compared with DNA sequences from S.
cerevisiae using the BLAST alignment program found at the
Saccharomyces Genome Database Website
(http://genome-www.stanford.edu/Saccharomyces/).
The interacting genes identified were YSC84 [17 isolates
(Dewar et al., 2002)], SLA2 (7
isolates), SED5 (3 isolates), RAD52 (1 isolate) and non-coding DNA (3
isolates).
Sucrose gradients
Linear sucrose gradients (12 ml of 3-30%) in UBT buffer (50 mM KHEPES, pH
7.5, 100 mM KCl, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100) were
prepared in Beckman UltraClear 13 ml centrifuge tubes. Yeast extracts were
prepared from 100 ml cultures of KAY303 cells, at an OD600 of 1.0, by liquid
nitrogen grinding after harvesting cells at 3000 g for 4
minutes at 4°C and resuspending the pellet in cold 2xUBT buffer.
Ground extracts were thawed at 4°C then subjected to a 3000
g spin, and the resulting supernatant samples (500 µl) were
layered on top of the sucrose gradient spun at 35,000 g for 18
hours at 4°C. Fractions (250 µl) were removed manually from the bottom
of the tube and then assayed by SDS-PAGE. Immunoblotting was used to determine
the position of various proteins. For immunoblots, antibodies used were
anti-cofilin (1/500), anti-Abp1p (1/1000), anti Sla2p (1/500) all
gifts from D. Drubin (Berkeley, CA); anti-Chc1 (1/100), a gift from Sandra
Lemmon (Cleveland, Ohio); anti-actin (1/500), a gift from John Cooper
(Washington University, St. Louis); and anti-Sla1 (1/500)
(Warren et al., 2002).
Quantitation of band intensity was determined using NIH Image 1.6.1
Software.
Fluorescence microscopy
Endocytosis of the fluid phase marker lucifer yellow was performed
according to the published method (Dulic
et al., 1991), and analysis of the uptake of lipophylic dye FM4-64
was performed as described previously
(Vida and Emr, 1995
).
Rhodamine-phalloidin (Molecular Probes) staining of actin was performed as
described elsewhere (Hagan and Ayscough,
2000
). Cells were processed for immunofluorescence microscopy as
described previously (Ayscough and Drubin,
1998
). Antibodies were used to detect Sla2p, Abp1p, Sac6p (a gift
from D. Drubin, UC Berkeley, CA), Rvs167p (a gift from H. Friesen, University
of Toronto, CA) and the myc epitope (for Sla1p-myc; Santa Cruz Biotech, CA)
for immunofluorescence microscopy at 1:100 dilution. Secondary antibodies used
were FITC-conjugated goat anti-rabbit (Vector Laboratories) at dilutions of
1:1000. Cells were viewed with an Olympus microscope BX-60 fluorescence
microscope with a 100 W mercury lamp and an Olympus 100x Plan-NeoFluar
oil-immersion objective. Images were captured using a Roper Scientific
Micromax 1401E cooled CCD camera using IP lab software (Scanalytics, Fairfax,
VA) on an Apple Macintosh G4 computer.
Electron microscopy
Log phase cells were fixed using freshly prepared 2% potassium permanganate
for 45 minutes at room temperature. Following washing the pellets were
processed by dehydration through a series of ethanol from 50-100% and then in
propylene oxide. Samples were then incubated overnight in a resin (Durcapan):
propylene oxide 1:1 mix before embedding in resin and curing. Sections were
cut and stained with uranyl acetate and lead citrate before viewing them on a
Zeiss 902 transmission electron microscope.
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Results |
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Our antibodies did not prove useful for indirect fluorescence microscopy
so, in order to localise the mutant protein in cells, a myc tag was integrated
at the C-terminus of the mutant Sla1118-511p. The myc-tagged protein
was also detectable by western blotting and had no observable phenotypes in
addition to those of the mutant itself (data not shown). By indirect
fluorescence microscopy, Sla1
118-511p was observed to localise to small
punctate patches at the cell cortex, which indicates that the protein still
contains signals required for its localisation to discrete cortical sites in
the cell (Fig. 1C). The pattern
of staining was not well polarised and does not show the same organisation as
actin, which is in large cortical chunks in these cells
(Ayscough et al., 1999
)
(Fig. 5B).
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As well as showing that Sla1118-511p is expressed and localises to
appropriate regions of the cell, we were also able to show that cells
expressing the mutant are able to rescue a phenotype associated with the
complete sla1 deletion. Cells lacking sla1 cannot grow in
the absence of abp1 expression (deletion of both genes is
synthetically lethal). However, a combination of
sla1
118-511 and
abp1 is not lethal to
cells, which demonstrates, firstly, that the Sla1
118-511 protein must
be partly functional and, secondly, that the overlapping function with Abp1p
lies in another part of the protein. Other work in the laboratory has
indicated that this redundancy is attributable to the presence of the
C-terminal repeat region of Sla1p (Ayscough
et al., 1999
). Further analysis of the
sla1
118-511 strain showed that it has similar
resistance to the effects of the actin-disrupting drug latrunculin-A to cells
in which the entire sla1 gene is deleted. Again, this indicates that
the links with the actin cytoskeleton are mediated through this domain of the
protein (data not shown).
A two-hybrid screen with the Sla1 118-511 region detects Sla2p as a
binding partner
To investigate the role of the Sla1(118-511) region further, a two-hybrid
screen was conducted using this sequence as bait. Details of the constructs
used are given in Materials and Methods. Of 31 positive interactors, seven
were subsequently sequenced and shown to encode Sla2p. Within the seven
isolates two different start sites at amino acids 204 and 310 were identified,
but they all carried the same end site corresponding to amino acid 768 in
Sla2p. The minimum interacting sequence was therefore the region between amino
acids 310 and 768. This region of Sla2p corresponds to its central domain,
which is predicted to be largely coiled-coil in nature
(Fig. 1A,
Fig. 2A,B)
(Wesp et al., 1997;
Yang et al., 1999
).
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To ascertain whether the SH3 domain of Sla1p is important for the interaction with Sla2p, a modified bait plasmid was generated in which the SH3#3 domain is deleted. The two-hybrid transformations were repeated, and an interaction was still found to occur, which indicates that the sequence important for the Sla1p:Sla2p interaction lies outside the third SH3 domain of Sla1p (Fig. 2). Interestingly, this interaction appeared to be stronger than with the construct containing the SH3 domain, which suggests that the domain might normally partly interfere with, or regulate, an Sla1p:Sla2p interaction. In addition, an Sla1p bait plasmid containing the sequence from residue 1 to 361 also interacted with the Sla2p activation plasmid, indicating that the region of Sla1p involved in the interaction with Sla2p lies between residues 118 and 361. Further interactions between Sla1p and Sla2p central domain fragments were also tested (Fig. 2A,B), but none of these yielded a clear positive interaction, which might suggest that the interaction sites require a tertiary structure that cannot be generated by any of the Sla2p fragments.
Sla1p and Sla2p interact in vitro
Although an interaction between Sla1pand Sla2p has been shown through a
two-hybrid approach, it is possible that this could occur through an
intermediary protein. To investigate this possibility a plasmid expressing
GST-Sla1p was transformed into yeast cells and purified from whole cell
lysates. Following purification of Sla1p, cell extracts were passed over the
protein on a column, and proteins in bound and unbound fractions were
separated by SDS-PAGE and transferred to PVDF for analysis. Western blots were
probed with antibodies raised against several proteins known to associate with
cortical patches. Fig. 3A shows
that under these conditions, an interaction was detected between GST-Sla1p and
Sla2p.
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To establish whether the interaction between Sla1p and Sla2p is likely to be direct and not mediated by an accessory, we purified 6xHis-tagged Sla2p and performed a direct in vitro binding assay. The GST-Sla1p beads were blotted prior to Sla2p binding to verify that no Sla2p co-purified in the GST-Sla1p preparation (lower panel Fig. 3B). Fig. 3B clearly demonstrates that Sla1p and Sla2p interact in vitro. The controls presented demonstrate that the pulldown of purified Sla2p by Sla1p-coated beads is not due to the presence of the associated His or GST tags nor is it attributable to the presence of glutathione beads. In both GST pulldown experiments, Coomassie-stained gels showed that proteins other than Sla1p and Sla2p were not present at detectable levels in the purified protein extracts (data not shown).
Finally, an interaction between bacterially expressed GST-Sla1 fragment (118-511) and His-tagged Sla2p was demonstrated (Fig. 3C). Beads carrying Sla2p were washed stringently (in 2 M NaCl) prior to incubating with the GST or GST-Sla1 fragment. This wash removes any interacting proteins that can be detected on gels or by western blotting for specific proteins including Sla1p, Sac6p, actin, cofilin or Abp1p. In fact, a lower stringency wash (0.3 M) is sufficient to disrupt the Sla1p:Sla2p interaction (data not shown). Either GST alone or the GST-Sla1p fragment was incubated with His-tagged Sla2p. As shown in Fig. 3C, the Sla1p fragment, but not GST alone, is able to bind to Sla2p. All other bands on the Coomassie-stained gel were detectable by western blotting using antibodies against GST (for the GST-Sla1 preparation) or against Sla2p (His-tagged Sla2p preparation), which indicates that the Sla1p:Sla2p interaction is direct.
Fractionation of cell extracts indicates that Sla1p and Sla2p form a
complex in vivo
Evidence that Sla1p and Sla2p may form a complex in vivo was produced by
carrying out subcellular fractionation of yeast total protein extracts on a
3-30% sucrose gradient (Fig.
4). Fractions taken from the velocity gradient were probed with
antibodies raised against Sla2p, Abp1p, actin, cofilin, clathrin heavy chain
(Chc1p) and Sla1p. Identical fractionation was also performed on cell extracts
from strains lacking sla1. A number of significant findings were
made. Firstly, there are two peaks that are highly enriched for actin, Abp1p
and cofilin (fractions 6-10 and 16-20), indicating these proteins are found in
both small and large complexes in the cell. In extracts from wild-type cells,
Sla1p and Sla2p are not found in the fraction 6-10 peak but do co-fractionate
in the 16-20 peak, which represents larger protein complexes. Clathrin is also
found in these same dense fractions. Interestingly, in the absence of Sla1p,
the major Sla2p peak is shifted to a less dense fraction, indicating that it
may have been part of an Sla1p complex, which is consequently smaller in the
absence of this 140 kDa protein. A significant proportion of Sla2p has also
moved out of the dense fractions into less dense fractions; 72% of Sla2p is in
fractions 14-20 in wild-type cells compared with 43% in sla1
cells. This indicates that Sla1p is important for maintaining the integrity of
the Sla2p complex.
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In the wild-type extracts 30% of actin was found in fractions 16-20, this falls to 20% in the absence of sla1, also suggesting a role for Sla1p in association of actin with the large complex. These proportional changes in actin and Sla2p fractionation patterns in the presence and absence of Sla1p were reproducible over the three experiments performed. Abp1p is found in two distinct peaks, both in the presence and absence of Sla1p, though in the absence of Sla1p the denser fraction is reduced in size, which indicates that it may have contained Sla1p in the wild-type cell. Therefore Abp1p localisation with both complexes appears to be independent of Sla1p. Interestingly, the small peak of cofilin that co-fractionates with the denser actin complexes completely disappears in the absence of Sla1p.
Localisation of Sla1p and Sla2p in wild-type and mutant strains
Sla1p and Sla2p localise to cortical patches, a subset of which contain
actin (Ayscough et al., 1999;
Yang et al., 1999
). In order
to examine the distribution of Sla1p-myc and Sla2p, a co-immunofluorescence
experiment was carried out (Fig.
5A). Cells are shown that represent different stages within the
cell cycle of budding yeast. Both Sla1p-myc- and Sla2p-containing patches were
polarised to the site of bud emergence, to the enlarging bud and to the bud
neck (Fig. 5A). Furthermore,
Sla1p and Sla2p showed substantial colocalisation throughout the cell cycle.
However, it was also apparent that a number of cortical patches contained only
Sla1p-myc and rather fewer appeared to contain Sla2p alone, which suggests
that colocalisation was not always complete.
Deletion of the entire SLA1 coding region abrogated the
interaction of Sla2p with the actin cytoskeleton
(Ayscough et al., 1999), but
our work detailed above shows that the interaction is mediated in vitro and in
a two-hybrid assay by amino acids 118-511 of Sla1p. To demonstrate the
importance of this interaction in vivo, Sla2p was localised in cells
expressing mutant Sla1p (sla1
118-511). As shown in
Fig. 5B, while Sla2p is still
able to localise to the cell cortex, it no longer shows an overlapping
localisation with the cortical actin patches. Thus, both in vivo and in vitro,
the 118-511 region of Sla1p mediates an interaction between Sla2p and the
actin cytoskeleton.
Sla1p is required for the localisation of Sla2p to actin-containing
cortical patches and for its correct polarisation during growth
(Ayscough et al., 1999), and so
here we examined the distribution of Sla1-myc in an
sla2-null
background by immunofluorescence microscopy
(Fig. 5C). We found that
instead of Sla1p being localised to areas of cell growth as is seen in
wild-type cells, its distribution was depolarised and Sla1p-containing patches
appeared evenly distributed around the mother cell and bud. Co-staining with
rhodamine-phalloidin revealed that Sla1p-myc still localises to a subset of
cortical patches that contain actin (Fig.
5C, right). Co-staining for Sla1p-myc and actin in cells lacking
sla2 also revealed the existence of novel cortical structures.
Normally actin and Sla1p, when colocalised, show an apparently tight overlap
(Warren et al., 2002
). In the
structures shown in Fig. 5C
Sla1p and actin only partially overlap and Sla1p appears to be mostly
localised at the cortex of the cell, whereas actin appears to localise on the
cytoplasmic side of the structures (Fig.
5C, inset).
Overexpression of Sla1(118-511) causes defects in endocytosis and
membrane trafficking
The coiled-coil domain of Sla2p interacts with clathrin light chain
(Henry et al., 2002). Because
Sla1p also interacts with this region of Sla2p, we reasoned that
overexpression of Sla1(118-511
SH3#3) might interfere with other
functions of Sla2p requiring this domain. Cells induced by growth in galactose
to overexpress Sla1(118-511
SH3#3) were assessed for their ability to
endocytose the dye lucifer yellow. As shown in
Fig. 6A,B, a very marked
phenotype is observed. There is a reduction in uptake of lucifer yellow to the
vacuole coupled with an increase in staining of smaller organelles,
potentially endosomes, as well as increased plasma membrane staining. Thus
Sla1(118-511
SH3#3) overexpression causes reductions in both endocytosis
and, interestingly, defects in subsequent trafficking to the vacuole. In order
to determine whether this trafficking phenotype is largely due to
Sla1(118-511) binding Sla2p but no longer being open to the regulation
afforded by the presence of the rest of the molecule, two controls were
performed. Firstly, full-length Sla1p was overexpressed. This caused a
complete inhibition of endocytosis, and no endosomal staining was seen at all
(Fig. 6D). Second, a mutant
Sla1(118-511
SH3#3)* was expressed. This sequence contains a mutation
rendering the domain unable to interact with Ysc84p, the other Sla1p-binding
partner identified in the original two-hybrid screen
(Dewar et al., 2002
) (see also
Materials and Methods and two-hybrid screen results). Cells expressing this
mutant form of Sla1p sequence exhibited the same trafficking defect as in the
Sla1(118-511
SH3) cells (Fig.
6C).
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Over the time course of overexpression the actin phenotype was also monitored. Despite the endocytic defect, cells overexpressing either of the Sla1 fragments did not have an obviously disrupted actin phenotype, and the majority of actin was polarised in small punctate patches, which suggested that these cells are producing partially functional proteins. However, overexpression of the full-length sequence causes actin to aggregate into large cortical chunks, which produces an almost complete block in fluid phase endocytosis.
The Sla2p:Rvs167p interaction requires the presence of Sla1p
Work from other laboratories has demonstrated a two-hybrid interaction
between Sla2p and one of the yeast amphiphysin homologues Rvs167p
(Wesp et al., 1997). This
interaction was postulated to facilitate the role of Sla2p in endocytosis.
Rvs167p has also been reported to be an actin-associated protein
(Cid et al., 1995
). To address
whether Rvs167p association with Sla2p occurred in the absence of Sla1p, we
observed the localisation of Rvs167p in wild-type cells and in cells
expressing the sla1 deletion mutant
(Fig. 7). In wild-type cells
there was a partial overlap between Rvs167p and actin localisation, whereas in
the
sla1 cells Rvs167p colocalises completely with F-actin
structures. From our previous data
(Ayscough et al., 1999
) and the
data in Fig. 5B, we have shown
that Sla2p does not colocalise with actin in the sla1 deletion
strain. Therefore, because Rvs167p colocalises with actin under these
conditions, its association with Sla2p is likely to occur after the
interaction between Sla1p and Sla2p.
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Characterisation of sla1
sla2 double mutant cells
To characterise the combined role played by Sla1p and Sla2p further, yeast
strains were generated that did not expression both proteins, and the growth
and morphology of these cells was analysed.
sla1
sla2 double mutant cells appeared to grow
at a similar rate and possessed a similar temperature-sensitive phenotype to
that of
sla2 cells. However, comparisons between
sla1
sla2 double mutants with wild-type,
sla1 or
sla2 cells revealed a unique
cold-sensitive phenotype only when both Sla1p and Sla2p are absent (results
are summarised in Table 3). The
significance of this double mutant phenotype is not known at present, although
cold sensitivity has been associated with mutations in proteins involved in
forming multi-subunit complexes (Moir et
al., 1982
). It was also noted that the
sla1
sla2 cells were increased in cell volume
compared with either
sla1 or
sla2 cells. The
volume of cells grown at 29°C was measured using a Schärfe
CASYTM cell counter and the mean volume of wild-type cells is 5.76 fl and
sla1
sla2 cells is 13.8 fl. These data are
summarised in Table 3.
|
We have shown previously that the deletion of sla1 and
sla2 individually influences the sensitivity of yeast to the
actin-disrupting drug Latrunculin-A (LAT-A). Because LAT-A binds to actin
monomers, sensitivity to the drug can be related to whether a protein
increases or decreases the stability of F-actin in cells. When LAT-A was
administered to cells in culture, as described previously, deletion of
sla1 reduces the sensitivity to LAT-A whereas sla2
cells are more sensitive to the effects of the drug
(Ayscough et al., 1999
;
Warren et al., 2002
). However,
the double mutant cells displayed an intermediate phenotype, being more
sensitive than
sla1 cells but less so than the
sla2 mutant (data not shown). A similar result was obtained
when LAT-A sensitivity was assessed by a halo assay approach. A summary of the
halo assay data is shown in Fig.
8A.
|
Actin patch organisation in sla1
sla2 cells
The most striking phenotype of sla1
sla2
double mutant cells is observed when the actin cytoskeleton is stained with
rhodamine-phalloidin. Fig. 8B
shows F-actin staining in wildtype, cells with deletions of either
sla1 or
sla2 or both
sla1 and
sla2. Wild-type cells demonstrated polarisation of the actin
cytoskeleton to sites of cell growth. In
sla1 cells, cortical
actin patches appeared larger than those in wildtype, were fewer and were less
well polarised. In
sla2 mutant cells the documented phenotype
of smaller, more numerous, actin patches that were distributed evenly
throughout the mother cell and bud was seen
(Holtzman et al., 1993
). In
sla1
sla2 double mutant cells a novel phenotype
was observed. Cortical actin patches were primarily polarised to the distal
end of the mother cell. In these cells, small, less brightly stained patches
could often be seen at polarised regions of the cell (arrows) and an actin
ring could be seen at the site of cytokinesis (arrowheads), but this
represented a small fraction of actin that was visualised
(Fig. 8B lower panel). The
organisation of actin in the
sla1
sla2 cells
was unexpected as it appears to be opposite from the cell polarity as defined
by the sites of bud growth. However, in these cells, budding appears to occur
relatively normally and tubulin staining of cytoplasmic microtubules and the
mitotic spindle is not aberrant (data not shown).
To determine whether the distally localised F-actin patches contained a
normal complement of actin-binding proteins we used immunofluorescence
microscopy to determine the localisation of the yeast fimbrin homologue Sac6p
and the actin-binding protein Abp1p (Fig.
8C,D). Both of these proteins normally localise to cortical actin
patches, and their localisation to the distally located actin patches in the
sla1
sla2 cells indicates that this actin is
likely to be folded correctly.
Endocytosis and membrane trafficking in sla1
sla2
cells
Defects in endocytosis have been reported in both sla1 and
sla2 single mutant strains. The effect of sla1
deletion on fluid phase uptake of the fluorescent dye lucifer yellow is not
absolute, with about 30% of cells showing no uptake and 60-70% of cells
showing a reduction in uptake of the dye
(Warren et al., 2002
). Sla2p
appears to play a more critical role in endocytosis with most cells showing
only a low level of lucifer yellow uptake
(Fig. 9A)
(Raths et al., 1993
). The
sla1
sla2 cells show a phenotype that is more
severe than the
sla2 strain, and we observed less than 1% of
cells with any fluid phase uptake of the dye. The percentage of cells showing
vacuolar staining was assessed for all strains; this is summarised graphically
in Fig. 9B and all experiments
were performed three times, counting 200 cells of each strain on every
occasion.
|
Uptake of the dye FM4-64 was also monitored in wild-type and mutant cells.
This dye can be used to study mutants that have defects in membrane
trafficking in addition to in the initial internalisation step of endocytosis
(Vida and Emr, 1995). All of
the mutant strains showed a kinetic delay in FM4-64 uptake, although by 60
minutes, labelling was similar to that found in wild-type cells. Wild-type
cells showed vacuolar staining after 15 minutes,
sla1 and
sla2 after 25 minutes and
sla1
sla2 cells showed vacuolar staining after
35 minutes, although this was weaker than in the wild-type or single mutant
strains.
Finally we used electron microscopy to investigate whether there were any
marked changes in number or organisation of membrane structures. Previously,
sla2 cells were shown to accumulate vesicles in the bud
(Mulholland et al., 1997
). As
shown in Fig. 9C-F, there is a
clear increase in the number of vesicles in both
sla2 and
sla1
sla2 cells. One difference however between
these two mutants is that the vesicles in the double mutant appear to
accumulate in both the mother and bud, whereas the vesicles in the
sla2 mutant appear mostly to localise to the bud.
![]() |
Discussion |
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On the basis of the data shown above, previous work in our laboratory and work from other laboratories, we believe that current data are consistent with the following model for the role of the interaction of Sla1p and Sla2p in coupling cortical actin to endocytosis. It should be noted, however, that this model does not aim to be all inclusive and that both proteins are also likely to make interactions with other proteins at the cell cortex that may also play a role in the endocytic process. Our model is summarised in Fig. 10. Numbers indicate potentially sequential interactions.
|
In sla2 cells, Sla1p cannot localise to the endocytic
sites, which in turn means there is no actin recruitment to these sites. This
results in a very significant reduction in levels of endocytosis. The loss of
sla1 is rather more subtle, possibly because there are
proteins with roles that partially overlap with that of Sla1p. In the absence
of Sla1p there is an increase in F-actin, visualised as fewer larger chunks of
actin in cells (Holtzman et al.,
1993
). This correlates to a reduction but not total abrogation of
endocytosis (Warren et al.,
2002
). The effect of sla1 deletion on F-actin
organisation indicates that the Sla1p interaction with the Arp2/3 activators
Abp1p and Las17/Bee1p may be inhibitory. In wild-type cells, Sla1p may
downregulate actin polymerisation and thereby result in net depolymerisation.
Thus, when Sla1p is absent, there is an imbalance in actin assembly and
disassembly rates, and this leads to accumulation of actin chunks, which are
relatively stable However, there is possibly still some level of actin
dynamics that means that endocytosis is not completely prevented. It is
interesting to speculate from the sucrose density gradient analysis
(Fig. 4) that the absence of
cofilin from large protein complexes may serve to partly explain the lack of
depolymerisation in the absence of Sla1p.
The observation that Sla1p and Sla2p proteins have opposite effects on
actin dynamics in cells even though they are found in overlapping complexes is
likely to be crucial (Fig. 5).
As mentioned above, deletion of sla1 causes cells to become resistant
to the effects of LAT-A, which indicates that it normally has a destabilising
effect on F-actin, whereas deletion of sla2 causes a marked increase
in sensitivity to LAT-A, suggesting a role for Sla2p in stabilising F-actin
(Ayscough et al., 1997)
(Fig. 8A). Actin rearrangements
require both disassembly of existing structures (therefore a
destabilising/disassembling activity is required) and subsequently reassembly
into new structures (requiring nucleating/stabilising properties). Talin has
been shown to stabilise F-actin structures
(Hemmings et al., 1996
;
Priddle et al., 1998
) and so
it is possible that the talin homology region of Sla2p may perform a similar
role to this mammalian protein. The Sla2p actin-binding function appears to be
masked by intramolecular interactions that have been demonstrated to be due to
sequences within the central domain (Yang
et al., 1999
). This Sla2p domain, however, appears to be involved
in a number of interactions. As well as interacting with itself to form a
dimer, associations have also been reported with clathrin light chain
(Henry et al., 2002
), the
actin-regulating kinase Ark1p (Cope et al.,
1999
) and, as described here, with Sla1p. It is possible that
association with one or more of these proteins leads to unmasking of the
actin-binding site, which then facilitates formation of actin structures that
favour endocytosis.
We also show that overexpression of the Sla1-Sla2 interaction domain
(Sla1-118-511) can block both endocytosis and membrane trafficking to the
vacuole. This suggests that Sla1p and Sla2p must normally become dissociated
for endocytosis to proceed. Work by Zeng and colleagues has indicated that
phosphorylation of the C-terminal region of Sla1p by the actin-regulating
kinase Prk1p causes it to dissociate from cortical complexes
(Zeng et al., 2001). This may
then be a pre-requisite for endocytosis. In our experiment the fragment
Sla1(118-511) cannot be regulated by Prk1p because it lacks the C-terminal
motifs that become phosphorylated. It may therefore continue to associate with
the endocytic complex, presumably blocking interactions that are required for
endocytosis to proceed. Interestingly, there appears to be a low level of
fluid phase uptake of the dye, into small organelles, which may be endosomes.
Alternatively, these structures could be similar to the previously reported
finger-like invaginations, which may not have yet pinched off from the plasma
membrane (Wendland et al.,
1996
). This result may suggest that proteins with which
Sla1p/Sla2p interact also act downstream of the initial endocytosis
events.
Finally, we report the consequences of combining the sla1
and
sla2 mutations in cells. This leads to a complete block in
fluid phase endocytosis and also to a novel actin phenotype in which the
majority of F-actin is localised to the distal pole of the cell. The reason
for this organisation of cortical actin is not clear. It does however
illustrate that lack of polarised cortical actin does not abrogate polarised
growth of cells and adds further support to the importance of actin cables
rather than cortical actin patches in generating and maintaining cell polarity
through directed cell growth (Evangelista
et al., 2002
; Sagot et al.,
2002
)
The role of actin in the process of endocytosis is not yet clear. However,
studies in mammalian cells have shown that depolymerisation of F-actin can
increase the lateral movement of clathrin-coated pits, which suggests that the
actin cytoskeleton provides a scaffold at the plasma membrane for the
endocytic machinery (Gaidarov et al.,
1999). Other ideas for the role of actin include a role in
invagination of the membrane, fission of the vesicle or movement of the
vesicle into the cell (reviewed in
Qualmann et al., 2000
). The
data we present here suggest that actin is not required for the initial
marking of the site of endocytosis. Rather it is required to be dynamic and is
rearranged at sites of endocytosis. Potentially it is these rearrangements
that drive formation of the vesicle or its inward movement into the cell.
![]() |
Acknowledgments |
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References |
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