1 Institute of Molecular and Cell Biology, The National University of Singapore,
Singapore, 117609, Singapore
2 Department of Biochemistry, Faculty of Medicine, The National University of
Singapore, Singapore, 119260, Singapore
3 Department of Physiology and Biophysics, University of Iowa, Iowa City, 52242,
IA, USA
4 Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD,
4072, Australia
Author for correspondence (e-mail:
a.munn{at}imb.uq.edu.au)
Accepted 7 July 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: AAA-ATPase, Endocytosis, Endosome, Lysosome, LYST/beige, Chediak-Higashi syndrome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One subset of VPS genes (class E VPS) is required for MVB
sorting. Class E vps mutants accumulate newly synthesized soluble and
membrane-associated vacuolar proteins and late Golgi proteins (e.g. Vps10p) in
an enlarged endocytic compartment adjacent to the vacuole (known as the `class
E compartment') (Raymond et al.,
1992; Davis et al.,
1993
; Piper et al.,
1995
; Cereghino et al.,
1995
; Rieder et al.,
1996
; Odorizzi et al.,
1998
; Babst et al.,
1997
; Babst et al.,
1998
). A key player in MVB sorting, and the only class E Vps
protein with known enzymatic activity, is the VPS4 gene product
(Vps4p). Vps4p is a member of the AAA (ATPase associated
with a variety of cellular activities)-family of ATPases that also
includes other membrane transport proteins such as NEM-sensitive fusion
protein (NSF/Sec18p) (Babst et al.,
1997
; Finken-Eigen et al.,
1997
). Several other class E VPS genes encode small
coiled-coil proteins that are cytosolic in wild-type cells but accumulate on
endosomes in mutant cells lacking Vps4p ATPase activity
(Babst et al., 1998
;
Babst et al., 2002
). By analogy
with NSF/Sec18p, which uses ATP hydrolysis to disassemble SNARE complexes on
the surface of various membrane compartments, Vps4p may disassemble a
coiled-coil class E Vps protein complex on the surface of endosomes
(Babst et al., 1998
).
We previously isolated a vps4 mutant (end13, renamed
vps4-E13) in a screen for mutants unable to survive loss of the 60
kDa subunit of vacuolar ATPase (Vma2p/Vat2p) and defective in fluid-phase
endocytosis. In vps4-E13 receptor-mediated internalisation is only
slightly affected, but subsequent transport of internalised cargo through
early and late endosomes to the vacuole is strongly delayed
(Munn and Riezman, 1994;
Zahn et al., 2001
). We report
here that the class E Vps protein Vps20p and the product of a novel open
reading frame (ORF) VTA1/YLR181c interact with Vps4p. We
show that binding of each protein to Vps4p is direct and does not require ATP,
and identify the domains of each protein that mediate interaction. Loss of
Vps20p or Vta1p leads to class E vps phenotypes similar to those
caused by loss of Vps4p. We also show that whereas transport of other membrane
proteins through the class E compartment to the vacuole is only delayed, the
vacuole resident protein Sna3p cannot exit the class E compartment in cells
lacking Vps20p, Vta1p or Vps4p.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Genetic techniques
Genetic crosses and tetrad analysis were performed as described in Adams et
al. (Adams et al., 1997).
Transformation of yeast with plasmid DNA was either by a modification of the
lithium acetate protocol (Munn et al.,
1995
) or using the lithium acetate protocol recommended by
Clontech for strain EGY48. Genomic DNA was prepared from S.
cerevisiae essentially as described by Adams et al.
(Adams et al., 1997
) and PCR
amplification was performed with Pfu polymerase (Stratagene) or
Taq polymerase (Stratagene). Plasmid DNA was isolated from S.
cerevisiae using the method of Adams et al.
(Adams et al., 1997
) and
introduced into E. coli by electroporation
(Dower et al., 1988
). The
BAR1 gene was disrupted using a bar1::LYS2 construct (pEK3)
as described previously (Kübler and
Riezman, 1993
).
For functional studies, a congenic set of wild-type, vps20
and vta1
strains were analysed. Wild-type and vps20
cells were described previously in the parental strains SF838-9D
(Raymond et al., 1992
). The
vps20 mutant strain was originally isolated as the vpl10-7
mutant (vps20-7). The vps20-7 strain was complemented by a
low copy plasmid carrying the wild-type VPS20 gene, but not the
vps20 gene isolated from the vps20-7 strain (data not
shown). Sequencing of the VPS20 ORF (YMR077c) from the
vps20-7 strain revealed a deletion of the first nucleotide (G) in
codon 20 (GTA to TA), causing the protein product to be translated out of
frame (data not shown). This confirms that YMR077c is the ORF
corresponding to VPS20, consistent with previous reports
(Kranz et al., 2001
;
Howard et al., 2001
;
Forsberg et al., 2001
). Thus,
for simplicity, we refer to the vps20-7 strain as
vps20
. Y04130 (vta1
::KanMx) and the
isogenic wild-type strain Y10000 were obtained from Euroscarf (European
Saccharomyces Cerevisiae ARchives for Functional Analysis, Frankfurt,
Germany). To introduce vta1
into the SF838-9D strain
background, a PCR fragment encoding the KanMx ORF flanked by 400 bp either
side of the YLR181c coding sequence was amplified from Y04130 genomic
DNA and used to transform SF838-9D cells. G418-resistant colonies were
verified for loss of YLR181c by PCR analysis of genomic DNA, and one
isolate was retained for phenotypic analysis (PLY3046).
For -factor assays we prepared vps20
and
vta1
MATa strains that lack Bar, a secreted
protease responsible for degradation of
-factor (bar1). DNA
fragments corresponding to 301 to 1 nucleotide upstream and 5 to 230
nucleotides downstream of the VPS20 coding sequence were amplified by
PCR and subcloned into pFA6a-KanMx6 either side of the KanMx gene
(Longtine et al., 1998
) to
create pAM399. pAM399 was digested with KpnI and PstI to
release a fragment containing the KanMx cassette with VPS20
flanking sequences and used to disrupt the VPS20 gene in the
wild-type MATa bar1 strain RH1800. G418-resistant
transformants were selected on YPUAD/G418 medium and the presence of the
disruption was confirmed by PCR analysis of genomic DNA (data not shown). One
vps20
haploid (AMY174) was retained for further analysis.
Complementation analysis was performed with several vps mutant
strains including the vps2-7 (vpl2-7), vps20-7
(vpl10-7), vps22-6 (vpl14-6), vps25-1
(vpl12-1) and vps37-2 (vpl16-2) mutants defined in
previous studies (Raymond et al.,
1992
) (Table 1).
These studies showed that only vps20-7 failed to complement
vps20
(AMY174). Introduction of a low copy VPS20
plasmid corrected the CPY secretion defect in AMY174.
To make congenic vta1 and wild-type strains suitable for
-factor assays, Y04130 was crossed with Y10000 and the resulting
diploid subjected to tetrad dissection. Two haploids from this cross were
AMY149 (MATa lys2 vta1
) and AMY158
(MATa lys2 VTA1). The BAR1 gene was deleted
in AMY149 and AMY158, yielding AMY162 and AMY165, respectively, which were
then used for
-factor assays. The CPY missorting defect in AMY162 was
complemented by the introduction of a low copy VTA1 plasmid.
Yeast two-hybrid analysis
A bait construct expressing full-length Vps4p as a fusion to the
DNA-binding domain of LexA, pLexA-Vps4 (pAM333), was constructed and
introduced into yeast strain EGY48 containing the reporter plasmid p8op-LacZ
(Clontech). Transformation of this strain with the pB42AD transcription
activation domain vector alone did not confer significant expression of the
two-hybrid reporter genes LEU2 or lacZ (data not shown). An
S. cerevisiae two-hybrid library containing genomic DNA inserts in
vector pB42AD (pJG4-5) (Gyuris et al.,
1993) (a gift from U. Surana, IMCB, Singapore) was transformed
into this strain and colonies exhibiting expression of the LEU2
interaction reporter gene were selected on synthetic galactose/raffinose (SG)
complete medium-Leu. The equivalent of
5x104-1x105 colonies were screened (as
assessed by plating one sample of the transformed cells on SD complete medium
selecting only for pLexA-Vps4 and the pB42AD library plasmids). Positive
colonies were subsequently tested for blue colouration on SG complete medium
containing X-gal. The library plasmid was isolated from positive colonies and
retransformed into EGY48 containing p8op-LacZ and either pLexAVps4 or pLexA
vector alone. Library plasmids that were reproducibly able to confer growth on
SG complete-Leu and blue colouration on SG complete + X-gal upon
retransformation into EGY48/p8op-LacZ cells containing pLexA-Vps4, but not
when introduced into EGY48/p8op-LacZ cells containing pLexA vector only, were
retained for further analysis. ß-galactosidase activity was assayed using
a kit as recommended by the manufacturer.
To identify the domains within Vps20p and Vta1p that mediate two-hybrid interaction with Vps4p, we constructed a series of plasmids expressing different fragments of Vps20p or Vta1p fused in-frame with B42AD (in pB42AD) (Table 2). To identify the domains within Vps4p that mediate two-hybrid interaction with Vps20p and Vta1p, we constructed a series of plasmids expressing different fragments of Vps4p fused in-frame with LexA (in pLexA) (Table 2). We then tested interaction of both the longer fragments of Vps20p and Vta1p encoded by the original library clones (pAM349 and pAM398, respectively) and the shorter fragments encoded by the pB42AD-based plasmids described above with full-length Vps4p (pAM333) in EGY48 carrying p8op-LacZ. We also tested interaction of full-length Vps4p (pAM333) and the shorter fragments encoded by the pLexA-based plasmids described above with the original library clones of Vps20p and Vta1p (pAM349 and pAM398, respectively) in EGY48 carrying p8op-LacZ. The strength of interaction was assessed by blue colouration on SG complete medium containing X-gal.
Construction of VPS20 and VTA1 complementing
plasmids
The wild-type VPS20 and VTA1 full-length genes were
amplified by PCR using Pfu polymerase from RH1800 yeast genomic DNA.
An amount (400 bp) of upstream sequence was included for VPS20 and
1kb for VTA1. These fragments were cloned into the low-copy plasmids
YCplac33 and YCplac111, respectively
(Gietz and Sugino, 1988). The
inserts of YCp-VPS20 (pAM214) and YCp-VTA1 (pAM272) were
confirmed by sequencing. YCp-VPS20 and YCp-VTA1 were able to
fully complement the Vps- defect of vps20
and
vta1
, respectively.
In vitro binding assays
To test whether Vps20p and Vta1p can bind Vps4p in vitro we expressed
Vps20p and Vta1p as fusions to glutathione S-transferase (GST). The
full-length VPS20 and VTA1 coding sequences were amplified
by PCR from pAM214 and pAM272 and cloned into pGEX5X-1 (Amersham/Pharmacia
Biotech) in-frame and 3' of GST. vps4E233Q-6HIS and
vps4K179A-6HIS were constructed by amplifying VPS4 by PCR
from yeast genomic DNA using internal mutagenic primers and 5' and
3' VPS4 flanking primers including NdeI and
BamHI sites, respectively. Wild-type VPS4-6HIS was
constructed by amplifying VPS4 using the flanking primers only. The
3' flanking primers also encoded six histidine residues fused in-frame
with the Vps4p C-terminus. Wild-type VPS4-6HIS, vps4E233Q-6HIS and
vps4K179A-6HIS were cloned into pET11a between the NdeI and
BamHI sites and 3' of the T7 promoter. Inserts and frame were
confirmed by sequencing. The GST- and 6HIS-tagged proteins were expressed in
BL21-CodonPlusTM (DE3) E. coli and purified using
glutathione-agarose or Ni2+-NTA agarose beads, respectively. The
VPS4 gene was amplified without the terminator codon from RH1800
genomic DNA and ligated into a YCplac111-based plasmid encoding yEGFP (a gift
of B. Winsor, IBMC, University of Strasbourg, France) to create a fusion in
which the C-terminus of VPS4 is fused to yEGFP (pAM352). pAM352 fully
complemented the Vps- phenotype of vps4 and
therefore encodes a functional fusion protein (data not shown).
The binding of Vps4p in yeast lysates to recombinant Vps20p and Vta1p was
assayed as follows. vps4 (RH2906) cells expressing Vps4p-GFP
(pAM352) or Vps4p with no tag (pEND13.1)
(Zahn et al., 2001
) were grown
in SD minimal medium and subjected to glass bead lysis in extraction buffer
(20 mM HEPES, 200 mM sorbitol, 100 mM potassium acetate, 1 mM EDTA, pH7.5)
containing protease inhibitors (10 µg/ml aprotinin, 5 µg/ml leupeptin, 8
µg/ml pepstatin, 1 mM phenylmethylsulphonylfluoride) and 1 mM DTT.
Low-speed centrifugation (700 g) was used to remove unbroken
cells and the resulting supernatant (S1) was fractionated by differential
centrifugation into 16,000 g and 100,000 g
pellets (P2, P3) and a 100,000 g supernatant (S3). The S3
fraction was supplemented with 20 mM MgCl2 and divided in two. One
sample was incubated with apyrase (5.7 U/ml final) for 10 minutes at room
temperature to deplete endogenous ATP, whereas the other was incubated without
apyrase. The apyrase activity under these buffer and temperature conditions
was approximately 25% of that specified by the manufacturer (i.e. 1.4 U/ml
final) (data not shown). Both ATP-depleted and untreated lysates were then
incubated with beads bearing GST-Vps20p, GST-Vta1p or GST only at 4°C for
12 hours. Unbound protein was precipitated with trichloroacetic acid,
dissolved in Laemmli sample buffer, and neutralised with 1 M Tris base. The
beads were washed with extraction buffer prior to elution of the bound
proteins by heating in Laemmli sample buffer. Proteins in each sample were
resolved by SDS-PAGE, transferred to Immobilon-PSQ PVDF membranes
and Vps4p-GFP was detected with an anti-GFP antiserum and enhanced
chemiluminescence.
To test direct binding of Vps4p to Vps20p and Vta1p, 6HIS-tagged wild-type or mutant Vps4p were expressed in and purified from E. coli as described above. Vps4p-6HIS was eluted from the beads with 250 mM imidazole in PBS containing 1mM ß-mercaptoethanol and 0.05% Tween 20 and dialysed against extraction buffer containing 1 mM DTT. For binding assays, 5-10 µg of purified wild-type or mutant Vps4-6HIS in extraction buffer containing 1 mM DTT and 1 mM phenylmethylsulphonylfluoride were used. Each sample was supplemented to 20 mM MgCl2 and 0.1% Triton X-100 and incubated with beads bearing GST-Vps20p (500 µg), GST-Vta1p (100 µg) or GST (500 µg) in the presence or absence of 1 mM ATP at 4°C for 12 hours. The samples were processed as above (with the slight modification that unbound samples were supplemented with bovine serum albumin as carrier prior to precipitation with trichloroacetic acid). After SDS-PAGE Vps4p-6HIS was detected by immunoblotting using a pentaHIS-specific monoclonal antibody and enhanced chemiluminescence.
Endocytosis assays
Fluid-phase endocytosis was measured by vacuolar accumulation of the
membrane-impermeant fluorescent dye LY carbohydrazide following incubation for
1 hour at 24°C as described in Munn et al.
(Munn et al., 1999).
Endocytosis of plasma membrane was assayed using the lipid-soluble styryl dye
FM4-64 (Vida and Emr, 1995
).
Cells were incubated with 2 µM FM4-64 at 0°C for 30 minutes to label
the cell surface. Then the cells were washed on ice, resuspended in fresh
medium at 30°C (0'), and transport of the dye from the cell surface to the
vacuole was assessed at various time points. [35S]
-factor
internalisation assays were performed at 30°C using the continuous
presence protocol (Dulic et al.,
1991
). [35S]
-factor degradation assays were
performed at 30°C using the pulse-chase protocol
(Dulic et al., 1991
).
Carboxypeptidase Y missorting test
To assess maturation of newly synthesised CPY and processing of the
receptor for soluble vacuolar proteins (Vps10p) we immunoprecipitated CPY and
Vps10p from cells labeled with [35S]Methionine/cysteine as
described (Piper et al.,
1995). For this analysis, strains were converted to
Pep+ by transformation with pTS18 (PEP4 centromeric
plasmid).
Multivesicular body sorting assay
Localisation of Ste3-GFP, Fth1-GFP-Ub, Sna3-GFP and NBD-PC were performed
as previously described (Bilodeau et al.,
2002). The Sna3-GFP reporter used here was made by amplifying
SNA3 without a termination codon by PCR using RH1800 genomic DNA and
then subcloning it into a YCplac111-based plasmid 5' and in-frame with
yEGFP (pAM397). This places the GFP at the Sna3p C-terminus.
Fluorescence microscopy
All microscopy was performed using an Olympus BX-60 microscope fitted with
Differential Interference Contrast (DIC) light filters and appropriate
fluorescence light filters.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We next used the yeast two-hybrid system to map the domains of Vps4p, Vps20p and Vta1p that mediate the interactions. Two-hybrid plasmids expressing various fragments of Vps4p, Vps20p and Vta1p were constructed (Fig. 1A, Table 2). The results of two-hybrid analyses using these constructs as well as pLexA-Vps4 (pAM333) and the original Vps20p and Vta1p library clones (pAM349 and pAM398, respectively) are shown in Tables 3 and 4. The N-terminal coiled-coil domain of Vps4p interacted very strongly with Vps20p, but not with Vta1p, whereas the C-terminal acidic domain of Vps4p interacted with both proteins, but more strongly with Vta1p. The AAA-ATPase domain did not interact with either Vps20p or Vta1p. A fragment containing the central coiled-coil domain plus the C-terminal domain of Vps20p was necessary and sufficient for interaction with full-length Vps4p. In the case of Vta1p, the C-terminal domain was sufficient for interaction with Vps4p.
|
|
Vps20p and Vta1p directly bind Vps4p in vitro
We next tested whether Vps20p or Vta1p bind Vps4p in vitro. Vps20p and
Vta1p were expressed as GST fusions in bacteria and purified. Beads bearing
GST-Vps20p or GST-Vta1p, but not beads bearing GST alone, precipitated
GFP-tagged Vps4p from yeast lysates (Fig.
2). We also tested the ability of Vps4p-GFP to associate with
GST-Vps20p and GST-Vta1p after depletion of ATP from the lysates. The
association of Vps4p-GFP with GST-Vps20p, but not GST-Vta1p, was slightly
enhanced (approximately twofold) by ATP depletion
(Fig. 2). Strong association of
Vps4p-GFP with both GST-Vps20p and GST-Vta1p was also observed after depletion
of free Mg2+ (data not shown). Hence, the in vitro association of
Vps4p with Vps20p and Vta1p does not require ATP, and in the case of Vps20p it
is slightly sensitive to ATP hydrolysis. Similar results were obtained using a
Vps4p construct with a C-terminal myc-epitope (data not shown).
|
In order to test whether Vps4p directly binds Vps20p and Vta1p in vitro and to further examine the role of ATP binding, we expressed wild-type Vps4p-6HIS, Vps4pE233Q-6HIS (ATP hydrolysis defective) and Vps4pK179A-6HIS (ATP binding defective) in bacteria. Each protein was affinity purified and used in pulldown assays using beads bearing GST-Vps20p, GST-Vta1p or GST only (Fig. 3). Wild-type Vps4p-6HIS bound both GST-Vps20p and GST-Vta1p, but not GST alone, in vitro, indicating that the association of Vps4p with Vps20p and Vta1p is direct and not mediated by other proteins. Binding was observed in the presence and in the absence of added ATP, in agreement with the results described above for Vps4p-GFP in yeast lysates. Binding of Vps4p-6HIS to GST-Vta1p was not affected by addition of ATP, but binding to GST-Vps20p was stronger when ATP was omitted. These findings are in agreement with the results described above that association of Vps4p-GFP in yeast lysates with GST-Vps20p is stronger after depleting the lysate of ATP. Vps4pE233Q-6HIS and Vps4pK179A-6HIS also bound to both GST-Vps20p and GST-Vta1p, but not to GST alone, indicating in another way that ATP binding is not necessary for association of Vps4p with Vps20p or Vta1p. In the case of both mutant Vps4p-6HIS proteins, binding to GST-Vps20p and GST-Vta1p was significantly enhanced compared with wild-type Vps4p-6HIS. As expected, binding of the mutant Vps4p-6HIS proteins to GST-Vps20p and GST-Vta1p was not significantly affected by addition of ATP.
|
Vps20p and Vta1p are required for efficient post-internalisation
transport of -factor
We next determined whether Vps20p or Vta1p are required for
receptor-mediated endocytosis. Receptor-mediated uptake of
[35S]-factor was assayed at 30°C in
vps20
and vta1
and the corresponding wild-type
strains (Fig. 4).
[35S]
-factor was internalised by all four strains, although
vps20
and vta1
showed slightly slower kinetics
and this difference was reproducible. To investigate whether
post-internalisation transport of [35S]
-factor to the
vacuole is affected in the mutants, we assayed the kinetics of
[35S]
-factor degradation at 30°C. Both the
vps20
and vta1
mutations significantly delayed
the vacuolar delivery and consequent degradation of the internalised
[35S]
-factor (as assessed by loss of intact
[35S]
-factor spots and appearance of degraded
[35S]
-factor spots) (Fig.
5).
|
|
Loss of Vps20p or Vta1p result in CPY secretion and accumulation of
Ste3-GFP in an endosomal compartment
To characterize the role of Vps20p and Vta1p in vacuolar protein sorting
and MVB sorting, we constructed congenic vps20 and
vta1
mutant strains using the SF838-9D MAT
parental strain. SF838-9D has been extensively used for analysis of these
processes (Raymond et al.,
1992
) and carries the pep4-3 mutation that allows lumenal
vesicles to accumulate in the vacuole.
Ste3p is the plasma membrane receptor for the secreted yeast mating
pheromone a-factor. In wild-type cells, Ste3-GFP travels to the cell
surface and is then rapidly endocytosed and delivered to the vacuole. The
Ste3-GFP protein typically accumulates to high levels in the lumen of
wild-type vacuoles indicative of proper MVB sorting to intralumenal vesicles
(Urbanowski and Piper, 2001).
To test whether Vps20p and Vta1p are required for MVB sorting of Ste3p, we
localised Ste3-GFP in wild-type cells and in vps20
and
vta1
cells carrying the low copy YCp-VPS20 plasmid or
YCp-VTA1 plasmid or empty vector only
(Fig. 6). In both
vps20
and vta1
cells Ste3-GFP was found on the
limiting membrane of the vacuole as well as in 2-3 punctate structures
adjacent to the vacuole, but not in the vacuole lumen. The punctate structures
adjacent to the vacuole were similar to the class E compartments observed for
other class E vps mutants
(Raymond et al., 1992
). The
defects in MVB sorting of Ste3-GFP were corrected when the
vps20
strain or vta1
strain was transformed
with the corresponding wild-type gene borne on a low-copy plasmid
(Fig. 6).
|
To test whether Vta1p, like Vps20p, is required for efficient vacuolar
protein sorting, we compared the sorting of the soluble vacuolar hydrolase
carboxypeptidase Y (CPY) to the vacuole in cells lacking Vps20p or Vta1p. Both
the vps20 and vta1
mutants secreted
40%
of CPY after a 60-minute chase (Fig.
7). Previous studies have shown that CPY secretion is because of
depletion of the CPY receptor (Vps10p) from the Golgi and its accumulation
within the class E endosomal compartment. In Pep+ cells the class E
compartment is proteolytically active and the delivery and accumulation of
Vps10p in the class E compartment can be monitored by pulse/chase labeling and
immunoprecipitation of Vps10p (Piper et
al., 1995
). In vps20
and vta1
, a
significant amount of newly synthesised Vps10p undergoes a
PEP4-dependent cleavage after a 60-minute chase consistent with what
has been observed for other class E vps mutants
(Cereghino et al., 1995
).
|
Vps20p and Vta1p are required for MVB formation
The defects in MVB sorting of Ste3-GFP in vps20 and
vta1
mutants was consistent with a role of Vps20p and Vta1p in
MVB formation. This process incorporates a variety of membrane proteins into
intralumenal vesicles that accumulate in the vacuoles of pep4 mutant
yeast (Piper and Luzio, 2001
).
For some proteins, their delivery to the vacuole interior is dependent on
attachment of ubiquitin, whereas the delivery of other proteins is
ubiquitin-independent (Katzmann et al.,
2002
). The ubiquitin-dependent MVB sorting mechanism is
exemplified by the chimeric Fth1-GFP-Ub reporter protein. Fth1p is an iron
transporter whose distribution is restricted to the limiting membrane of the
vacuole, but the Fth1-GFP-Ub chimera is a substrate for ubiquitin-dependent
MVB sorting and in wild-type cells localises to vacuolar intralumenal vesicles
(Urbanowski and Piper, 2001
).
The ubiquitin-independent sorting mechanism is exemplified by the Sna3-GFP
reporter protein. Sna3p is an integral membrane protein first identified as a
component of vacuolar intralumenal vesicles. MVB sorting of Sna3p is not
affected by substitution of its two cytoplasmic lysines (potential
ubiquitination sites) or by lowered free ubiquitin levels (e.g. in a
doa4
mutant) and is thus ubiquitin-independent
(Reggiori and Pelham, 2001
).
In wild-type cells, we found that both Fth1-GFP-Ub and Sna3-GFP were localised
to the vacuole lumen (Fig. 8).
However, in either vps20
or vta1
cells,
Fth1-GFP-Ub accumulated on both the limiting membrane of the vacuole as well
as in large `class E' structures similar to where Ste3-GFP accumulated (Figs
6,
8). Sna3-GFP was also excluded
from the vacuole interior, but far less was observed on the limiting membrane
of the vacuole. Rather, Sna3-GFP was found almost exclusively within class E
compartments. Morphometric analysis of fluorescence intensity between
Ste3-GFP, Fth1-GFP-Ub and Sna3-GFP confirmed that the exclusive localisation
of Sna3-GFP to the class E compartment was not because of the overall level of
these proteins or limits in fluorescence detection.
|
Aside from the inability to sort membrane proteins into intralumenal
vesicles, at least some class E vps mutants (including vps4)
are also unable to perform lipid sorting events required to make lumenal
vesicles. Previously, NBD-PC has been shown to be a lipid marker of the
intralumenal vesicles (Bilodeau et al.,
2002; Hanson et al.,
2002
). To test whether Vps20p and Vta1p are (like Vps4p) required
for sorting of lipids into intralumenal vesicles, we compared the distribution
of internalised NBD-PC in wild-type, vps20
and
vta1
mutants (Fig.
9). In wild-type cells, NBD-PC was sorted into vesicles in the
vacuole lumen. In vps20
or vta1
cells,
however, NBD-PC was not sorted into intralumenal vesicles, but remained on the
limiting membrane of the vacuole as well as in class E endosomal compartments.
Thus sorting of lipids (as well as proteins) to form intralumenal vesicles
requires both Vps20p and Vta1p.
|
Vps20p and Vta1p are required for efficient endocytosis of
fluid-phase and membrane markers from the cell surface to the vacuole
We next tested whether Vps20p and Vta1p play a role in endocytic membrane
traffic of bulk fluid to the vacuole. Wild-type, vps20 and
vta1
cells were incubated in the presence of the fluid-phase
endocytic marker LY and accumulation of the dye in the vacuole was examined
(Fig. 8). Low levels of LY did
accumulate in the vacuoles of vps20
and vta1
cells, but markedly less than in the vacuoles of wild-type cells. Therefore,
both Vps20p and Vta1p are important for fluid-phase transport to the vacuole,
although not essential.
We next examined whether Vps20p and Vta1p are important for bulk membrane
transport from the cell surface to the vacuole. The lipid dye FM4-64 is a
membrane-soluble dye that binds to the plasma membrane and is internalised by
endocytosis and delivered to the vacuole membrane
(Vida and Emr, 1995). Cell
surface membranes of wild-type, vps20
and vta1
cells were labeled with FM4-64 at 0°C, and cells were then warmed to
30°C and assessed for distribution of FM4-64 at various times
(Fig. 10). At early times
after shift to 30°C, FM4-64 labeled small punctate structures and at later
times it accumulated in 1-2 large late-endosomal/prevacuolar structures
adjacent to the vacuole. By 30 minutes, FM4-64 could clearly be seen on the
vacuole limiting membrane in wild-type cells. In contrast, the bulk of FM4-64
remained in large endosomal structures in both the vps20
and
vta1
cells, indicating a delay in transport from the class E
compartment to the vacuole.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vps20p and two other class E Vps proteins, Snf7p/Vps32p/Did1p (30%
identity) and Mos10p/Vps60p/Chm5p (15% identity) comprise a gene family
(Babst et al., 1998;
Amerik et al., 2000
;
Kranz et al., 2001
;
Howard et al., 2001
). Hspc177
(Genbank Acc. No. BC016698) encodes a mammalian protein with strong homology
(21% identity) to yeast Vps20p over its full length. Other yeast family
members include the class E Vps proteins Vps24p/Did3p, Did2p/Chm1p/Fti1p and
Vps2p/Did4p/Ren1p/Chm2p (Davis et al.,
1993
; Babst et al.,
1998
; Amerik et al.,
2000
; Kranz et al.,
2001
; Howard et al.,
2001
). Proteins in this family all have extensive coiled-coil
domains that can potentially mediate protein-protein interaction
(Babst et al., 1998
;
Kranz et al., 2001
;
Howard et al., 2001
); for
example, in Vps20p the coiled-coil domain comprises residues 61-169 as
predicted using the COILS algorithm (Lupas
et al., 1991
).
Although this is the first report of interaction between Vps4p and Vps20p,
there have been two earlier reports of interactions between Vps4p and other
family members: (1) a genomic two-hybrid screen identified an interaction
between Vps4p and Snf7p/Vps32p (Uetz et
al., 2000), and (2) a yeast two-hybrid screen using CHMP1 (the
mammalian homologue of yeast Did2p/Chm1p) as bait led to the identification of
human Vps4-A/SKD1 as a CHMP1 interactor
(Howard et al., 2001
).
Interestingly, Did2p/Chm1p is among the other Vps4p two-hybrid interactors we
identified in our screen (M. Wagle and A. Munn, unpublished).
Four of the six known coiled-coil class E Vps proteins (viz. Vps20p, Snf7p,
Vps2p and Vps24p) have recently been shown to form a large protein complex
known as ESCRTIII implicated in concentration and sorting of cargo proteins at
the MVB prior to incorporation into intralumenal vesicles
(Babst et al., 2002). In
vps4
mutants, all four ESCRTIII proteins redistribute from the
cytoplasm to the surface of endosomal membranes
(Babst et al., 1998
;
Babst et al., 2002
). Based on
this in vivo data, it has been suggested that Vps4p uses the energy of ATP
hydrolysis to break coiled-coil interactions and release ESCRTIII proteins
from the surface of endosomes into the cytoplasm. ATP-dependent release of
ESCRTIII proteins from endosomal membranes by Vps4p has yet to be demonstrated
in vitro, however a mutant form of human Vps4-A (E228Q) that is locked in the
ATP-bound state exhibits enhanced association with CHMP1
(Howard et al., 2001
). This
data supports the conclusion that in the ATP-bound form Vps4p associates with
class E Vps proteins and breaks coiled-coil interactions.
We have shown here that binding of Vps4p to Vps20p in vitro is independent
of ATP. A Vps4p ATP hydrolysis mutant (E233Q) exhibited increased binding to
Vps20p in vitro compared with wild-type Vps4p, however an ATP binding mutant
(K179A) also exhibited increased association
(Fig. 3). Thus, enhanced
binding to coiled-coil proteins may be a feature of non-functional (rather
than ATP-bound) Vps4p. This is consistent with the observation that an
ATP-binding mutant of mammalian Vps4-A (KQ) behaves like an ATP hydrolysis
mutant (EQ) in exhibiting enhanced localisation to endosomes
(Bishop and Woodman, 2000).
ATP-independent association with coiled-coil domain proteins may distinguish
Vps4p from other AAA-ATPases, such as NEM-sensitive fusion protein
(NSF/Sec18p). NSF/Sec18p forms a 20S complex with soluble NSF attachment
protein (
-SNAP) and
-SNAP-receptors (SNARES) and uses ATP
hydrolysis to break coiled-coil interactions between SNARES. An NSF/Sec18p
mutant protein with a mutation in the first of its two AAA-domains (D1) that
prevents ATP binding is unable to associate with the
-SNAP-SNARE
complex in vitro (Nagiec et al.,
1995
). Nevertheless, our analysis strongly supports the view that
ATP hydrolysis by Vps4p dissociates coiled-coil interactions as proposed by
Babst et al. (Babst et al.,
1998
; Babst et al.,
2002
). Interaction of Vps4p with Vps20p appears to involve
coiled-coil interactions (Tables
3,
4) and is sensitive to ATP
hydrolysis (Fig. 3).
ESCRTIII comprises Vps20p-Snf7p and Vps2p-Vps24p subcomplexes
(Babst et al., 2002). The
Vps2p-Vps24p subcomplex has been proposed to mediate recruitment of Vps4p to
endosomes based on the finding that accumulation of ATP-locked Vps4p-E233Q
mutant protein on endosomes is affected in vps2 and vps24
mutants (vps20 and snf7 mutants were not tested)
(Babst et al., 2002
). Our data
suggest that Vps20p may also be important for recruitment of Vps4p to
membranes. Vps20p is myristoylated and associates strongly with membranes
(Ashrafi et al., 1998
;
Babst et al., 2002
).
Interestingly, myristoylation is not required for Vps20p interaction with
Vps4p as Vps20p fusions lacking the myristoylation motif (MG, residues 1-2)
still exhibit strong two-hybrid interaction with Vps4p. Furthermore,
bacterially expressed Vps20p binds Vps4p in vitro and bacteria lack the
ability to perform myristoylation. Our data show that Vps20p interacts with
the N-terminal coiled-coil domain of Vps4p essential for Vps4p association
with endosomal membranes (Babst et al.,
1998
). That Vps4p-E233Q and Vps4p-K179A mutant proteins exhibit
increased association with Vps20p correlates well with reports that both
mutant proteins also exhibit increased accumulation on endosomes in vivo
(Babst et al., 1998
;
Bishop and Woodman, 2000
). The
strength of Vps4p-Vps20p interaction may be an important determinant of Vps4p
subcellular localization.
YLR181c (VTA1) is a novel class E VPS gene.
Although Vta1p does not share significant homology to other class E Vps
proteins, a putative mammalian homologue is encoded by dopamine-responsive
gene (Drg-1) (GenBank Acc. No. AF271994) [also known as LYST-interacting
protein 5 (Lip5) (GenBank Acc. No. AF141341)]. Drg-1/Lip5 has strong homology
to Vta1p over N- (24% identity) and C-terminal (59% identity) sequences
representing 43% of the total length of the protein. LYST is the protein
affected in the human inherited immune and neurological disorder
Chediak-Higashi Syndrome (CHS) and in beige mutant mice. Defects in
endosomal membrane transport and lysosome morphology have been reported in CHS
(Tchernev et al., 2002). The
best yeast homologue of LYST is Bps1p, however deletion of BPS1 in
the SF838-9D background gave no class E vps phenotypes (R. C. Piper,
unpublished).
Vta1p has two predicted coiled-coil domains (1stCC and 2ndCC comprising
residues 35-63 and 232-263, respectively). The latter is encoded by all of our
positive two-hybrid library clones, but our interaction domain analysis shows
that Vps4p does not interact with the 1stCC or the 2ndCC domain of Vta1p.
Instead, Vps4p interacts specifically with a short (65 residue) domain located
at the extreme Vta1p C-terminus that lacks predicted coiled-coil structure.
Furthermore, Vps4p binds to Vta1p via its acidic C-terminal domain (Tables
3,
4). Hence, coiled-coil
interaction does not appear to mediate Vps4p interaction with Vta1p. This
suggests that Vps4p may interact quite differently with Vta1p compared with
coiled-coil Vps20p-family proteins. Vta1p may represent a stable subunit
rather than a substrate of the Vps4p complex. Consistent with this, Vps4p
binding to Vps20p in vitro is sensitive to ATP hydrolysis, whereas binding to
Vta1p is unaffected by the presence or absence of ATP (Figs
2,
3). Little is known about the
role of the C-terminal acidic domain in Vps4p function. In the case of another
AAA-family ATPase, Hsp104p, the acidic C-terminal domain regulates ATP
hydrolysis by the AAA domain (Cashikar et
al., 2002). Although this is not known for Vps4p, it is intriguing
to speculate that Vta1p regulates the ATPase activity of Vps4p.
Loss of Vps20p or Vta1p confers classical class E Vps- phenotypes similar to loss of Vps4p, including: 1) a block in MVB sorting of endocytosed surface proteins, vacuolar membrane proteins and the lipid NBD-PC; 2) accumulation of endocytosed surface proteins (e.g. Ste3p), lipid-soluble endocytic dyes (e.g. FM4-64) and vacuolar membrane proteins (e.g. CPS, Sna3p) in the `class E' compartment; and 3) secretion of Golgi-modified p2CPY. As in other class E vps mutants, p2CPY secretion is associated with degradation of Vps10p. In wild-type cells Vps10p is stable and cycles from the late Golgi to the PVC during the sorting of soluble vacuolar hydrolases to the PVC. In class E vps mutants Vps10p and vacuolar hydrolases are trapped in the class E compartment and Vps10p is degraded. Some phenotypic characterisation has already been reported for vps20, but this is the first report that vta1 mutants have class E vps phenotypes.
Our results suggest that vacuolar delivery of Sna3p may have unique
requirements. Although Ste3-GFP, Fth1-GFP-Ub (Figs
6,
8) and GFP-Cps1p (data not
shown) localise to both the class E compartment and the vacuole limiting
membrane in class E vps mutants, Sna3-GFP appears to exclusively
localise to the class E compartment (Fig.
8). Unlike the ubiquitin-dependent MVB sorting of other proteins
(Urbanowski and Piper, 2001;
Katzmann et al., 2001
), MVB
sorting of Sna3p is ubiquitin-independent. It has been proposed that MVB
sorting of Sna3p occurs via spontaneous partitioning of the Sna3p
transmembrane domains into subdomains of the MVB limiting membrane that form
intralumenal vesicles (Reggiori and
Pelham, 2001
). The unique properties of the Sna3p transmembrane
domains may thus prevent Sna3p from entering endosome to vacuole transport
intermediates, leading to Sna3p retention in endosomes. Alternatively, Sna3p,
but not CPS, Ste3p or Fth1-Ub, could possess the ability to recycle from the
vacuole to the MVB in wild-type cells. If exit from the vacuole is relatively
unaffected compared with exit from the class E compartment in class E
vps mutants, Sna3p would redistribute from the vacuole to the class E
compartment. Ultimately, an understanding of these sorting differences
requires insight into the biological function of Sna3p, however a function for
Sna3p has not yet been ascribed.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
* Present address: Biosciences Program, University of Iowa, Iowa City, 52242,
IA, USA
Present address: Temasek Life Sciences Laboratory, Singapore
Present address: Genome Damage and Stability Centre, School of Biological
Sciences, University of Sussex, UK
Present address: Institute for Molecular Bioscience, University of
Queensland, Brisbane, QLD, 4072, Australia
¶ Present address: Deutsche Krebsforschungszentrum, Heidelberg, Germany
** Present address: University of Edinburgh, Edinburgh, UK
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, A., Gottschling, D. E., Kaiser, C. A. and Stearns, T. (1997). Methods in Yeast Genetics. A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.
Amerik, A. Y., Nowak, J., Swaminathan, S. and Hochstrasser,
M. (2000). The Doa4 deubiquitinating enzyme is functionally
linked to the vacuolar protein-sorting and endocytic pathways. Mol.
Biol. Cell 11,
3365-3380.
Ashrafi, K., Farazi, T. A. and Gordon, J. I.
(1998). A role for Saccharomyces cerevisiae fatty acid
activation protein 4 in regulating protein N-myristoylation during entry into
stationary phase. J. Biol. Chem.
273,
25864-25874.
Babst, M., Sato, T. K., Banta, L. M. and Emr, S. D.
(1997). Endosomal transport function in yeast requires a novel
AAA-type ATPase, Vps4p. EMBO J.
16,
1820-1831.
Babst, M., Wendland, B., Estepa, E. J. and Emr, S. D.
(1998). The Vps4p AAA ATPase regulates membrane association of a
Vps protein complex required for normal endosome function. EMBO
J. 17,
2982-2993.
Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T. and Emr, S. D. (2002). ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting. Dev. Cell 3, 271-282.[Medline]
Bilodeau, P. S., Urbanowski, J. L., Winistorfer, S. C. and Piper, R. C. (2002). The Vps27p-Hse1p complex binds ubiquitin and mediates endosomal protein sorting. Nat. Cell Biol. 4, 534-539.[Medline]
Bishop, N. and Woodman, P. (2000).
ATPase-defective mammalian VPS4 localizes to aberrant endosomes and
impairs cholesterol trafficking. Mol. Biol. Cell
11,
227-239.
Bryant, N. J. and Stevens, T. H. (1998).
Vacuole biogenesis in Saccharomyces cerevisiae, protein transport
pathways to the yeast vacuole. Microbiol. Mol. Biol.
Rev. 62,
230-247.
Cashikar, A. G., Schirmer, E. C., Hattendorf, D. A., Glover, J. R., Ramakrishnan, M. S., Ware, D. M. and Lindquist, S. L. (2002). Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein. Mol. Cell 9, 751-760.[Medline]
Cereghino, J. L., Marcusson, E. G. and Emr, S. D. (1995). The cytoplasmic tail domain of the vacuolar protein sorting receptor Vps10p and a subset of VPS gene products regulate receptor stability, function, and localization. Mol. Biol. Cell 6, 1089-1102.[Abstract]
Davis, N. G., Horecka, J. L. and Sprague, G. F., Jr (1993). Cis- and transacting functions required for endocytosis of the yeast pheromone receptors. J. Cell Biol. 122, 53-65.[Abstract]
Dower, W. J., Miller, J. F. and Ragsdale, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16, 6127-6145.[Abstract]
Dulic, V., Egerton, M., Elguindi, I., Raths, S., Singer, B. and Riezman, H. (1991). Yeast endocytosis assays. Methods Enzymol. 194, 697-710.[Medline]
Finken-Eigen, M., Rohricht, R. A. and Kohrer, K. (1997). The VPS4 gene is involved in protein transport out of a yeast pre-vacuolar endosome-like compartment. Curr. Genet. 31, 469-480.[CrossRef][Medline]
Forsberg, H., Hammar, M., Andreasson, C., Moliner, A. and
Ljungdahl, P. O. (2001). Suppressors of ssy1 and
ptr3 null mutations define novel amino acid sensor-independent genes
in Saccharomyces cerevisiae. Genetics
158,
973-988.
Gietz, R. D. and Sugino, A. (1988). New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527-534.[CrossRef][Medline]
Gyuris, J., Golemis, E., Chertkov, H. and Brent, R. (1993). Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75, 791-803.[Medline]
Hanson, P. K., Grant, A. M. and Nichols, J. W.
(2002). NBD-labeled phosphatidylcholine enters the yeast vacuole
via the pre-vacuolar compartment. J. Cell Sci.
115,
2725-2733.
Hicke, L., Zanolari, B., Pypaert, M., Rohrer, J. and Riezman, H. (1997). Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein. Mol. Biol. Cell 8, 13-31.[Abstract]
Howard, T. L., Stauffer, D. R., Degnin, C. R. and Hollenberg, S.
M. (2001). CHMP1 functions as a member of a newly defined
family of vesicle trafficking proteins. J. Cell Sci.
114,
2395-2404.
Katzmann, D. J., Babst, M. and Emr, S. D. (2001). Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-1. Cell 106, 145-155.[Medline]
Katzmann, D. J., Odorizzi, G. and Emr, S. D. (2002). Receptor downregulation and multivesicular-body sorting. Nat. Rev. 3, 893-905.
Kranz, A., Kinner, A. and Kolling, R. (2001). A
family of small coiled-coil-forming proteins functioning at the late endosome
in yeast. Mol. Biol. Cell
12,
711-723.
Kübler, E. and Riezman, H. (1993). Actin and fimbrin are required for the internalisation step of endocytosis in yeast. EMBO J. 12, 2855-2862.[Abstract]
Longtine, M. S., McKenzie, A., DeMarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P. and Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953-961.[CrossRef][Medline]
Lupas, A., Van Dyke, M. and Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 1162-1164.[Medline]
Mellman, I. (1996). Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575-625.[CrossRef][Medline]
Mulholland, J., Konopka, J., Singer-Krüger, B., Zerial, M.
and Botstein, D. (1999). Visualization of receptor-mediated
endocytosis in yeast. Mol. Biol. Cell
10,
799-817.
Munn, A. L. and Riezman, H. (1994). Endocytosis is required for the growth of vacuolar H+-ATPase-defective yeast, identification of six new END genes. J. Cell Biol. 127, 373-386.[Abstract]
Munn, A. L., Stevenson, B. J., Geli, M. I. and Riezman, H. (1995). end5, end6, and end7, mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae. Mol. Biol. Cell 6, 1721-1742.[Abstract]
Munn, A. L., Heese-Peck, A., Stevenson, B. J., Pichler, H. and
Riezman, H. (1999). Specific sterols required for the
internalization step of endocytosis in yeast. Mol. Biol.
Cell 10,
3943-3957.
Munn, A. L. (2000). The yeast endocytic membrane transport system. Microsc. Res. Tech. 51, 547-562.[CrossRef][Medline]
Nagiec, E. E., Bernstein, A. and Whiteheart, S. W.
(1995). Each domain of the N-ethylmaleimide-sensitive fusion
protein contributes to its transport activity. J. Biol.
Chem. 270,
29182-29188.
Odorizzi, G., Babst, M. and Emr, S. D. (1998). Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell 95, 847-858.[Medline]
Piper, R. C., Cooper, A. A., Yang, H. and Stevens, T. H. (1995). VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae. J. Cell Biol. 131, 603-617.[Abstract]
Piper, R. C. and Luzio, J. P. (2001). Late endosomes: sorting and partitioning in multivesicular bodies. Traffic 2, 612-621.[CrossRef][Medline]
Prescianotto-Baschong, C. and Riezman, H.
(1998). Morphology of the yeast endocytic pathway.
Mol. Biol. Cell 9,
173-189.
Prescianotto-Baschong, C. and Riezman, H. (2002). Ordering of compartments in the yeast endocytic pathway. Traffic 3, 37-49.[CrossRef][Medline]
Raymond, C. K., Howald-Stevenson, I., Vater, C. A. and Stevens, T. H. (1992). Morphological classification of the yeast vacuolar protein sorting mutants, evidence for a prevacuolar compartment in class E vps mutants. Mol. Biol. Cell 3, 1389-1402.[Abstract]
Reggiori, F. and Pelham, H. R. (2001). Sorting
of proteins into multivesicular bodies: ubiquitin-dependent and -independent
targeting. EMBO J. 20,
5176-5186.
Rieder, S. E., Banta, L. M., Kohrer, K., McCaffery, J. M. and Emr, S. D. (1996). Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Mol. Biol. Cell 7, 985-999.[Abstract]
Tchernev, V. T., Mansfield, T. A., Giot, L., Kumar, A. M., Nandabalan, K., Li, Y., Mishra, V. S., Detter, J. C., Rothberg, J. M. and Wallace, M. R. et al. (2002). The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins. Mol. Med. 8, 56-64.[Medline]
Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M. and Pochart, P. et al. (2000). A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623-627.[CrossRef][Medline]
Urbanowski, J. L. and Piper, R. C. (2001). Ubiquitin sorts proteins into the intralumenal degradative compartment of the late-endosome/vacuole. Traffic 2, 622-630.[CrossRef][Medline]
Vida, T. A. and Emr, S. D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779-792.[Abstract]
Zahn, R., Stevenson, B. J., Schröder-Köhne, S.,
Zanolari, B., Riezman, H. and Munn, A. L. (2001).
End13p/Vps4p is required for efficient transport from early to late endosomes
in Saccharomyces cerevisiae. J. Cell Sci.
114,
1935-1947.