1 Department of Molecular, Cellular and Developmental Biology, University of
Colorado, Campus Box 347, Boulder, CO 80309, USA
2 Department of Cellular and Molecular Medicine, Howard Hughes Medical
Institute, Campus Box 0668, School of Medicine, University of California, San
Diego, La Jolla, CA 92093, USA
Present address: Department of Biochemistry and Molecular Biology, 1611
Guggenheim Building, Mayo Clinic, Rochester, MN 55902, USA
Present address: Microgenomics, 5935 Darwin Court, Carlsbad, CA 92008,
USA
* Author for correspondence (e-mail: odorizzi{at}colorado.edu)
Accepted 28 January 2003
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Summary |
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Key words: Multivesicular, Vesicle, Transport, Vacuole
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Introduction |
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Many proteins that are critical to cell growth and development are sorted
into MVB vesicles. For example, activated epidermal growth factor receptors in
mammalian cells are downregulated through endocytosis and MVB sorting en route
to being degraded in the lysosome (Haigler
et al., 1979; McKanna et al.,
1979
). A similar pathway is followed by Ste2, a G protein-coupled
pheromone receptor that is downregulated and degraded in the vacuole of the
budding yeast Saccharomyces cerevisiae
(Hicke et al., 1997
;
Odorizzi et al., 1998
). These
receptors as well as many other cell-surface proteins that are targeted via
the MVB pathway are modified by the attachment of ubiquitin to their
cytoplasmic domains. Ubiquitin is a highly conserved 76-amino acid polypeptide
that is covalently linked to specific protein substrates by a cascade of
ubiquitin-conjugation enzymes. Ubiquitin was originally known for its
attachment as a chain of four or more subunits (polyubiquitination) to soluble
protein substrates that are targeted for degradation by the proteasome
(Weissman, 2001
). In contrast,
a single ubiquitin or a short chain of less than four ubiquitin subunits is
covalently linked to the cytoplasmic domains of cell-surface proteins that are
targeted for degradation in the lysosome/vacuole
(Hicke, 2001
). Prior to
proteasomal or vacuolar degradation, ubiquitin is removed from protein
substrates by a de-ubiquitinating enzyme, thereby enabling the cell to
maintain a constant pool of ubiqiutin
(Weissman, 2001
).
A direct role for ubiquitin in the MVB pathway has been demonstrated in
studies of a yeast vacuolar hydrolase, carboxypeptidase S (CPS). The precursor
form of CPS is synthesized as an integral membrane protein that is transported
to endosomes directly from the Golgi rather than via endocytosis from the
plasma membrane (Cowles et al.,
1997). The sorting of CPS into MVB vesicles results in its
delivery into the vacuole lumen (Odorizzi
et al., 1998
), where CPS is proteolytically cleaved from its
transmembrane anchor to produce the soluble mature form of the enzyme
(Spormann et al., 1992
). CPS
is mono-ubiquitinated on its cytoplasmic domain after exiting the Golgi, and
mutations in CPS that block this modification result in its mislocalization to
the limiting vacuolar membrane (Katzmann
et al., 2001
). Ubiquitination, therefore, can function as a
sorting signal for entry into the MVB pathway.
Ubiquitinated CPS is bound by ESCRT-I (Endosomal Sorting Complex Required
for Transport), a cytoplasmic protein complex that transiently associates with
endosomal membranes (Katzmann et al.,
2001). ESCRT-I is comprised of Vps23, Vps28 and Vps37, all three
of which are encoded by class E VPS (Vacuolar Protein Sorting) genes,
a set of 17 genes that are required for the sorting of both CPS and Ste2 into
the MVB pathway (reviewed by Katzmann et
al., 2002
). Two additional ESCRT complexes consisting of class E
Vps proteins have been described recently. Similar to ESCRT-I, both ESCRT-II
(Vps22, Vps25 and Vps36) and ESCRT-III (Vps2, Vps20, Vps24 and Snf7/Vps32)
transiently associate with endosomes (Babst
et al., 2002a
; Babst et al.,
2002b
). Although their precise functions are not yet known,
genetic data suggest that ESCRT-II functions downstream of ESCRT-I and
initiates the recruitment and assembly of ESCRT-III at the endosomal membrane
(Babst et al., 2002b
). Another
class E Vps protein, Vps4, is an ATPase that catalyzes the dissociation and
disassembly of all three ESCRT complexes from endosomal membranes
(Babst et al., 2002a
;
Babst et al., 2002b
;
Babst et al., 1998
).
One of the class E VPS genes that has not been characterized as
encoding a component of the ESCRT complexes is VPS31. The
VPS31 gene is allelic to BRO1, which was originally
implicated in the protein kinase C/MAP kinase signaling pathway
(Nickas and Yaffe, 1996).
Recently, mutations in BRO1 were shown to block the
ubiquitin-dependent downregulation of the general amino acid permease, Gap1,
from the plasma membrane (Springael et
al., 2002
). Similarly, mutations in BRO1 were found to
restore amino acid uptake in cells harboring defects in the plasma membrane
amino acid sensor complex, presumably by blocking the downregulation and
degradation of amino acid permeases
(Forsberg et al., 2001
). These
observations are consistent with a role for BRO1 in the sorting of
cell-surface proteins to the vacuole.
We show that BRO1 encodes a soluble cytoplasmic protein that
associates with endosomes. Similar to the ESCRT complexes, Bro1 accumulates on
endosomal membranes in vps4 mutant cells, suggesting a role for the
Vps4 ATPase in regulating the endosomal dissociation of Bro1. Furthermore,
biochemical fractionation and fluorescence microscopic studies indicate that
Bro1 association with endosomes requires the ESCRT-III component, Snf7.
Interestingly, unlike Gap1 (Springael et
al., 2002), the ubiquitination of CPS is not blocked by a deletion
of the BRO1 gene, suggesting that ubiquitination of cargo proteins
downregulated from the plasma membrane may be subject to different
requirements than the ubiquitination of cargo proteins sorted directly from
the Golgi.
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Materials and Methods |
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The HIS3 gene was used to replace the open-reading frame (ORF) of
BRO1 in SEY6210, MBY3 and PBY34 by homologous recombination
(Longtine et al., 1998),
resulting in GOY65, GOY66 and DBY11, respectively. The same method was used to
replace the ORF of VPS4 in PBY34 with HIS3, resulting in
DBY12. To construct GOY54 and GOY55, the wild-type BRO1 gene was
amplified by PCR using as template the genomic DNA from SEY6210 together with
primers GO104p (containing a SpeI restriction site) and GO106p
(containing a SalI restriction site), which anneal 500 base pairs
upstream and downstream, respectively, of the BRO1 ORF. The resulting
PCR product was ligated as a SpeI-SalI fragment into
SpeI-SalI-digested pRS416
(Sikorski and Hieter, 1989
),
resulting in plasmid pGO187. The PCR method of gene splicing by overlap
extension (gene SOE) (Yon and Fried,
1989
) was used to construct a unique EcoRI restriction
site at the start codon of BRO1 within pGO187, resulting in plasmid
pGO221, which was then digested with HindIII, blunted with
T4 DNA polymerase, and subsequently digested with EcoRI.
In parallel, a plasmid consisting of the pBluescriptSK
vector (Stratagene) containing the wild-type TRP1 gene from
Saccharomyces cerevisiae was digested with BamHI, blunted
with T4 DNA polymerase, then digested with EcoRI to yield
a BamHIblunt-EcoRI fragment containing the
TRP1 gene that was ligated with the
HindIIIblunt-EcoRI fragment of pGO221, resulting
in the replacement of BRO1 codons 1-770 with TRP1. This
plasmid served as a PCR template using primers GO104p and GO106p, and the
resulting PCR product was transformed into CBY31 and TVY614 to construct GOY54
and GOY55, respectively.
The HIS3-CPS fusion was constructed by gene SOE PCR using plasmid
templates pRS415 (Sikorski and Hieter,
1989) for HIS3 and pDP83
(Cowles et al., 1997
) for
CPS1. The resulting PCR product was ligated into pRS416, resulting in
plasmid pGO96. SEY6210 cells transformed with pGO96 were mutagenized with
ethyl methanesulfonate, resulting in
25% viability. The mutagenized cells
were diluted in rich growth medium and grown at room temperature for 2 hours,
then harvested by centrifugation, resuspended in double-distilled water, and
spread at a concentration of 5000 cells/petri dish onto 200 petri dishes
consisting of agar growth medium that lacks supplemental histidine. The growth
medium contained 30 mM 3-amino-1,2,4-triazole, a competitive inhibitor of the
His3 enzyme, in order to suppress growth of wild-type cells because of the
activity of newly synthesized His3-CPS fusions being transported through the
early secretory pathway. After 10 days of incubation at room temperature,
surviving colonies were transformed with pGO45 and examined by fluorescence
microscopy in order to determine the localization of green fluorescence
protein (GFP)-CPS.
The GFP-BRO1 fusion was constructed by PCR amplification of
BRO1 using pGO187 as a template, together with primers GO98p (which
places a unique EcoRI restriction site at the start codon of
BRO1) and GO106p. (Primer sequences are available upon request.) The
resulting PCR product was digested with EcoRI and SalI, then
ligated into EcoRI-SalI-digested pGO36, which is identical
to the pGOGFP plasmid that was described in Cowles et al.
(Cowles et al., 1997), except
that pGO36 has a pRS416 vector backbone rather than a pRS426 vector backbone
(Sikorski and Hieter, 1989
).
The resulting GFP-BRO1-containing plasmid is pGO249. Plasmids
containing the vps4K179A (pMB24) and
vps4E233Q (pMB49) alleles were described in Babst et al.
(Babst et al., 1997
). The
plasmid containing GFP-CPS (pGO45) was described in Odorizzi et al.
(Odorizzi et al., 1998
).
Fluorescence microscopy
Cells expressing GFP fusion proteins were pulse-labeled or labeled
continuously with FM 4-64 at 30°C as previously described
(Vida and Emr, 1995). GFP and
FM 4-64 fluorescence as well as Nomarski optics were observed using a Leica
DMRXA fluorescence microscope equipped with a Cooke Sensicam digital camera
(Applied Scientific Instruments). Images were processed using Slidebook
software (Intelligent Imaging Innovations).
Immunoprecipitations and subcellular fractionations
For immunoprecipitation of CPS, 2 sets of 5 A600 units of cells
grown at 30°C to mid-logarithmic phase in liquid synthetic medium were
harvested by centrifugation at 500 g and resuspended in 1 ml
of medium. Fifty microcuries of Tran35S-label (ICN Biochemicals)
was added, and cells were shaken for 10 minutes at 30°C. Labeling of newly
synthesized proteins was terminated by adding 5 mM methionine, 1 mM cysteine,
and 0.2% yeast extract, and cultures were shaken for an additional 0- or
30-minute chase period at 30°C. CPS was subsequently immunoprecipitated
using rabbit anti-CPS antiserum (Cowles et
al., 1997) and Protein A-sepharose (Pharmacia) from total cell
extracts that had been precipitated by the addition of 10% (vol/vol)
trichloroacetic acid (TCA) and washed twice with icecold acetone.
Immunoprecipitates were treated with endoglycosidase H to remove carbohydrate
modifications as previously described
(Cowles et al., 1997
), then
resolved by SDS-PAGE and examined by fluorography. For immunoprecipitation of
CPY, 5 A600 units of cells were labeled with
Tran35S-label as described above, then chased for 30 minutes. The
cells were then harvested and converted to spheroplasts, then CPY was
immunoprecipitated from the intracellular fraction and external medium using
rabbit anti-CPY antiserum and Protein A-sepharose as previously described
(Darsow et al., 1997
).
Following SDS-PAGE, the amount of internal and secreted radioactive CPY was
quantitated using a Storm 860 phosphorimager (Molecular Dynamics).
For subcellular fractionation, 10 A600 units of cells grown at
30°C to mid-logarithmic phase were converted to spheroplasts
(Darsow et al., 1997), then
harvested by centrifugation at 500 g. Spheroplasts were
resuspended gently in 1 ml ice-cold lysis buffer [200 mM sorbitol, 50 mM
potassium acetate, 20 mM Hepes, pH 7.2, 2 mM EDTA, supplemented with a
protease inhibitor cocktail (Roche)], then subjected to 12 strokes in an
ice-cold Dounce tissue homogenizer. Lysates were divided into two 0.5-ml
aliquots and centrifuged at 4°C for 15 minutes at 13,000 g
to generate P13 pellets enriched for vacuole, endosome, endoplasmic reticulum
and plasma membranes. The 13,000 g supernatant fractions were
centrifuged at 100,000 g for 1 hour at 4°C in a Beckman
TLA100.3 rotor, resulting in S100 supernatant fractions containing soluble
proteins and P100 pellet fractions enriched for membranes of the Golgi and
small transport vesicles. Protein samples of each fraction were
TCA-precipitated and acetone-washed. One-half A600 unit-equivalent
of each fraction was resolved by SDS-PAGE, transferred to nitrocellulose and
examined by western blotting using antibodies against Bro1 (see below), Vps24
(Babst et al., 1998
), Snf7
(Babst et al., 1998
), Vps4
(Babst et al., 1997
), ALP
(Molecular Probes) and PGK (Molecular Probes).
For detection of Ub-CPS, denaturing immunoprecipitations using anti-CPS
antiserum and Protein A-sepharose were performed as previously described
(Katzmann et al., 2001).
Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose,
and two A600 units of each sample were examined by SDS-PAGE and
western blotting using anti-ubiquitin antibodies (Zymed Laboratories). In the
wild-type (TVY614), vps4
(MBY52) and bro1
(GOY55) strains that were examined, the PEP4, PRB1 and PRC1
genes encoding vacuolar proteases had been deleted in order to reduce the
non-specific cleavage of ubiquitin from CPS after cell lysis.
For sucrose density gradient fractionation, the P13 fraction from
vps4 cells was resuspended in 1 ml of lysis buffer
supplemented with 60% sucrose, then loaded beneath 2 ml of 55% and 2 ml of 35%
sucrose/lysis buffer solutions. After a 14-hour spin at 200,000
g, 3 fractions were collected: the top 3 ml (F), the remaining
2 ml (NF), and the sediment (P). Each fraction was TCA-precipitated and washed
in acetone, and one-half A600 unit was examined by SDS-PAGE and
western blotting.
Preparation of antiserum against Bro1
A 908-bp DNA fragment of BRO1 encompassing codons 542-844 was
subcloned into pGEX-KG, and the resulting glutathione S-transferase
fusion protein was inducibly expressed in Escherichia coli, purified
from bacterial extracts following SDS-PAGE, then used to immunize New Zealand
White rabbits as previously described
(Cowles et al., 1997).
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Results |
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The isolation of class E vps mutants using His3-CPS reinforced the
idea that class E VPS genes have a central role in the MVB pathway.
One of the class E VPS genes that had not previously been
characterized in detail for its role in Golgi-to-vacuole protein sorting is
VPS31. Analysis of the VPS31 nucleotide sequence revealed
that it is allelic to the BRO1 gene. BRO1 stands for
BCK1-like Resistance to Osmotic shock and was originally identified
in a study showing that a bro1 mutation worsens the viability of
cells that are mutant for several components of the protein kinase C/MAP
kinase signaling pathway, including the MEK kinase Bck1
(Nickas and Yaffe, 1996). A
similar genetic interaction has been found between the bck1 mutation
and other class E vps mutations, including vps24, vps28 and
vps36 (M. Nickas, personal communication). These observations may
indicate a functional relationship between class E VPS genes and
signaling through the protein kinase C/MAP kinase pathway. Alternatively,
class E vps mutations may non-specifically exacerbate the relatively
poor viability caused by mutations in MAP kinase components.
Similar to other class E VPS genes, BRO1 is required for
the sorting of GFP-CPS via the MVB pathway. In wild-type cells, the delivery
of GFP-CPS into the vacuole lumen is in sharp contrast to FM 4-64
(Fig. 2A), a fluorescent
lipophilic compound that intercalates into the plasma membrane and is
delivered to the vacuole membrane by endocytosis
(Vida and Emr, 1995). However,
bro1
cells and other class E vps mutants, such as
vps4
, mislocalize GFP-CPS to the vacuole membrane and to the
class E compartment, an aberrant late endosomal structure located adjacent to
the vacuole (Fig. 2A).
|
BRO1 and other class E VPS genes are also required for
the efficient proteolytic maturation of the native CPS enzyme. The precursor
form of CPS (pCPS) is synthesized as a type II integral membrane protein that,
upon delivery to the vacuole, is proteolytically cleaved at a site adjacent to
its transmembrane domain, resulting in the mature vacuolar form of the enzyme
(mCPS) (Spormann et al.,
1992). This proteolytic maturation event can be monitored by
immunoprecipitation of CPS from cells that have been pulsed-labeled with
[35S]methionine/cysteine and chased in non-radioactive medium for 0
or 30 minutes (Cowles et al.,
1997
). In wild-type cells, pCPS is detected as a 73-kDa
polypeptide immediately after synthesis (0-minute chase) and is completely
converted to its 69-kDa mature form after a 30-minute chase period
(Fig. 2B). In
bro1
and vps4
mutants, however,
50% of
the pool of newly synthesized CPS fails to be proteolytically matured
(Fig. 2B). The partial
conversion of CPS to its mature form in class E vps mutant cells
probably occurs in the class E compartment, as newly synthesized vacuolar
hydrolases required for CPS maturation also accumulate at this aberrant
structure; the pH-dependent activation of these enzymes is thought to be
because of the mislocalization of the vacuolar ATPase at the class E
compartment (Piper et al.,
1997
; Piper et al.,
1995
).
BRO1 is not required for ubiquitination of CPS
Recently, mutations in BRO1 were found to impair ubiquitination of
the general amino acid permease, Gap1, which undergoes ubiquitin-dependent
downregulation from the plasma membrane
(Springael et al., 2002).
Similar to Gap1, CPS is ubiquitinated on its cytoplasmic domain, which is
required for the sorting of CPS via the MVB pathway
(Katzmann et al., 2001
).
Therefore, we investigated whether CPS ubiquitination requires BRO1
by immunoprecipitating CPS from bro1 mutant cells, followed by
western blotting using anti-ubiquitin antibodies. CPS is ubiquitinated before
its arrival at endosomes but is de-ubiquitinated prior to being sorted into
MVB vesicles (Katzmann et al.,
2001
). As a result, ubiquitinated CPS (Ub-CPS) is difficult to
detect in wild-type cells, but is easily observed upon deletion of the
PEP12 gene (Katzmann et al.,
2001
), which encodes a t-SNARE required for the fusion of
post-Golgi transport vesicles with endosomes
(Becherer et al., 1996
). As
shown in Fig. 2C, the amount of
Ub-CPS observed in pep12
cells is not diminished if
BRO1 has also been deleted in this strain, indicating that
BRO1 is not required for CPS ubiquitination. Interestingly, Ub-CPS
can be detected in class E vps mutants, including
bro1
and vps4
cells, even if the
PEP12 gene has not been deleted
(Fig. 2C). A similar
stabilization of Ub-CPS has been observed in vps23
(Katzmann et al., 2001
) and
vps27
cells (Shih et al.,
2002
). The de-ubiquitination of CPS, therefore, appears to require
the functions of many class E Vps proteins.
BRO1 is not essential for sorting CPY
In addition to mislocalizing CPS to the vacuole membrane, all class E
vps mutant cells aberrantly secrete a soluble vacuolar enzyme,
carboxypeptidase Y (CPY). Newly synthesized CPY is transported from the
endoplasmic reticulum to the Golgi, where it receives oligosaccharide
modifications, resulting in the 69-kDa p2CPY precursor
(Stevens et al., 1982). A
vacuolar targeting sequence within the N-terminal pro-peptide region of p2CPY
is bound by Vps10, a transmembrane receptor in the late Golgi
(Marcusson et al., 1994
). The
Vps10-p2CPY complex is sorted from the Golgi to endosomes, whereupon p2CPY
dissociates from Vps10, and the receptor recycles back to the Golgi, whereas
p2CPY is transported further toward the vacuole
(Cereghino et al., 1995
;
Cooper and Stevens, 1996
). The
pro-peptide region in p2CPY is proteolytically removed upon vacuolar delivery,
resulting in mCPY, the mature 61-kDa form of the enzyme
(Stevens et al., 1982
).
To monitor the requirement for BRO1 in CPY sorting, wild-type
bro1 and vps4
cells were pulse-labeled with
[35S]methionine/cysteine for 10 minutes, then chased in
non-radioactive medium for 30 minutes. Afterward, the cells were converted to
spheroplasts, separated into intracellular (I) and extracellular (E)
fractions, and CPY was recovered by immunoprecipitation. Virtually all newly
synthesized CPY was found intracellularly in its mature form in wild-type
cells, reflecting its efficient delivery to the vacuole
(Fig. 3A). In contrast,
50% of CPY was secreted in its p2 form by vps4
cells
(Fig. 3A)
(Babst et al., 1997
).
Similarly, other class E vps mutants have been shown to secrete
30-50% of newly synthesized CPY (Li et
al., 1999
; Piper et al.,
1995
; Raymond et al.,
1992
; Rieder et al.,
1996
). However, <10% of CPY was secreted to the extracellular
medium by bro1
cells (Fig.
3A). Indeed, an earlier study that compared the sorting defects in
class E vps mutants showed that vps31/bro1 mutant cells
secrete much less CPY than all of the other class E vps mutants that
were examined (Raymond et al.,
1992
). Thus, the BRO1 gene product appears to have a
relatively minor role in CPY sorting compared to other class E VPS
gene products.
|
The aberrant secretion of CPY caused by mutations in class E VPS
genes is thought to be because of a defect in the recycling of the CPY
receptor, Vps10, back to the Golgi. Previous indirect immunofluorescence
microscopic studies showed that in wild-type cells Vps10 is localized to
multiple punctate structures corresponding to Golgi and endosomal
compartments. However, upon deletion of VPS27, a class E VPS
gene, Vps10 was observed exclusively at the class E compartment
(Piper et al., 1995). Because
bro1
cells secrete very little CPY compared to other class E
vps mutants, we examined the localization of a Vps10-GFP fusion
protein expressed in wild-type bro1
and vps4
cells. The Vps10-GFP chimera consists of GFP fused to the C-terminal
cytoplasmic domain of Vps10 (Burda et al.,
2002
). As shown in Fig.
3B, Vps10-GFP was localized to multiple punctate structures in
wild-type cells, consistent with its steady-state distribution in Golgi and
endosomal compartments (Burda et al.,
2002
; Piper et al.,
1995
). In contrast, Vps10-GFP in vps4
cells
colocalized entirely with FM 4-64 at the class E compartment
(Fig. 3B). Thus, as observed
previously in vps27
cells
(Piper et al., 1995
), a
deletion of VPS4 causes a severe defect in the recycling of Vps10
back to the Golgi. In bro1
cells, however, Vps10-GFP not only
colocalized with FM 4-64 at the class E compartment but was also observed on
additional punctate structures, the majority of which were not stained with FM
4-64 (Fig. 3B). The
localization of Vps10-GFP in bro1
cells, therefore, is very
similar to its localization in wild-type cells. These observations explain the
relatively mild CPY sorting defect observed in bro1 mutant cells
(Fig. 3A)
(Raymond et al., 1992
), and
indicate that the recycling of Vps10 back to the Golgi is not strongly
dependent on BRO1.
BRO1 encodes a soluble cytoplasmic protein that associates
with endosomes
BRO1 is predicted to encode an 844-amino acid polypeptide (Bro1)
that has a molecular weight of 97.3 kDa. Amino acid sequence alignments
predict that Bro1 has a highly conserved N-terminal domain (termed the `Bro1
domain' by the Pfam protein domain database)
(Bateman et al., 2002), a
central coiled-coil region and a C-terminal proline-rich domain
(Fig. 4B). In order to
investigate the intracellular distribution of Bro1, we raised a polyclonal
antiserum against its C-terminal 304 amino acids (see Materials and Methods).
We then used this antiserum for western blot analysis of yeast cell lysates
that were separated by differential centrifugation to yield a 13,000
g pellet fraction (P13) that contains membranes of the
vacuole, endosomes, plasma membrane and endoplasmic reticulum, a 100,000
g pellet fraction (P100) that contains membranes of the Golgi
and small transport vesicles, and a 100,000 g supernatant
fraction (S100) that contains soluble proteins
(Marcusson et al., 1994
). As
shown in Fig. 4A, Bro1 was
located predominantly in the S100 fraction of wild-type cells, although a
small amount (
5%) was also detected in the P13 and P100 pellets.
|
We also examined the intracellular localization of Bro1 in vivo using a
GFP-Bro1 chimera in which GFP had been fused to the N terminus of Bro1
(Fig. 4B). GFP-Bro1 rescued the
sorting of both CPS and CPY when expressed from a low-copy plasmid in
bro1 cells (data not shown), indicating that the fusion
protein is fully functional. As shown in
Fig. 4B, wild-type cells (in
which GFP-Bro1 was expressed in place of endogenous Bro1) exhibited a diffuse
cytoplasmic fluorescence which probably corresponds to the distribution of
Bro1 in the S100 fraction of cell lysates
(Fig. 4A). In addition, a few
punctate structures that were positive for GFP fluorescence were also observed
(Fig. 4B). These structures
probably correspond to endosomal compartments, as they are also stained by FM
4-64 (Fig. 4B, inset).
Together, the subcellular fractionation and fluorescence microscopic data
indicate that in wild-type cells, Bro1 is predominantly soluble and
cytoplasmic, with a portion of its total cellular pool associated with
endosomes.
Bro1 accumulates at the class E compartment in the absence of Vps4
ATPase activity
Vps4 is an AAA-type ATPase required for normal endosomal sorting and
morphology (Babst et al.,
1997). In general, members of the AAA family (ATPases associated
with a variety of cellular activities) function as chaperones that disrupt
molecular or macromolecular structures, and many AAA proteins assemble into
ring-shaped homo-oligomers, which appear to regulate their ATPase activities
and mechanism of action (Ogura and
Wilkinson, 2001
). The ATP-bound form of Vps4 assembles as a
homo-oligomer that associates with endosomal compartments, and upon ATP
hydrolysis, the Vps4 oligomer disassembles and dissociates from endosomes
(Babst et al., 1998
).
In addition to regulating its own endosomal localization, Vps4 ATPase
activity is required for the endosomal dissociation of several class E Vps
proteins, including Vps24 and Snf7/Vps32
(Babst et al., 1998). Vps24 and
Snf7 exist in cytoplasmic and membrane-associated pools in wild-type cells
(Babst et al., 1998
) and are,
therefore, distributed among the P13, P100 and S100 fractions
(Fig. 4A). In
vps4
cells, however, both Vps24 and Snf7 accumulate at the
class E compartment (Babst et al.,
1998
), resulting in both proteins being found almost entirely in
the P13 fraction (Fig. 4A).
Similarly,
50% of the total cellular pool of Bro1 was located in the P13
pellet in vps4
cells, whereas the remainder was found in the
S100 fraction (Fig. 4A). As in
the case of Vps24 and Snf7 (Babst et al.,
1998
), the P13 enrichment of Bro1 appeared to be because of its
accumulation at the class E compartment, as GFP-Bro1 was shifted from being
primarily cytoplasmic in wild-type cells to being concentrated at the class E
compartment in vps4
cells
(Fig. 4B).
To confirm that the P13 pool of Bro1 in vps4 cells was
indeed membrane-associated, this fraction was resuspended in buffer and loaded
at the bottom of a sucrose step gradient. Following centrifugation, the
samples were divided into the top fraction containing the membrane-associated
floating material (F), the load fraction containing non-floating material
(NF), and the pellet fraction (P) that corresponds to large
non-membrane-associated material (Babst et
al., 1998
). Western blot analysis indicated that the
P13-associated pool of Bro1, together with Vps24 and Snf7, floated to the top
of the gradient along with alkaline phosphatase (ALP), an integral membrane
protein of the vacuole (Fig.
4C). Thus, similar to Vps24 and Snf7
(Babst et al., 1998
), a
membrane-associated pool of Bro1 accumulates at the class E compartment in
vps4
cells.
The accumulation of Vps24 and Snf7 at the class E compartment in
vps4 mutant cells is specifically because of the loss of Vps4 ATPase
activity (Babst et al., 1998).
Thus, both proteins shift to the P13 fraction in cells expressing mutant forms
of Vps4 that are either unable to bind ATP (Vps4K179A) or unable to
hydrolyze the bound nucleotide (Vps4E233Q)
(Fig. 4D). Similarly, Bro1 was
shifted to the P13 fraction in both vps4KA and
vps4EQ mutant cells
(Fig. 4D). Therefore, both
ATP-binding and ATP-hydrolysis by Vps4 appear to be required for Bro1 to
dissociate from endosomes.
The endosomal association of Bro1 requires Snf7
Both Vps24 and Snf7 are components of ESCRT-III, a large, hetero-oligomeric
complex that also contains two other class E Vps proteins, Vps2 and Vps20
(Babst et al., 2002a).
ESCRT-III associates with endosomal compartments and appears to be comprised
of two functionally distinct subcomplexes: a membrane-proximal Vps20-Snf7
subcomplex and a peripheral Vps2-Vps24 subcomplex
(Babst et al., 2002a
). Vps2 and
Vps24 are required for the recruitment of ATP-bound Vps4 and, in turn, ATP
hydrolysis by Vps4 catalyzes the dissociation of all four ESCRT-III components
from endosomes (Babst et al.,
2002a
).
Because the data described above suggest that Bro1 also associates with
endosomal compartments, we examined whether the distribution of Bro1 is
dependent upon the ESCRT-III complex. We found that if either the
VPS2 gene or the VPS24 gene had been deleted, Bro1 was
enriched in the P13 pellet (Fig.
5A), and GFP-Bro1 was concentrated at the class E compartment
(Fig. 6). Similarly, Snf7 was
shifted almost entirely to the P13 fraction in the absence of either Vps2 or
Vps24 (Fig. 5A). This shift in
the distributions of Bro1 and Snf7 occurred in vps2 and
vps24
cells regardless of whether the VPS4 gene had
also been deleted, which is consistent with the Vps2-Vps24 subcomplex being
required for the endosomal recruitment of Vps4
(Babst et al., 2002a
). Thus, as
found previously for ESCRT-III components, Bro1 appears unable to undergo
Vps4-dependent dissociation from endosomes in the absence of either Vps2 or
Vps24.
|
|
Interestingly, we found that if the SNF7 gene had been deleted
either in VPS4+ or vps4 cells, Bro1
remained in the S100 fraction (Fig.
5A) and GFP-Bro1 was mostly cytoplasmic
(Fig. 6). However, there was
only a modest decrease in the endosome-associated pool of Bro1 upon deletion
of the VPS20 gene in vps4
cells
(Fig. 5A,
Fig. 6). Snf7 had similarly
been shown to be largely capable of localizing to endosomes independently of
Vps20 (Babst et al., 2002a
)
(Fig. 5A). Thus, the
association of Bro1 with endosomal compartments appears to be specifically
dependent upon Snf7.
In the absence of Vps4 function, both Bro1 and Snf7 accumulated at the
class E compartment and were, therefore, enriched in the P13 fraction
(Fig. 4A). As shown in
Fig. 5B, however, Snf7 was
still found in the P13 fraction in vps4 bro1
cells, and the relatively equal distribution of Snf7 in soluble and
membrane-associated fractions of wild-type cells was not altered upon deletion
of BRO1 (compare Fig.
4A with Fig. 5B).
Similarly, the distribution of Vps24 in VPS4+ and
vps4
cells was not affected if BRO1 had been deleted
(Fig. 5B). To investigate
further, we resuspended the P13 fraction from vps4
cells in
buffer containing 1% Triton X-100, then subjected the extract to
centrifugation at 100,000 g. Consistent with previous results
(Babst et al., 1998
), we found
that both Snf7 and Vps24 were insoluble upon detergent extraction
(Fig. 5C), which is presumably
because of their oligomerization into the very high molecular weight ESCRT-III
complex (Babst et al., 2002a
).
In contrast, Bro1 was completely soluble under these conditions
(Fig. 5C). Altogether, these
observations suggest that Bro1 does not assemble as a core component of the
ESCRT-III complex.
![]() |
Discussion |
---|
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---|
Bro1 is unable to dissociate from endosomes if either VPS2 or
VPS24 has been deleted, which is consistent with the observation that
Vps2 and Vps24 are required for the recruitment of ATP-bound Vps4 to endosomes
(Babst et al., 2002a). Vps2 and
Vps24 are recruited as a subcomplex to endosomes by Snf7 and Vps20, which may
associate directly with endosomal membranes
(Babst et al., 2002a
).
Interestingly, if SNF7 has been deleted, Bro1 is primarily
cytoplasmic with only minimal localization to the class E compartment, even in
the absence of Vps4 ATPase activity. In contrast, the accumulation of Bro1 at
the class E compartment is only mildly reduced in vps4
cells
upon deletion of the VPS20 gene. Similar to Bro1, Snf7 localizes to
the class E compartment in vps4
vps20
double-mutant cells (Babst et al.,
2002a
). Thus, our data suggest that among the ESCRT-III
components, Snf7 has a specific role in the endosomal recruitment of Bro1.
However, Bro1 is unlikely to form a stable complex with Snf7 at endosomes, as
Bro1 is completely solubilized upon detergent extraction of endosomal
membranes, whereas Snf7 remains insoluble under these conditions because of
its oligomerization into the ESCRT-III complex
(Babst et al., 2002a
).
Furthermore, only
50% of the cellular pool of Bro1 is trapped at the
class E compartment in the absence of Vps4 function, whereas almost 100% of
Snf7 (and Vps24) localizes to the class E compartment under these conditions.
Altogether, these results suggest that the endosomal association of Bro1 (and
possibly its Vps4-dependent dissociation) occurs downstream of ESCRT-III
complex assembly. However, it is possible that Bro1 is a peripheral component
of ESCRT-III that associates with Snf7-Vps20 less tightly than does
Vps2-Vps24. In this scenario, Bro1 could function together with Vps2-Vps24 in
the same ESCRT-III complex or, alternatively, either Bro1 or Vps2-Vps24 could
bind interchangeably to the membrane-proximal Snf7-Vps20 subcomplex in order
to confer distinct ESCRT-III activities.
ESCRT-III functions downstream of two additional ESCRT complexes consisting
of class E Vps proteins. ESCRT-I is a 350-kDa cytoplasmic complex
comprised of Vps23, Vps28 and Vps37
(Katzmann et al., 2001
).
ESCRT-I binds ubiquitinated CPS via the ubiquitin conjugation-like domain of
Vps23, suggesting that this complex functions in the recognition of
ubiquitinated MVB cargo proteins (Katzmann
et al., 2001
). ESCRT-II is a
155-kDa cytoplasmic complex
consisting of Vps22, Vps25 and Vps36, and is necessary for the recruitment of
ESCRT-III to the endosomal membrane (Babst
et al., 2002b
). Consistent with these observations, we have found
that ESCRT-II is also required for the association of Bro1 with endosomes
(G.O., unpublished).
Further evidence that Bro1 functions downstream of other class E Vps
proteins is the relatively mild CPY sorting defect observed in bro1
mutant cells. Although vps4 and other class E vps
mutants secrete 30-50% of newly synthesized CPY from the Golgi to the plasma
membrane (Li et al., 1999
;
Piper et al., 1995
;
Raymond et al., 1992
;
Rieder et al., 1996
),
bro1
cells secrete only
10% of CPY
(Raymond et al., 1992
) (this
study). The aberrant secretion of CPY by class E vps mutants stems
from a defect in the recycling of the CPY receptor, Vps10, from endosomes back
to the Golgi (Cereghino et al.,
1995
; Piper et al.,
1995
). Accordingly, we have found that the normal Golgi/endosomal
distribution of a Vps10-GFP fusion observed in wild-type cells is not
significantly altered upon deletion of the BRO1 gene. In contrast,
Vps10-GFP is concentrated exclusively at the class E compartment in
vps4
cells, which has also been observed for the native Vps10
distribution in vps27
cells
(Piper et al., 1995
).
Altogether, these data indicate that the recycling of Vps10 to the Golgi is
more dependent upon other class E Vps proteins than it is dependent upon Bro1.
One possibility is that Bro1 functions after most of the CPY receptors have
recycled out of the endosome, whereas other class E Vps proteins may be
required at an earlier stage in endosomal sorting. Alternatively, Bro1 could
have a more specific role in sorting MVB cargoes, whereas the activities of
other class E Vps proteins could be required in general to maintain normal
endosomal function.
The BRO1 gene was originally identified in a study showing that a
bro1 mutation worsens the viability of bck1 mutant cells
(Nickas and Yaffe, 1996).
BCK1 encodes a MEK kinase that functions in the protein kinase C/MAP
kinase pathway (Lee and Levin,
1992
). bck1 mutant cells also exhibit reduced viability
upon deletion of other class E VPS genes (M. Nickas, personal
communication). Although a functional relationship may exist between class E
VPS genes and signaling through the protein kinase C/MAP kinase
pathway, it is also possible that mutations in class E VPS genes
result in a non-specific exacerbation of the relatively poor viability caused
by mutations in MAP kinase components.
Bro1 is clearly required for the MVB pathway, as a deletion of the
BRO1 gene blocks the sorting of GFP-CPS, which is transported to
endosomes from the Golgi, as well as Ste2-GFP, an MVB cargo protein that is
endocytosed from the plasma membrane
(Odorizzi et al., 1998). Amino
acid sequence comparisons indicate that Bro1 has orthologs in a wide range of
eukaryotic species, including the mammalian Alix/Aip1 protein that has been
implicated in apoptosis (Missotten et al.,
1999
; Vito et al.,
1999
), and the Xenopus Xp95 protein, which was identified as a
phosphoprotein during meiosis (Che et al.,
1999
). Bro1 and its putative orthologs have a highly conserved
N-terminal
150-amino acid domain, a central coiled-coil region, and a
C-terminal
150-amino acid proline-rich domain. Interestingly, the
RIM20 gene in yeast, which has been implicated to function in the pH
response pathway (Xu and Mitchell,
2001
), encodes a protein that is similar to the N-terminal and
central regions of Bro1 but which lacks the proline-rich C-terminal domain.
However, our observations indicate that Rim20 does not have a role in vacuolar
protein sorting, as a deletion of the RIM20 gene does not cause
defects in the sorting of CPS or CPY, and a double deletion of both
RIM20 and BRO1 does not worsen the sorting defects observed
upon deletion of BRO1 alone (data not shown).
Although the precise function of Bro1 in the MVB pathway is not yet clear,
our observations indicate it is not required for the ubiquitination of CPS.
CPS ubiquitination is not impaired upon deletion of BRO1 or other
class E VPS genes, including VPS27
(Shih et al., 2002),
VPS23 or VPS4 (Katzmann
et al., 2001
). In contrast, mutations in BRO1 have been
found to block ubiquitination of the Gap1 amino acid permease, which undergoes
ubiquitin-dependent downregulation from the plasma membrane
(Springael et al., 2002
).
Similarly, deletion of VPS27 has been shown to block ubiquitination
of the uracil permease, Fur4, which is also downregulated from the cell
surface (Dupre and Haguenauer-Tsapis,
2001
). One possible reason why class E vps mutations
affect ubiquitination of Gap1 and Fur4 but not CPS could be because of the
findings that plasma membrane protein cargoes in yeast are ubiquitinated at
the cell surface, whereas CPS is ubiquitinated during its transit from the
Golgi to endosomes (reviewed by Katzmann
et al., 2002
). The ubiquitination of Gap1 and Fur4 requires the
ubiquitin ligase, Rsp5 (Hein et al.,
1995
), the function of which could be sensitive to the membrane
trafficking defects that occur upon mutation of class E VPS genes.
For instance, Rsp5 or other components of the ubiquitination machinery could
be mislocalized in class E vps mutant cells. In contrast, the
ubiquitination of CPS has been proposed to require Tul1, a putative ubiquitin
ligase that resides in the Golgi (Reggiori
and Pelham, 2002
); the function and/or localization of Tul1 may be
independent of class E Vps protein activities. Whether Bro1 or other class E
Vps proteins have a direct or indirect role in ubiquitination of plasma
membrane proteins such as Gap1 and Fur4 awaits further investigation.
![]() |
Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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. (2002a). ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting. Dev. Cell 3,272 -282.
Babst, M., Katzmann, D. J., Snyder, W. B., Wendland, B. and Emr, S. D. (2002b). Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev. Cell 3,283 -289.[Medline]
Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L.,
Eddy, S. R., Griffiths-Jones, S., Howe, K. L., Marshall, M. and Sonnhammer, E.
L. (2002). The Pfam protein families database.
Nucleic Acids Res. 30,276
-280.
Becherer, K. A., Rieder, S. E., Emr, S. D. and Jones, E. W. (1996). Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. Mol. Biol. Cell 7,579 -594.[Abstract]
Burd, C. G., Peterson, M., Cowles, C. R. and Emr, S. D. (1997). A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast. Mol. Biol. Cell 8,1089 -1104.[Abstract]
Burda, P., Padilla, S. M., Sarkar, S. and Emr, S. D.
(2002). Retromer function in endosome-to-Golgi retrograde
transport is regulated by the yeast Vps34 PtdIns 3-kinase. J. Cell
Sci. 115,3889
-3900.
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]
Che, S., El-Hodiri, H. M., Wu, C. F., Nelman-Gonzalez, M., Weil,
M. M., Etkin, L. D., Clark, R. B. and Kuang, J. (1999).
Identification and cloning of xp95, a putative signal transduction protein in
Xenopus oocytes. J. Biol. Chem.
274,5522
-5531.
Cooper, A. A. and Stevens, T. H. (1996). Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J. Cell Biol. 133,529 -541.[Abstract]
Cowles, C. R., Snyder, W. B., Burd, C. G. and Emr, S. D.
(1997). Novel Golgi to vacuole delivery pathway in yeast:
identification of a sorting determinant and required transport component.
EMBO J. 16,2769
-2782.
Darsow, T., Rieder, S. E. and Emr, S. D.
(1997). A multispecificity syntaxin homologue, Vam3p, essential
for autophagic and biosynthetic protein transport to the vacuole.
J. Cell Biol. 138,517
-529.
Dupre, S. and Haguenauer-Tsapis, R. (2001).
Deubiquitination step in the endocytic pathway of yeast plasma membrane
proteins: crucial role of Doa4p ubiquitin isopeptidase. Mol. Cell
Biol. 21,4482
-4494.
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.
Futter, C. E., Pearse, A., Hewlett, L. J. and Hopkins, C. R. (1996). Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol. 132,1011 -1023.[Abstract]
Haigler, H. T., McKanna, J. A. and Cohen, S. (1979). Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J. Cell Biol. 81,382 -395.[Abstract]
Hein, C., Springael, J. Y., Volland, C., Haguenauer-Tsapis, R. and Andre, B. (1995). NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol. Microbiol. 24,607 -616.
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]
Hicke, L. (2001). Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2, 195-201.[CrossRef][Medline]
Hirsch, J. G., Fedorko, M. E. and Cohn, Z. A.
(1968). Vesicle fusion and formation at the surface of pinocytic
vacuoles in macrophages. J. Cell Biol.
38,629
-632.
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-I. Cell 106,145 -155.[Medline]
Katzmann, D. J., Odorizzi, G. and Emr, S. D. (2002). Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell Biol. 3, 893-905.[CrossRef][Medline]
Lee, K. S. and Levin, D. E. (1992). Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. Mol. Cell Biol. 12,172 -182.[Abstract]
Lemmon, S. K. and Traub, L. M. (2000). Sorting in the endosomal system in yeast and animal cells. Curr. Opin. Cell Biol. 12,457 -466.[CrossRef][Medline]
Li, Y., Kane, T., Tipper, C., Spatrick, P. and Jenness, D.
D. (1999). Yeast mutants affecting possible quality control
of plasma membrane proteins. Mol. Cell Biol.
19,3588
-3599.
Longtine, M. S., McKenzie, A., III, 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]
Losko, S., Kopp, F., Kranz, A. and Kolling, R.
(2001). Uptake of the ATP-binding cassette (ABC) transporter Ste6
into the yeast vacuole is blocked in the doa4 mutant. Mol. Biol.
Cell 12,1047
-1059.
Marcusson, E. G., Horazdovsky, B. F., Cereghino, J. L., Gharakhanian, E. and Emr, S. D. (1994). The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell 77,579 -586.[Medline]
McKanna, J. A., Haigler, H. T. and Cohen, S. (1979). Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor. Proc. Natl. Acad. Sci. USA 76,5689 -5693.[Abstract]
Missotten, M., Nichols, A., Rieger, K. and Sadoul, R. (1999). Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein. Cell Death Differ. 6,124 -129.[CrossRef][Medline]
Nickas, M. E. and Yaffe, M. P. (1996). BRO1, a novel gene that interacts with components of the Pkc1p-mitogen-activated protein kinase pathway in Saccharomyces cerevisiae. Mol. Cell Biol. 16,2585 -2593.[Abstract]
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]
Ogura, T. and Wilkinson, A. J. (2001). AAA+
superfamily ATPases: common structure-diverse function. Genes
Cells 6,575
-597.
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., Bryant, N. J. and Stevens, T. H.
(1997). The membrane protein alkaline phosphatase is delivered to
the vacuole by a route that is distinct from the VPS-dependent pathway.
J. Cell Biol. 138,531
-545.
Raymond, C. K., Howald-Stevendon, 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. (2002). A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies. Nat. Cell Biol. 4, 117-123.[CrossRef][Medline]
Rieder, S. E., Banta, L. M., Köhrer, 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]
Robinson, J. S., Klionsky, D. J., Banta, L. M. and Emr, S. D. (1988). Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell Biol. 8,4936 -4948.[Medline]
Shih, S. C., Katzmann, D. J., Schnell, J. D., Sutanto, M., Emr, S. D. and Hicke, L. (2002). Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nat. Cell Biol. 4,389 -393.[CrossRef][Medline]
Sikorski, R. S. and Hieter, P. (1989). A system
of shuttle vectors and yeast host strains designed for efficient manipulation
of DNA in Saccharomyces cerevisiae. Genetics
122, 19-27.
Sotelo, J. R. and Porter, K. R. (1959). An
electron microscope study of the rat ovum. J. Biophys. Biochem.
Cytol. 5,327
-342.
Spormann, D. O., Heim, J. and Wolf, D. H.
(1992). Biogenesis of the yeast vacuole (lysosome) the
precursor forms of the soluble hydrolase carboxypeptidase yscS are associated
with the vacuolar membrane. J. Biol. Chem.
267,8021
-8029.
Springael, J. Y., Nikko, E., Andre, B. and Marini, A. M. (2002). Yeast Npi3/Bro1 is involved in ubiquitin-dependent control of permease trafficking. FEBS Lett. 517,103 -109.[CrossRef][Medline]
Stevens, T., Esmon, B. and Schekman, R. (1982). Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell 30,439 -448.[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]
Vito, P., Pellegrini, L., Guiet, C. and D'Adamio, L.
(1999). Cloning of AIP1, a novel protein that associates with the
apoptosis-linked gene ALG-2 in a Ca2+-dependent reaction. J. Biol.
Chem. 274,1533
-1540.
Weissman, A. M. (2001). Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol. 2, 169-178.[CrossRef][Medline]
Xu, W. and Mitchell, A. P. (2001). Yeast
PalA/AIP1/Alix homolog Rim20p associates with a PEST-like region and is
required for its proteolytic cleavage. J. Bacteriol.
183,6917
-6923.
Yon, J. and Fried, M. (1989). Precise gene fusion by PCR. Nucleic Acids Res. 17, 24895.