From the Department of Molecular Medicine, and
§ Microbiology and Immunology, Cornell University,
Ithaca, New York 14830
Received for publication, September 14, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Rab proteins are small GTPases that are essential
elements of the protein transport machinery of eukaryotic cells. Each
round of membrane transport requires a cycle of Rab protein nucleotide binding and hydrolysis. We have recently characterized a protein, Yip1p, which appears to play a role in Rab-mediated membrane transport in Saccharomyces cerevisiae. In this study, we report the
identification of a Yip1p-associated protein, Yop1p. Yop1p is a
membrane protein with a hydrophilic region at its N terminus through
which it interacts specifically with the cytosolic domain of Yip1p.
Yop1p could also be coprecipitated with Rab proteins from total
cellular lysates. The TB2 gene is the human homolog
of Yop1p (Kinzler, K. W., Nilbert, M. C., Su, L.-K.,
Vogelstein, B., Bryan, T. M., Levey, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R.,
Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H. M., Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) Science 253, 661-665). Our data
demonstrate that Yop1p negatively regulates cell growth. Disruption of
YOP1 has no apparent effect on cell viability, while
overexpression results in cell death, accumulation of internal cell
membranes, and a block in membrane traffic. These results suggest that
Yop1p acts in conjunction with Yip1p to mediate a common step in
membrane traffic.
The Rab family encompasses a conserved group of key molecules
involved in membrane traffic and represents a distinct subgroup of the
Ras superfamily (2). Each stage of membrane traffic through both the
constitutive and regulated secretory pathways of all eukaryotic cells
is associated with a distinct Rab protein that regulates the cascade of
events that lead to SNARE-mediated membrane fusion (3). A hallmark of
Rabs is their localization to specific compartments of the transport
pathway. This distribution is consistent with the function of Rab
proteins in distinct intracellular transport processes. In every case
examined, the localization pattern of a Rab protein reflects the
membrane transport step that it regulates. In keeping with this view,
more than 30 members of the Rab family have been identified (2).
Rabs are stably prenylated at their C terminus, which mediates their
association with membranes (4). However, while the majority of Rabs are
membrane-associated, prenylated Rabs are also found in the cytosol
bound to the Rab GDP dissociation inhibitor (GDI).1 GDI shares sequence
homology with the Rab escort protein involved in presenting and
removing Rab proteins from the prenylation machinery (5, 6). GDI has
several properties that underscore its role in mediating Rab protein
function: (i) GDI binds preferentially to the GDP-bound conformation of
Rab proteins and slows the intrinsic rate of GDP nucleotide
dissociation (7), (ii) GDI requires the fully prenylated Rab protein
for interaction and binds in such a way so that the geranylgeranyl
groups are shielded in a hydrophobic pocket (8), (iii) GDI is a
pleiotropic factor interacting with many different Rab proteins
in vitro and in vivo; in Saccharomyces cerevisiae, a single gene encodes GDI function for all 11 Rab proteins (9). These properties enable the Rab protein to exist in the
aqueous environment of the cytoplasm as a soluble heterodimer with GDI
and facilitate recycling of the GDP-bound Rab back to the donor
compartment (10). Consistent with this model, Rab proteins are
complexed to GDI in the cytosol, and depletion of GDI in yeast causes
loss of the soluble pool of Rabs and a concomitant inhibition of
transport in the secretory pathway.
The specificity of Rab protein function, localization, and their
presence on the surface of vesicles suggests the existence of a
machinery that recruits Rab proteins to the proper target membrane.
However, identification of such a machinery has proven elusive. To
date, no factor mediating this process has been identified; however,
several features of Rab membrane recruitment have been established: (i)
Rabs are recruited to membranes in their inactive GDP-bound
conformation bound to GDI (11); (ii) membrane recruitment is
accompanied by the displacement of GDI (12); (iii) membrane recruitment
is specific, and the C-terminal hypervariable region of the Rab protein
mediates this specificity (13); (iv) prenylation of Rab proteins
is crucial for membrane recruitment in addition to the C-terminal ~35
amino acid residues; (v) membrane recruitment is followed by nucleotide
exchange, and the two processes can be distinguished kinetically (14,
15); and (vi) for Rab4, the existence of a membrane protein that acts
as a specific Rab receptor has been demonstrated, although the precise
identity of this receptor is unknown (16).
We have characterized a membrane protein in yeast, Yip1p, which appears
to mediate the dissociation of the Rab heterodimer from GDI.
YIP1 is an essential gene (17) that is highly conserved in
evolution.2 However, Yip1p is
a pleiotropic factor and lacks specificity for interaction with any
particular Rab GTPase (17). We have therefore searched for a protein
accessory factor that may act in conjunction with Yip1p, and we report
the identification of a novel membrane protein, Yop1p, which physically
interacts with Yip1p. Disruption of YOP1 has no apparent
effect on cell viability, while overexpression results in cell death
and accumulation of internal cell membranes. These results suggest that
Yop1p acts in conjunction with Yip1p to mediate a common step in
membrane traffic. Because of the essential nature of Rab recruitment
for the activation and recycling of Rabs, characterization of Yop1p may
provide crucial insight into the action of Rab proteins in mediating
membrane transport.
Yeast Strains and Media--
The S. cerevisiae
strains used in these studies are listed in Table
I. All yeast strains were manipulated as
described by Guthrie and Fink (18). YOP1 gene deletion was
carried out using the KANR module (19) as a
selectable marker and the primers
CAAAGACATAACCGCACTCCAATCATGTCCGAATATGCATCTAGTATTCACTCTCCGTACGCTGCAGGTCGAC and
GAGGATATAGGTGAGTTGCCTCTTAATGAACAGAAGCACCTGTAGCCTTAGAAGCCTATCGATGAATTCGAGCTCG to precisely eliminate the YOP1 ORF. Genomic PCR using
an internal deletion primer and the flanking primer
CTTGAAGCTTGTTATTCCGA was performed to verify gene disruption. Yeast
expressing GST-Yop1p under the control of the GAL1/10
promoter (RCY423) and GST alone (RCY427) were created by digesting
pRC494 and pRC337, respectively, with ClaI to direct
integration at the LEU2 locus of NY605. Strains RCY425,
RCY428, and RCY462 were created by transforming pRC695 into RCY423,
RCY427, and NY605, respectively. In the same manner, RCY429 was created
by transforming pNB632 into RCY423. RCY460 was created by transforming
the hemagglutinin (HA)-tagged Yop1p protein expression vector pRC778
into RCY407. For immunofluorescence, RCY469 was created by
digesting pRC833 with EcoRV to direct integration of the
plasmid at the URA3 locus of RCY407.
Yeast strains were streaked out on a selective plate and
incubated at 30 °C. Liquid media cultures were grown at room
temperature. A single colony from each strain was inoculated into 5 ml
of selective medium and grown to stationary phase. The day prior to the
experiment, medium was inoculated with aliquots of stationary culture
at room temperature to obtain cells in logarithmic phase growth.
Turbidity measurements were made using a Beckman model DU-40
spectrophotometer at 600 nm.
Plasmids and DNA Constructs--
The genomic YOP1 ORF
contains a single intron. For convenience, this intron was removed for
the majority of YOP1 constructs by overlap PCR with the
primers RNC66
(GGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGGAAGAGTTGCATAGATAGGATGGGTGA) and RNC78 (CGATACCAAGTACTCTGGTAATAGAATTTTACAGC) together with RNC67
(CTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATGCTCAAAAGCTAACACTAGGCCAG) and
RNC79 (TATCCATGGGTAAGTACTCTGGTAATAGAATTTTACAGC). Full-length YOP1 fusion constructs were constructed by PCR with
oligonucleotides RNC44 (TGGTACCTCATGAGCGAATATGCATCTAGTATTCACTCTC) and
RNC80 (AATAGGATCCTTAATGAACAGAAGCACCTGTAG). The
NcoI/BamHI-digested PCR product was subcloned
into pAS2-1 and pACT2 to create two-hybrid vectors expressing
full-length YOP1, p121 to create HA-tagged YOP1
under the control of the GAL1/10 promoter (pRC393) and
pRC337 to create GST-tagged YOP1 under the control of the
GAL1/10 promoter (pRC494). C-terminal YOP1
constructs containing amino acids 18-180 were created in a similar
manner with the oligonucleotides RNC79
(TATCCATGGGTAAGTACTCTGGTAATAGAATTTTACAGC) and RNC80. The PCR product
was subcloned into p121 to create pRC439 expressing HA-tagged
YOP1 C terminus under the control of the GAL1/10
promoter. pRC581 containing yEGFP-tagged YOP1 under the control of its own gene regulatory elements in pRS406 was created by
overlap PCR with the oligonucleotides RNC66, RNC67, RNC179 (CAAAGACATAACCGCACTCCAATCATGTCTAAAGGTGAAGAATTATTC), RNC180
(AGAGTGAATACTAGATGCATATTCGGATTTGTACAATTCATCCATACC), RNC181
(CATGATTGGAGTGCGGTTATG), and RNC182
(TCCGAATATGCATCTAGTATTCACTCTCAAATGAAAC). pRC695 expressing
Myc9-YIP1 in pRS426 (20) was created by
overlap PCR placing a cassette containing Myc9 (gift of Y. Barral, ETH, Zurich) in frame behind the start codon of YIP1
with the primers 9× oligo 1 YIP1
(GCAAGACAACTATTAGTCCCTCTCGAGATGCTCCACCGCGGTGGC) and 9× oligo 2 YIP1 (TGTTACTAGTATTGTAGAAAGACATAATTCCTGCAGCCCGGGGGAT). pNB632, a
URA3 multicopy plasmid containing
DSS4-Myc3, has been described previously (21).
pRC337 was created by subcloning GST in front of the
GAL1/10 promoter of vector pNB527 digested with
BamHI/XhoI using primers RNC177
(CTAGACTAGATCTTCATGAGTTCCCCTATACTAGGTTATTGGAAAATTAAG) and RNC178
(GACTGACCTCGAGTAGGATCCAGTCACCATGGTCAGATCCGATTTTGGAGGATG) and
digesting the PCR product with BglII/XhoI. pRC778
containing a single HA epitope at the N terminus of Yop1p expressed at
wild-type levels in the vector pRS315 was created by PCR overlap with
the oligonucleotides RNC157
(TACGACGTCCCAGACTACGCTTCCGAATATGCATCTAGTATTCAC) and HA 1 (AGCGTAGTCTGGGACGTCGTATGGGTACATCTCGAGAGGGACTAATAGTTGTC). The
insert of pRC778 was removed with SalI/HindIII
and ligated into the vector pRS306 digested with
XhoI/HindIII to create pRC833, a
URA3-integrating vector expressing wild-type levels of
HA-tagged Yop1p. pRC693 containing green fluorescent protein
(GFP)-tagged Yip1p under the control of its own promoter and terminator
in pRS315 was constructed by placing a cassette containing
yeast-enhanced GFP mut3 (22) in frame behind the start codon of
YIP1 with the primers GFP oligo 1 (GACAACTATTAGTCCCTCTCGAGATGTCTAAAGGTGAAGAATTATTCAC) and GFP
oligo 2 (GTTACTAGTATTGTAGAAAGACATTTTGTACAATTCATCCATACCAT). pRC650
and pRC556 containing GFP-tagged Ypt6p and Sec4p, respectively, in pRS315 were created in a similar fashion. pRC903 was created by
subcloning a cassette containing GST-tagged YOP1 under the control of the GAL1/10 promoter from pRC494 into the 2µ
URA3 vector pRS426. pRC940 containing YIP1 was
constructed by genomic PCR with the oligonucleotides YF YIP1
(GTACCGGGCCCCCCCTCGAGGTCGACGTAGTGCTTGTTACGTTAG) and YR
YIP1 (CCACCGCGGTGGCGGCCGCTCTAGAACTCTATGCTTTCCTTATTTACCTCTGGA) and
inserted into pRS426 to create a multicopy URA3 vector.
Electrophoresis and Western Blotting--
For electrophoresis,
samples were boiled for 5 min in gel loading buffer (60 mM
Tris, pH 6.8, 10% sucrose, 2% SDS, 5% Coprecipitation Assays--
Yeast strains were grown in minimal
medium containing 2% galactose. 10 OD units from each culture
were harvested and washed in 1 ml of ice-cold TAZ buffer (10 mM Tris, pH 7.5, 10 mM NaN3). Cell
pellets were then resuspended in 100 µl of ice-cold lysis buffer (20 mM KPi, 80 mM KCl, 1 mM EDTA, 2%
glycerol, 0.1% Tween 20) containing protease inhibitors (10 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A),
and an equal volume of glass beads was added. The cells were then lysed
by vortexing for 2 min in a Turbo-Beater (Fisher) at 4 °C. A total
detergent-solubilized lysate was generated by incubating lysates
end-over-end with an additional 1 ml of lysis buffer for 10 min at
4 °C. Detergent-solubilized lysates were cleared by two sequential
centrifugation steps in a microcentrifuge for 5 min at 13,000 rpm. 20 µl of glutathione S-transferase 4B beads (GST-beads;
Amersham Pharmacia Biotech) was added to the lysates and incubated with
constant mixing for 30 min at 4 °C. After four washes with 0.6 ml of
lysis buffer, the GST beads were boiled with SDS-PAGE sample buffer,
and the samples were analyzed by SDS-PAGE and Western blot. Pull-down experiments from yeast strains RCY509 and RCY508 used the lysis buffer
25 mM KPi, pH 7.5, 160 mM KCl, 2 mM
EDTA, 2% glycerol, and 0.4% Triton X-100. Pull-down experiments fom
yeast strains RCY455, RCY456, RCY457, RCY465, RCY467, and RCY464 used
the lysis buffer, 25 mM KPi, pH 7.5, 160 mM
KCl, 2 mM EGTA, 2% glycerol, and 0.5% Tween 20. Primary
antibodies used were rabbit polyclonal Subcellular Fractionation--
Yeast strain RCY460 containing
wild-type levels of HA-tagged Yop1p as the only source of
YOP1 was used for this experiment. 25 OD units were
harvested and washed in 1 ml of TAZ buffer. Cells were broken by glass
bead lysis in a Turbo-Beater at 4 °C in fractionation buffer with
protease inhibitors (PBS containing 0.2 M sorbitol and 1 mM EDTA, 10 mM phenylmethylsulfonyl fluoride,
10 µg/ml pepstatin). A postnuclear supernatant (PNS) was generated by
two sequential centrifugation steps for 5 min at 500 × g. 2.7 mg of PNS was then spun sequentially at 10,000 × g for 15 min and at 100,000 × g for 12 min to generate P10 and P100 fractions. For Triton X-100 solubilization, the P100 membrane pellet was resuspended in
fractionation buffer containing 1% Triton X-100. Samples were
incubated for 10 min on ice and recentrifuged at 100,000 × g. For high salt treatment, the P100 membrane pellet was
resuspended in fractionation buffer containing 1 M NaCl.
Samples were incubated for 10 min on ice and recentrifuged at
100,000 × g. Pellets and supernatants were resuspended
in sample buffer and analyzed by SDS-PAGE and Western blot. The
HA-Yop1p was detected with mouse monoclonal 12CA5 antibody followed by
anti-mouse alkaline phosphatase-conjugated secondary antibody.
Triton X-114 Phase Separation--
Triton X-114 (Roche Molecular
Biochemicals) was purified by precondensation as described (25). 25 OD
units of yeast strain RCY460 were harvested and washed in 1 ml of TAZ
buffer. Postnuclear supernatants were generated as described above. 1.8 mg of PNS was added to the same volume of PBS containing 2% Triton
X-114 with protease inhibitors (1 mM EDTA, 10 mM phenylmethylsulfonyl fluoride, and 10 µg/ml pepstatin
A). The samples were incubated for 20 min at 4 °C to solubilize
membrane proteins. The lysates were incubated for 3 min at 30 °C
followed by low speed centrifugation (700 × g) to
separate the detergent-enriched and the soluble phases. This cycle was
repeated a further two times with the detergent-enriched and soluble
phases individually. The detergent phase was washed twice with
PBS containing 0.05% Triton X-114 and the soluble phase with 10%
Triton X-114. Samples were analyzed by SDS-PAGE and Western blot.
Snc1/2p, an integral membrane protein, was used as a positive control and was detected with anti-Snc1/2p antisera (gift of P. Brennwald, Cornell University).
Carboxypeptidase Y Analysis--
Yeast strains RCY376 and
RCY377 containing HA-YOP1 full-length and C-terminal constructs
(respectively) behind the galactose promoter, were grown in sucrose
minimal medium to early log phase, before washing and
resuspending in galactose minimal medium. At the indicated
intervals, aliquots of 5 OD units were harvested for production of
lysates. For the sec18 experiments, cells were grown at room
temperature until log phase before shifting an aliquot to the
restrictive temperature (37 °C) for 1 h. Lysates were then boiled with SDS-PAGE sample buffer for 5 min and analyzed by SDS-PAGE and Western blot. The membrane was probed with polyclonal
anti-carboxypeptidase Y (CPY) (gift from P. Brennwald).
Immunofluorescence Experiments--
Yeast strains RCY469
containing HA-Yop1p and RCY407 (isogenic untagged control) were grown
to early log phase in YPD medium. 2× fixative (2× PBS, 4%
glucose, 40 mM EGTA, 7.4% formaldehyde) was added to an
equal volume of medium containing 3 OD units of cells and
incubated for 20 min at room temperature. Cells were then collected by
centrifugation, resuspended in 5 ml of 1× fixative, and incubated for
a further 1 h. The cells were washed twice in 2 ml of
spheroplasting buffer (100 mM KPi, pH 7.5, 1.2 M sorbitol) and then incubated in spheroplasting buffer
containing 0.2% 2-mercaptoethanol and 0.08 mg/ml of zymolyase for 30 min at 37 °C with gentle mixing. 20 µl of the cell suspension was
placed on individual wells of a polylysine-coated printed microscope
slides (Carlson Scientific, Inc.) for 10 min. The cells were then
washed three times with PBS/BSA (1 mg/ml BSA) and permeabilized for 5 min with either 0.1% SDS or 0.1% Triton X-100 in PBS/BSA. After
washing five times in PBS/BSA, cells were blocked for 30 min in
PBS/BSA. Polyclonal Electron Microscopy--
The cells were incubated for 14 h
in medium containing galactose as sole carbon source at a final
cell density (A600) of between 0.4 and 0.7. Cells were washed with 0.1 M cacodylate, pH 6.8, and then
fixed with 0.1 M cacodylate, pH 6.8, containing 3%
glutaraldehyde for 1 h at room temperature and then overnight
at 4 °C. The cell walls were removed by treatment with 0.1 M KPi buffer, pH 7.5, containing 0.2 mg/ml
zymolyase 100T. The cell pellet was incubated with 1.5 ml of cold 2%
OsO4 in 0.1 M cacodylate buffer for 1 h on
ice followed by incubation with 1.5 ml of filtered 2% uranyl acetate
(aqueous) at room temperature for 1 h. The cell pellets were
dehydrated with the following ethanol washes: 50, 70, 90, and 100%
followed by four washes from a fresh bottle of 200 proof ethanol and a
final rinse in 100% acetone. The pellet was then incubated with 50%
acetone, 50% SPURR resin (Electron Microscopy Sciences); this was
changed to 100% SPURR resin, and the sample was transferred to
beem capsules (Electron Microscopy Sciences) and baked at
80 °C for at least 24 h. Thin sections were cut onto Specimen
Grids (Veco) (3-mm diameter, 75 × 300 mesh copper),
contrasted with lead citrate and uranyl acetate, and then
examined in an FEI Philips TECHNAI 12 BioTwin electron microscope
at 100 or 80 kV.
Two-hybrid Experiments--
The ORF sequences were subcloned
into pAS1-CYH2 or pAS2-1 for "bait" and pACTII for "fish"
constructs, respectively. The yeast strain Y190 was used for to screen
the library for N-terminal Yip1p-interacting clones (27). The yeast
reporter strain Y190, which contains the reporter genes
lacZ and HIS3 downstream of the binding
sequences for Gal4, was sequentially transformed with the pACT2 and
pAS2-1 (CLONTECH) plasmids containing the genes of
interest. Double transformants were plated on selective medium (lacking tryptophan and leucine) and incubated for 2-3 days at 30 °C. Trp+ Leu+ colonies processed for the The Cytosolic Domain of Yip1p Interacts with a Novel Membrane
Protein--
To explore the role of Yip1p in membrane traffic, we
considered the possibility that it may exist in physical association with other proteins. Such a protein may perhaps act to provide a
specificity component to the Rab membrane recruitment reaction. To
identify such potential proteins, we performed a two-hybrid screen
using the cytosolic domain of Yip1p as bait. For this interaction screen, we used two-hybrid libraries constructed from short fragments (0.5-1 kilobase pair) of yeast genomic DNA (28). Since the yeast genome is relatively compact with few intron-containing genes, such a
library represents a collection of random protein fragments. The
rationale for such a strategy was that a Yip1p-interacting protein may
be a membrane protein interacting with Yip1p through exposed soluble
loops. Interactions may not be revealed by expressing full-length
cDNAs, but protein fragments of the isolated loops alone may
demonstrate interaction in the two-hybrid system. Analogous strategies
have been used successfully to explore interactions of multispanning
membrane proteins using the two-hybrid system (29). Using this screen,
we identified a previously uncharacterized membrane protein derived
from ORF YPR028W. The interacting clone identified contained
17 amino acids derived from the extreme N terminus of the protein fused
in frame with the GAL4 DNA activation domain. We have termed this gene
YOP1 (YIP one partner).
The interaction between the Yop1p fragment and Yip1p was recapitulated
with a full-length Yip1p construct in the two-hybrid system. The
interaction was also maintained whether or not the Yop1p fragment was a
GAL4 DNA binding domain plasmid or a GAL4 DNA activation domain fusion; i.e. if the "bait" construct is swapped with the
"fish" construct, the vast majority of false two-hybrid positives
will not interact in such a test. However, Yop1p full-length constructs
show no interactions with Yip1p in the two-hybrid system. These data
are summarized in Table II.
The primary sequence of Yop1p is predicted to have at least two
membrane-spanning domains (Fig. 1,
A and B). A BLAST search of GenBankTM
revealed that Yop1p is homologous to the human TB2 protein in addition
to several other proteins present in data bases (Fig. 1C).
Yop1p and human TB2 share 25.65% identity at the amino acid level, and
there is 22.4% identity between Yop1p and murine TB2. It is notable
that the overall structure of the mammalian and yeast protein is
conserved. Both proteins contain extensive hydrophobic domains with the
N terminus predicted to be exposed to the cytoplasmic face of the
membrane. No other Yop1p homologs could be identified in S. cerevisiae.
Yop1p Is an Integral Membrane Protein--
Sequence information
predicts Yop1p to be a 20-kDa protein with two membrane-spanning
segments that is likely oriented with its N terminus toward the
cytoplasm (Fig. 1). We examined whether Yop1p has the expected
properties of an integral membrane protein. First, Yop1p fractionated
exclusively in the pellet of a total postnuclear supernatant
centrifuged at 100,000 × g, indicating that it is
either membrane-associated or present in a large pelletable aggregate
(Fig. 2A). Second, we tested
whether Yop1p was a peripheral membrane protein and could be removed by
washing membranes in high salt-containing buffers. Yop1p could not be
extracted from membranes by incubation in buffer containing 1 M NaCl; however, Yop1p was quantifiably extracted in
Triton X-100 detergent-containing buffers (Fig. 2B).
Third, we performed Triton X-114 phase extraction experiments to
determine whether Yop1p has the physiochemical properties of an
integral hydrophobic membrane protein. In this technique, total
cellular proteins are first detergent solubilized at 0 °C. The
mixture is then warmed to 30 °C, exploiting the cloud point of
Triton X-114 to create two phases that can be separated by gentle
centrifugation: a detergent-rich phase containing membrane proteins and
an aqueous phase containing hydrophilic proteins. Yop1p partitioned
exclusively into the detergent-rich phase (Fig. 2C),
indicating that it contains hydrophobic domains that anchor it in the
lipid bilayer. As a control, fractions were also probed for a known
integral membrane protein, Snc1/2p (30), which partitioned into the
detergent phase as expected. Taken together, these data show that Yop1p
is an integral membrane protein.
Physical Association of Yip1p and Yop1p--
To confirm the
two-hybrid data, we performed biochemical studies of the Yip1p/Yop1p
interaction. For this purpose, we created the strain RCY425, which
expresses GST-Yop1p fusion protein under the control of the regulatable
GAL1/10 promoter and contains a multicopy plasmid
expressing Myc9-Yip1p. We also created an isogenic control strain, RCY428, which expresses GST alone together with Myc9-Yip1p. Tween 20 detergent-solubilized total lysates
were produced from mid-log phase cells grown in galactose and GST
fusion proteins were isolated on gluthathione-agarose beads followed by
SDS-PAGE and Western blotting to detect any associated Myc-tagged proteins. The results of this experiment are shown in Fig.
3A. Myc9-Yip1p was
detected in the GST-Yop1p pull-down but was not detected in the
pull-down of GST alone, showing that Myc9-Yip1p exists in
physical association with Yop1p. To rule out any possibility of
GST-Yop1p interacting with the Myc epitopes of Myc9-Yip1p, we repeated the experiment with RCY429, which expresses GST-Yop1p together with Dss4p-Myc3. In this experiment, the Western
blot was first probed with anti-Myc antibody and then reprobed with anti-GST antibody. The results are shown in Fig. 3B.
Dss4p-Myc3, Myc9-Yip1p, and GST-Yop1p are
expressed at equivalent levels in the detergent-solubilized lysates.
The GST pull-downs reveal that Myc9-Yip1p associated with
GST-Yop1p but Dss4p-Myc3 did not associate and could not be
detected in the pull-down, demonstrating that the biochemical
association of Yip1p and Yop1p is specific.
We also repeated the experiment with Yip1p expressed at wild-type
levels on a single-copy centromeric plasmid under the control of its
own promoter and terminator. For these experiments, Yip1p was tagged
with GFP, and lysates were produced with Triton X-100 detergent
solubilization. Western blot analysis of the glutathione resin
pull-downs (Fig. 3C) showed that GFP-Yip1p (RCY509) was specifically isolated with GST-Yop1p, while a control protein, GFP-Ypt7p (RCY508), was not. Western blots of the detergent-solubilized lysates confirmed that GST-Yop1p and the GFP fusion proteins were expressed at equivalent levels in both cases.
To further investigate the relationship between Yip1p and Yop1p, we
asked whether the interaction in our pull-down experiments occurred
in vivo prior to cell lysis or postlysis in
vitro. For these experiments, we performed the glutathione resin
pull-downs on lysates derived from cells coexpressing GST-Yop1p and
Myc9-Yip1p (RCY425) or by combining lysates from individual
strains RCY429 (containing GST-Yop1p and Dss4p-Myc3) or
RCY462, which contains Myc9-Yip1p only. These results can
be seen in Fig. 3D. We were only able to detect the
interaction of Yip1p and Yop1p from cells expressing both proteins
simultaneously. These results indicate that Yip1p and Yop1p interact
in vivo, in a complex that is formed prior to cell lysis.
Overexpression of YOP1 Is Dominant Negative and Can Be Suppressed
by Co-overexpression of YIP1--
We deleted the entire
YOP1 ORF in a diploid cell that was sporulated and dissected
into tetrads to study the phenotype of the haploid-disrupted strain.
The YOP1
The dominant negative phenotype of full-length Yop1p overexpression was
suppressed by co-overexpression of Yip1p from a multicopy plasmid (Fig.
5). However, multicopy YIP1
had no effect on the dominant negative phenotype of the Yop1p
C-terminal construct, which lacked the Yip1p interaction region. These
data further demonstrate that the interaction of Yip1p and Yop1p is a
bona fide physiological interaction and support the
identification of the Yop1p N terminus as the site of Yip1p
interaction.
Dominant Negative YOP1 Results in Alteration of Membrane Structures
and a Block in Membrane Traffic--
To investigate morphological
alterations in dominant negative YOP1 cells in detail, we
performed electron microscopy. Cells containing the full-length
dominant negative YOP1 construct and isogenic wild-type
cells were grown in galactose before being fixed with and processed for
electron microscopy. These results are shown in Fig.
6. Expression of the full-length
YOP1 dominant negative construct resulted in the
disappearance of large vacuoles normally seen in wild-type cells and
the appearance of smaller and aberrantly shaped compartments filled
with darkly stained material (Fig. 6, A-C). These cells
also contained numerous discontinuous ring-shaped structures; some
membrane structures resembled the cup-shaped "Berkeley bodies"
known to represent abnormal Golgi structures, while others had
pleiomorphic, clublike shapes. In some cells, an accumulation of ER
membranes, as judged by their connection to the nuclear envelope, was
also observed. Such aberrant membrane structures are not observed in
wild-type cells (Fig. 6D) and represent a gross distortion
of the normal pathways of membrane traffic in the YOP1
dominant negative cells.
To investigate the effect of YOP1 overexpression on membrane
traffic, we monitored the steady state level of newly synthesized precursors of the vacuolar protease CPY, the product of the
PRC1 gene. CPY is a soluble vacuole protein that undergoes
processing from a core-glycosylated ER form (p1, 67 kDa) to a modified
Golgi form (p2, 69 kDa) before being proteolytically cleaved in the vacuole to mature CPY (61 kDa). Using an anti-CPY antibody, we analyzed
total cell lysates for the relative levels of the precursor and mature
CPY forms under wild-type and YOP1 dominant negative conditions. As a
control, we used sec18 cells shifted to the restrictive temperature at which all stages of membrane traffic are blocked, resulting in the accumulation of the core-glycosylated p1 form of CPY
(shown by an asterisk). The results are shown in Fig.
7A. Cells overexpressing
full-length Yop1p show an accumulation of p1 CPY relative to isogenic
wild-type cells (RCY458), which is indicative of a block early in
exocytosis at the level of the ER. The observed accumulation is the
specific result of a block in membrane traffic and does not reflect a
generalized disruption of cellular function as the block can be
observed within the first 7 h of galactose induction, cell growth
rates are not affected until ~16.5 h after galactose induction (Fig.
7B). Dominant negative cells overexpressing Yop1p C terminus
(RCY377) do not show the same effect, and no accumulation of CPY is
observed.
Localization of YOP1--
We examined the localization of Yop1p by
subcellular fractionation and immunofluorescence. For this purpose, we
constructed the strain RCY460, which contains wild-type levels of
HA-tagged Yop1p as the sole cellular source of Yop1p. Separation of
postnuclear supernatants into P10 (after 10,000 × g
centrifugation) and P100 and S100 (after 100,000 × g
centrifugation) followed by immunoblotting with monoclonal anti-HA
antibody (12CA5) is shown in Fig.
8A. HA-Yop1p fractionates with
both light and heavy membranes (P10, P100) but not with cytosol
(S100).
By immunofluorescence microscopy, HA-Yop1p appears as a punctate
pattern that appears to be at, or near, the periphery of the cell,
roughly proportionally distributed between the mother and bud with a
greater concentration in the more actively growing region of the cell
(Fig. 8B). To identify the cellular location of Yop1p, we
performed double label immunofluorescence with Sec4p and with
GFP-Ypt6p. HA-Yop1p does not localize to the bud tip or at the neck
during cytokinesis and can be clearly distinguished from Sec4p
immunofluorescence, which is solely concentrated at the leading edge of
the cell (Fig. 9A). The Yop1p
signal partially overlapped with the Ypt6p fluorescence, especially
toward the leading edge of the cell, indicating the presence of Yop1p
on Golgi membranes (Fig. 9B). The HA-Yop1p pattern of
expression is identical whether or not the construct is integrated into
the genome or maintained as a centromeric plasmid; the expression pattern is also identical in diploids and haploids and on cells grown
in glucose, galactose, or glycerol carbon sources (data not
shown).
To further examine the subcellular localization of Yop1p and its
interactions with Yip1p, we performed confocal microscopy. Cells
coexpressing HA-Yop1p and GFP-Yip1p are shown in Fig. 9C. Substantial overlap of the Yop1p and Yip1p signal was observed toward
the growing edge of the cell, confirming our biochemical data
indicating that a physical interaction between Yop1p and Yip1p occurs
in vivo.
Interaction of Yop1p with Rab Proteins--
Since Yip1p is
required for secretory pathway function, presumably through its effects
on Rab proteins, we sought to investigate any possible interaction of
Yop1p with Rab proteins. There are 11 Rab protein family members in
S. cerevisiae; however, some of these represent closely
related isoforms (e.g. Vps21p, Ypt52p, and Ypt53p).
To gain as complete an insight as possible, we made GST fusions with
several Rab proteins encompassing representatives from each subset.
These proteins were expressed in cells behind the galactose promoter
and were tested for interaction by coprecipitation with Yop1p, which
was tagged with a single HA epitope and also expressed behind the
galactose promoter. The results of this analysis are shown in Fig.
10A. HA-Yop1p did not
coprecipitate with GST alone but was able to precipitate with GST fused
to the Rab proteins Ypt52p, Sec4p, Ypt6p, and Ypt7p. The observed
interaction was stable in buffers containing 0.5% Tween 20; however,
it could not be observed in 0.5% Triton X-100 containing buffers (data not shown). No interaction was observed with a Ypt1p construct lacking
its C-terminal cysteines, which are the sites of prenylation. The
interactions between Yop1p and Rab proteins are unlikely to be real
in vivo interactions; otherwise, the steady-state
localization of Yop1p would probably be more universally distributed
among subcellular membranes. It is more likely that the observed
interactions reflect a generalized biochemical ability of Yop1p to
interact with a common determinant of fully post-translationally
modified Rab proteins, an interaction that can be revealed by
overexpressing both proteins and performing coprecipitation assays as
shown in Fig. 10A. To reveal which Rab protein may be
important for Yop1p action in vivo, we performed suppression
analysis of the YOP1 dominant negative constructs with
multicopy plasmids encoding all 11 Rab proteins of S. cerevisiae. The full-length YOP1 dominant negative
construct, while able to be suppressed by multicopy YIP1 (Fig. 5), could not be suppressed by overexpression of any of the genes
encoding the yeast Rab proteins (data not shown). However, multicopy
YPT6 was able to suppress the dominant negative phenotype of
the YOP1 C-terminal construct. The suppression of the
YOP1 C-terminal construct (RCY377) by 2µ YPT6
together with YPT7 and DSS4 as a comparison is
shown in Fig. 10B. YPT6 was the only Rab gene
capable of causing in vivo suppression of RCY377; no other Rab gene tested (SEC4, YPT1, YPT31,
YPT32, VPS21, YPT52, YPT53, YPT10, YPT11, and YPT7; data not shown
except for YPT7, Fig. 10B), was able to restore
growth.
We have isolated YOP1 as a novel
YIP1-interacting clone in a yeast two-hybrid screen of a
yeast genomic library. Yop1p and Yip1p are both integral membrane
proteins. The interaction of a membrane protein in the two-hybrid
system is perhaps surprising and worthy of comment. Yip1p is not
alone in this regard; other membrane proteins have also been shown to
functionally interact in such a system (29). Although some membrane
proteins clearly cannot maintain their native structure and functional
interactions in the two-hybrid system, there are at least two factors
that might indicate whether or not the two-hybrid system will be useful for any given protein. (i) The GAL4 system contains a strong nuclear localization signal and so may dominate over other localization signals
present in the two-hybrid construct and be a better system for this
purpose than a system that relies on passive diffusion to enter the
nucleus (32). (ii) S. cerevisiae is probably more capable of
correctly folding endogenous yeast proteins rather than proteins from
other organisms. In addition, membrane channels have been observed in
the nucleus (33), and some viruses acquire membranes in the nucleus
(34), indicating that the ultrastructure of the nucleus may be more
complex than originally thought.
We have identified two functional domains of Yop1p that act in a
dominant manner to inhibit cell growth upon overexpression. The first
domain consists of the cytosolic N terminus of Yop1p that corresponds
to the first exon of the YOP1 gene. The second domain
comprises the C terminus of the molecule that is mainly hydrophobic and
corresponds to the second genomic exon. Overexpression of full-length
Yop1p leads to inhibition of cell growth and a phenotype of enlarged
cells that accumulate internal membrane structures. This phenotype can
be suppressed by co-overexpression of Yip1p. Presumably, Yip1p is able
to suppress the toxic effect of Yop1p by sequestration. These data
would suggest that YOP1 overexpression inhibits cell growth
by inhibiting the function of Yip1p, since Yip1p is an essential gene
required for secretion. Consistent with this interpretation is the
phenotype of YOP1 full-length overexpression, which results
in the accumulation of membrane structures and an accumulation of the
ER core-glycosylated form of CPY, indicating a block in ER to Golgi
traffic. Recently, a Yip1p homolog, Yif1p, has been identified (35)
that appears to act similarly to Yip1p in blocking ER to Golgi
transport. Although nothing is known about the precise function of
Yip1p, the identification of Yif1p and Yop1p as Yip1p binding partners
suggests that Yip1p may be involved in several different Rab-mediated
events through a combinatorial assortment with different binding partners.
The phenotype of the YOP1 C-terminal construct is distinct
from that of the full-length construct. Yip1p co-overexpression cannot
suppress the dominant negative effect of Yop1p C terminus overexpression. The Yop1p-C-terminal construct lacks the domain that is
both necessary and sufficient for Yip1p interaction by two-hybrid
analysis. The mechanism by which this construct inhibits growth cannot
be directly via an inhibition of Yip1p function. One clue may be
provided by the fact that YPT6 can suppress the dominant
negative YOP1 C-terminal construct but not that of the full-length construct, indicating that the action of Yop1p is intimately connected to Rab function. This finding further underscores our results, demonstrating that Yop1p can be specifically
coprecipitated with Rab proteins in cellular lysates. Since Yop1p shows
a restricted sub-cellular localization, we hypothesize that the
biochemical interaction of Yop1p with Rab proteins is limited in
vivo, possibly only to YPT6, which our suppression
analysis demonstrates to interact genetically with YOP1.
Consistent with this interpretation are our data demonstrating that the
steady-state immunofluorescent localization of Yop1p and Ypt6p shows
overlap in vivo.
The two domains of Yop1p may act antagonistically, or perhaps the
exposed Yop1p N terminus may constitute a signaling domain that acts in
a dose-dependent manner to negatively regulate membrane traffic. There is a growing appreciation that many proteins involved in
the regulation of intracellular membrane traffic may act as signal
transducers that coordinate membrane traffic with other cellular events
(36-38). Different branches of the Ras superfamily are ideally placed
to coordinate such cross-talk, and our data indicate that
YOP1 and possibly its human homolog TB2 may also play a role in the regulation of cell growth through its facilitation of membrane traffic. Our genetic data suggest that YOP1 is a
recessive gene that negatively regulates cell growth. Deletion of
YOP1 has no apparent effect on cell viability, and
full-length and C-terminal YOP1 constructs possess a
dose-dependent growth inhibitory effect.
Sequence comparison revealed that Yop1p is homologous to the human
TB2 gene with sequence similarities throughout the protein. The amino acid sequence conservation of Yop1p across species clearly points to its functional importance, and an interesting finding is that
TB2 is a human familial adenomatous polyposis locus (39) gene (40), adjacent to the tumor suppressor genes MCC
and APC (41). TB2 encodes a 197 amino acid
polypeptide (1). The deduced amino acid sequence predicts that Yop1p
contains at least two extensive membrane-spanning segments. The human
TB2 gene also contains a similar size and type of
membrane-spanning segments and would be predicted to have the same
topology. This similarity raises the possibility that Yop1p and TB2 may
share a common function in mediating vesicular transport. It is now
clear that the machinery and mechanisms of membrane traffic share much
in common between yeast and higher eukaryotes (42). For example, the
complex observed between Sec9p, Sso1/2p, and Snc1/2p, which is required
for exocytosis in yeast, is the structural and functional counterpart
of the neuronal SNARE complex (30). Rabs are also extremely well
conserved over evolution. In some cases, yeast and mammalian Rab
proteins are functionally interchangeable. For example, Vps21p/Ypt51p, a homolog of mammalian Rab5, is also required at an early step in
endocytic traffic (43). Remarkably, Ypt51p expression in animal cells
not only localizes to Rab5-positive early endosomes but also stimulates
endocytosis (44). This latter fact indicates that the machinery
involved in mediating Rab protein function is probably conserved across
diverse species. Our data indicate that Yop1p, probably in conjunction
with Yip1p, acts to facilitate a Rab-mediated event in membrane
traffic. It remains to be demonstrated whether TB2 has a role in
membrane transport.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S. cerevisiae strains used in this study
-mercaptoethanol, and
0.005% bromphenol blue), microcentrifuged for 5 min, and loaded onto
12 or 14% SDS-polyacrylamide gels (37.5:1
acrylamide/bisacrylamide). Prestained protein molecular weight
markers were from Life Technologies, Inc. For Western blotting, gels
were transferred to polyvinylidene difluoride membranes for 2 h at
200 mA. The membranes were stained with Ponceau S to observe the
quality of the transfer. Antigens on the membrane were detected by
incubating the filter with blocking buffer (5% nonfat dry milk in
TBST; 150 mM NaCl, 50 mM Tris, pH 7.5, and
0.2% Tween 20). Primary antibodies were incubated in TBST, followed by
three washes. Secondary alkaline phosphatase-conjugated antibodies were
added in blocking buffer, followed by three washes and chromogenic blot
development with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue
tetrazolium (both from Bio-Rad) substrates in AP buffer (100 mM Tris, pH 9.5, 100 mM NaCl, and 5 mM MgCl2).
-GST (gift of T. Fox, Cornell
University), mouse monoclonal
-Myc antibody (9E10; Ref. 23),
affinity-purified Rabbit
-GFP antibody (24) (gift of P. Silver,
Dana-Farber Cancer Institute), and mouse monoclonal
-HA 12CA5.
Alkaline phosphatase-conjugated anti-rabbit and anti-mouse secondary
antibodies were used (Bio-Rad) to detect the presence of
Myc9-Yip1p and either GST alone or GST-Yop1p.
-HA antibody (Y11; Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) was added to each well at a dilution of 1:5000
and incubated for 1 h at room temperature. Cells were washed 5 times in PBS/BSA and then incubated with Texas Red-labeled anti-rabbit
secondary antibody (Molecular Probes, Inc.) at a dilution of 1:200 for
30 min at room temperature. Monoclonal 1.2.3 antibody was used to detect Sec4p (26), and a monoclonal anti-GFP antibody (3E6; Molecular
Probes, Inc., Eugene, OR) was used to detect GFP. These were followed
by Oregon Green 514-labeled anti-mouse secondary antibody (Molecular
Probes) at a dilution of 1:250. To stain nuclei, 5 µg/ml Hoechst
33258 (Molecular Probes) in PBS/BSA was added to each well, and after
10 min at room temperature, cells were washed five times. Cells were
mounted in a small drop of mounting medium (Moviol), and the slides
were left to air dry in the dark for at least 30 min. Confocal
microscopy was performed using an Olympus FluoView confocal station
(Olympus). Oregon Green was excited with the 488-nm line of an argon
laser, and Texas Red was excited with the 568-nm line of a krypton laser.
-galactosidase
filter assay as described (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Pattern of two-hybrid interactions of YOP1 with various YIP1
constructs
-Galactosidase activity was determined by filter assay. Pairs were
coexpressed in the reporter strain Y190. Plus represents a positive
activity rated according to the following criteria: +++, activity
detected after 30 min; ++, activity detected after 90 min; and +,
activity detected after 5 h.
, a negative indication of
activity. At least 30 independent transformants were tested for each
pair. Yop1p N-terminal constructs contain amino acids 1-17, and Yop1p
C-terminal constructs contain amino acids 18-180. Yip1p N-terminal
constructs contain amino acids 1-117. ND, not detected.
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Fig. 1.
A, schematic representation of Yop1p.
Sequence data indicate a cytoplasmically oriented N terminus and a
hydrophobic C-terminal domain that spans the membrane twice. B,
Kyte-Doolittle hydrophobicity plot of Yop1p. This was generated using
the program Protean (DNASTAR) with a 9-residue parameter average and
shows the relative location of the two hydrophobic segments of the
protein. C, alignment of Yop1p with data base homologs.
Shown is the sequence of Yop1p and a comparison with human and murine
TB2 and full-length cDNAs from other organisms. T41634 is from
Schizosaccharomyces pombe, CG4960 and CG8331 are from
Drosophila melanogaster, AAF36016 is from
Caenorhabditis elegans, and CAN11144 is from
Plasmodium falciparum. Mammalian expressed sequence tag
fragments are not included in this alignment. The sequences were
aligned using MegAlign. Amino acid residues are numbered according to
the protein sequence. The shaded residues exactly
match the consensus sequence.
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Fig. 2.
A, membrane localization of Yop1p.
Differential centrifugation was performed on logarithmically growing
cells expressing HA-Yop1p at wild-type levels. Blots were probed for
HA-Yop1p, which is present exclusively in the pellet fraction (P100;
100,000 × g). PNS represents a total postnuclear
lysate (500 × g supernatant), and S100 is the
supernatant remaining after the 100,000 × g
centrifugation. B, high salt and Triton X-100 treatment of
Yop1p-containing membranes. Postnuclear supernatants were centrifuged
at 100,000 × g to obtain cytosolic and total membrane
fractions. Total membrane fractions were resuspended in buffer with and
without 1 M NaCl, 1% Triton X-100, or mock-treated before
recentrifugation at 100,000 × g. The pellets and
supernatants were dissolved in equivalent volumes of sample buffer and
run on SDS-PAGE gel, and HA-Yop1p was detected by Western blotting.
Relevant protein marker sizes are indicated. C, Triton X-114
phase separation of lysates expressing HA-tagged Yop1p. Triton X-114
fractionation generating a detergent-enriched phase and an aqueous
phase was performed as described under "Experimental Procedures" on
cells expressing HA-Yop1p at wild type levels. HA-Yop1p was detected by
Western blotting and fractionates in the detergent-enriched phase. PNS
represents total postnuclear supernatant. As a control, the fractions
were probed for the membrane protein Snc1/2p, which is contained in the
detergent-enriched phase. Relevant protein marker sizes are indicated
on the left.
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Fig. 3.
A, biochemical analysis of Yop1p and
Yip1p interaction: GST-Yop1p interacts specifically with
Myc9-Yip1p. Lysates were prepared from cells expressing
either GST-Yop1p and Myc9-Yip1p (RCY425) or GST and
Myc9-Yip1p (RCY428). Detergent-solubilized lysates were
incubated with GST beads for 30 min at 4 °C as described under
"Experimental Procedures." After four washes, the bead-bound
material was subjected to SDS-PAGE electrophoresis and analyzed by
Western blotting. Membranes were probed with both monoclonal 9E10
(1:500) to detect Myc9-Yip1p and polyclonal anti-GST
(1:800) to detect GST-Yop1p. Relevant protein marker sizes are
indicated. Myc9-Yip1p was detected on RCY425 but not on
RCY428 after GST pull-downs. B, GST-Yop1p and Myc9-Yip1p
interaction is specific to Yop1p and Yip1p. GST pull-down experiments
as in A were performed on yeast expressing either GST-Yop1p
and Myc9-Yip1p (RCY425) or GST-Yop1p and
Dss4p-Myc3 (RCY429). The membrane was first probed with
-Myc to detect expression of Myc-tagged proteins and was
subsequently probed with
-GST to confirm the presence of GST-Yop1p.
Myc9-Yip1p was detected after GST pull-down, but
Dss4p-Myc3 was not, indicating that the interaction is
specific to Yip1p and Yop1p. C, GST-Yop1p specifically
associates with Yip1p expressed at wild-type levels. Lysates were
prepared from yeast expressing GST-Yop1p and either GFP-Yip1p (RCY509)
or GFP-Ypt7p (RCY508) at single copy. Western blot analysis with
affinity-purified anti-GFP antibody showed that GFP-Yip1p specifically
associated with GST-Yop1p; however, a control protein (GFP-Ypt7p)
expressed at similar levels did not associate with GST-Yop1p. Western
blot of lysates demonstrates that the fusion proteins were expressed at
equivalent levels in both strains. D, the complex of Yop1p
and Yip1p interaction is formed in vivo. GST-Yop1p and
Myc9-Yip1p were either coexpressed in the same cell
(RCY425) or expressed in different strains that were mixed after lysis
(RCY429, RCY462). The GST-Yop1p and Myc9-Yip1p
interaction is only observed when the two constructs are expressed in
the same cell, indicating that the interaction occurs prelysis or
in vivo.
haploids were viable, indicating that
YOP1 is dispensable for vegetative growth. Furthermore, a
strain carrying the null allele has no apparent growth defect under
several conditions commonly used to detect phenotypes in S. cerevisiae (31): high temperature (37 °C), low temperature (15 °C), 2 mM caffeine, 2% formamide, high salt (1 M NaCl), and glycerol as carbon source (data not shown). We
next examined the phenotype of Yop1p overexpression. For this
experiment, we expressed both full-length Yop1p (Yop1p full-length,
RCY376) and Yop1p that lacks 17 amino acids at the N terminus
identified as the Yip1p-interacting region (Yop1p C terminus, RCY377).
Both constructs were expressed in yeast as HA-tagged proteins under the
control of the GAL1/10 promoter. Immunoblot analysis of the
lysates verified that both proteins were expressed in equivalent
amounts upon shift of the growth medium to galactose (Fig.
4A). Both of these constructs were dominant negative for growth upon overexpression (Fig.
4B) but resulted in different morphologies. Overexpression
of full-length Yop1p resulted in huge swollen cells of aberrant shape,
while overexpression of the Yop1p C-terminal construct gave rise to much smaller cells, similar in shape but considerably smaller than
cells harboring a control construct (Fig. 4C). To quantitate the observed effect, measurement of cell size was performed (Fig. 4D). Wild-type cells have an average width of 5.52 ± 0.90 µm; cells expressing full-length Yop1p have an average
width of 6.85 ± 0.97 µm; and cells expressing Yop1p C-terminal
construct have an average width of 3.37 ± 0.367 µm.
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Fig. 4.
Overexpression of both Yop1p full-length and
Yop1p C-terminal constructs results in a dominant negative
phenotype. A shows the ability of cells bearing
constructs as indicated for galactose-dependent expression
to grow when expression of the construct is induced by growth on
galactose-containing medium after 3.5 days of growth at
30 °C. B shows a Western blot of lysates derived from
cells shifted to galactose-containing medium for 10 h probed for
the presence of the HA-tagged construct. Lane 1 shows a lysate generated from RCY376 (Yop1p full-length construct), and
lane 2 shows a lysate generated from RCY377
(Yop1p C-terminal construct). C shows differential
interference contrast (DIC) images of cells expressing the
constructs as indicated and allowed to grow in galactose-containing
medium for 2 days. All images are shown at the same
magnification. Bar, 2.5 µm. D represents the
quantification of cell size illustrated in C; for each
condition, the width of randomly chosen cells was measured at the
widest point, and the average size is shown together with the
S.D.
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Fig. 5.
Multicopy YIP1 can rescue
the lethality associated with overexpression of full-length Yop1p but
not of the Yop1p C-terminal construct that lacks the Yip1p-interacting
domain. RCY376 expresses full-length Yop1p, and RCY377 expresses
the Yop1p C terminus in a galactose-dependent manner. The
control strain was transformed with the GAL1/10 HA-tagged
vector only (no insert). Growth of these strains is shown on both
glucose and galactose carbon sources with either multicopy
YIP1 or a control multicopy plasmid as indicated.
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Fig. 6.
A-D, thin section electron microscopy
of dominant negative YOP1. RCY376 cells expressing
full-length dominant negative Yop1p (A-C) and NY605 cells
expressing wild-type levels of Yop1p (D) were examined by
thin section electron microscopy. Representative examples of each
strain are shown. Bar in A, 1 µm. All
panels are shown at the same magnification.
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Fig. 7.
A, CPY immunoblot analysis of cells
expressing dominant negative YOP1 constructs. Shown is
immunoblot analysis of total cell lysates for the relative level CPY
processing in cells expressing dominant negative constructs containing
full-length YOP1 (RCY376), C-terminal YOP1
(RCY377), and an isogenic control strain (RCY458). At the time points
indicated after the switch from sucrose- to galactose-containing
medium, samples were taken and processed for total cell
lysates. The arrows indicate the relative migration of the
p1 (core-glycosylated ER), p2 (Golgi-modified), and m (mature vacuolar)
forms of CPY. sec18 cells are shown at room temperature
(permissive temperature) and after shift to 37 °C (restrictive
temperature) for 1 h as a control for the migration of the various
CPY forms and to provide a positive reference for the accumulation of
p1 CPY, marked with asterisk. B, cell growth of
dominant negative YOP1. Growth of cells expressing
full-length YOP1 dominant negative construct (RCY376)
relative to isogenic wild-type strain (RCY458). Cells were grown to log
phase in sucrose-containing selective medium before being
switched to galactose-containing medium to induce expression of
construct. At various times, as indicated, a turbidity
measurement was made as a record of cell growth. Cell concentration was
maintained in log phase for the duration of the experiment.
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Fig. 8.
A, intracellular localization of Yop1p.
RCY469 cells expressing wild type levels of a single HA epitope-tagged
Yop1p as the only source of Yop1p were grown to logarithmic phase,
disrupted with glass beads, and subjected to centrifugation at 500 × g to remove unbroken cells and cell debris. The PNS was
fractionated by differential centrifugation at 10,000 × g to give pellet fraction P10 and 100,000 × g to yield pellet fraction P100 and supernatant fraction
S100. Aliquots of fractions were subjected to SDS-PAGE and Western blot
analysis with anti-HA monoclonal antibody. B,
immunofluorescence localization of Yop1p. RCY469 cells expressing wild
type levels of HA-tagged Yop1p as the only source of Yop1p
(A) and an isogenic control strain, RCY407, expressing the
untagged protein (B and C) were examined by
immunofluorescence microscopy. HA-tagged Yop1p was localized with the
anti-HA tag antibody Y11 (A and B). Nuclei were
localized in the untagged control by Hoechst 33258 staining
(C). All panels are shown at the same
magnification. Bar, 5 µm.
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Fig. 9.
A-B, double label immunofluorescence
microscopy of Yop1p with Sec4p and Ypt6p. RCY469 cells expressing
wild-type levels of HA-tagged Yop1p as the only source of Yop1p
together with wild-type levels of either GFP-tagged Sec4p or Ypt6p were
examined by double label immunofluorescence microscopy. Cells were
labeled with the anti-HA tag antibody Y11 to visualize HA-Yop1p
(A and B (i)) and with anti-Sec4p
(A (ii)) or anti-GFP (B
(ii)). Nuclei were counterstained with the DNA stain Hoechst
33258 (A and B (iii)). A merge of all
three channels is shown in A and B
(iv). Note that under the processing conditions for
immunofluorescence, there was no interference from the intrinsic GFP
fluorescence. C, double label confocal immunofluorescence
microscopy of Yop1p with Yip1p. RCY496 cells expressing wild-type
levels of HA-tagged Yop1p as the only source of Yop1p together with
wild-type levels of GFP-tagged Yip1p were examined by double label
confocal microscopy. Cells were labeled with anti-HA tag antibody
(i) and with anti-GFP antibody (ii). A merge of
both channels is shown in iii.
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Fig. 10.
A, coprecipitation of Yop1p with Rab
proteins. Lysates were prepared from cells expressing either GST alone
or various GST-Rab constructs as indicated, together with
HA-tagged-Yop1p. Detergent-solubilized lysates containing 0.5% Tween
20 were incubated with GST beads for 30 min at 4 °C as described
under "Experimental Procedures." After four washes, the bead-bound
material was subject to SDS-PAGE electrophoresis and analyzed by
Western blotting. Membranes were probed with both polyclonal anti-GST
(1:800) to detect the bead-bound GST fusion proteins and monoclonal
12CA5 to detect any associated HA-Yop1p. Relevant protein marker sizes
are indicated. All constructs were under the control of the
GAL1/10 promoter and were expressed by inducing with
galactose for ~8 h. HA-Yop1p was detected associated with Ypt52p,
Ypt6p, Sec4p, and Ypt7p GST fusion proteins but not on GST alone or
Ypt1p lacking its C-terminal cysteines. B, growth of
dominant negative YOP1 C-terminal construct (RCY377) with various
plasmids. RCY377 expresses the Yop1p C terminus in a
galactose-dependent manner. The control strain was
transformed with the GAL1/10 HA-tagged vector only (no
insert). Growth of these strains is shown on both glucose and galactose
carbon sources with either multicopy YPT6, YPT7,
or a control multicopy plasmid as indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Yves Barral, Pat Brennwald, and Tom Fox for generous gifts of reagents and valuable discussion and P. Silver for the generous gift of GFP antibody. We thank Liz Wills for valuable technical assistance.
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FOOTNOTES |
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* This work was supported in part by the United States Department of Agriculture Animal Health and Disease Research Program, American Heart Association Grant 0030316T, and National Science Foundation Grant MCB-0079045 (to R. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of Army Predoctoral Fellowship DAMD17-00-1-0218.
¶ To whom correspondence should be addressed: Dept. of Molecular Medicine, C4-109 VMC, Cornell University, Ithaca, NY 14850. Tel.: 607-253-4123; Fax: 607-253-3659; E-mail: rnc8@cornell.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M008439200
2 R. Collins, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GDI, GDP dissociation inhibitor; GST, glutathione S-transferase; HA, influenza virus hemagglutinin epitope; PCR, polymerase chain reaction; ORF, open reading frame; PBS, phosphate-buffered saline; PNS, postnuclear supernatant; PAGE, polyacrylamide gel electrophoresis; ER, endoplasmic reticulum; CPY, carboxypeptidase Y.
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