From the Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405
Received for publication, August 14, 2000, and in revised form, September 27, 2000
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ABSTRACT |
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Nrf1p was first identified in a screen for
negative regulators of the Cdc42p GTPase. Overexpression of Nrf1p
resulted in dose-dependent lethality, with cells exhibiting
an ellipsoidal morphology and abnormal vacuolar phenotypes including an
increase in vacuolar fusion. Green fluorescent protein (GFP)-Cdc42p and
GFP-Nrf1p colocalized to vacuolar membranes and GFP-Nrf1p vacuolar
localization depended on Scd1p, the Schizosaccharomyces
pombe homolog of the Cdc24p guanine nucleotide exchange factor.
In this study, site-directed mutagenesis was conducted on Nrf1p to
determine its functional domains. Mutations in the three putative
transmembrane domains resulted in mislocalization of GFP-Nrf1p and an
inability to induce lethality, suggesting a loss of function. Mutations
in the second extramembranous loop of Nrf1p also resulted in a loss of
function and altered the ability of GFP-Nrf1p to localize to vacuolar
membranes. Analysis of Cdc42p is a Rho-like GTPase that is ubiquitously expressed in
eukaryotes and has been implicated in many cellular processes including
regulation of cellular polarity, transcriptional activation, and
phagocytosis of bacteria into mammalian cells (1). Cdc42p acts as a
binary switch, active in the GTP-bound state and inactive in the
GDP-bound state. The nucleotide-bound state is regulated by
guanine-nucleotide exchange factors that mediate the exchange of GDP
for GTP, thereby activating Cdc42p and GTPase-activating proteins,
which enhance the intrinsic GTPase activity of Cdc42p, thereby
inactivating the protein (1).
In fission yeast Schizosaccharomyces pombe, Cdc42p is
essential and regulates directed growth, with the S. pombe Nrf1p is a 122-amino acid protein with three
putative transmembrane domains that was previously identified in a
screen for negative regulators of Cdc42p (4). A To characterize the functional domains of Nrf1p, site-directed
mutations were generated. Mutations generated in the three putative
transmembrane domains affected GFP-Nrf1p localization, consistent with
the prediction that these transmembrane domains were essential for
membrane anchoring. Mutations created in predicted extramembranous
domains of Nrf1p had varying effects on GFP-Nrf1p localization and
function, with the second loop domain of Nrf1p having a role in
targeting Nrf1p to the vacuolar membrane. Studies were also conducted
to further examine the role of Scd1p in Nrf1p localization to the
vacuole. These studies led to the discovery that Strains, Media, and Growth Conditions--
S. pombe
cells were grown in yeast extract and supplements (YES) complex media
or in Edinburgh minimal media (EMM) lacking uracil, leucine, or both
(5). EMM and EMM agar were purchased from Bio101 (Vista, CA). Thiamine
was added to S. pombe growth media at 5 µg/ml to repress
transcription from the nmt1 promoter. The S. pombe strains are listed in Table I.
Yeast transformations were performed as described previously (5,
6).
Plasmids and DNA Manipulations--
Enzymes, polymerase chain
reaction kits, and other reagents were purchased from standard
commercial sources and used as specified by the suppliers.
Oligonucleotide primers were obtained from Genosys Biotechnologies Inc.
(The Woodlands, Texas). Standard methods were used for recombinant DNA
manipulations (7). The
pREP3X-GFP-A8-nrf1+
construct was generated by inserting a nrf1+
fragment isolated from
pREP41X-GFP-A8-nrf1+ (4)
into pREP3X-GFP-A8 (provided by A. Merla) at the
NotI and BamHI sites. Site-directed mutations
(Fig. 1A) were created in
pREP41X-GFP-A8-nrf1+ using the
QuikChange site-directed mutagenesis kit obtained from Stratagene (La
Jolla, CA). The DNA sequence of all polymerase chain reaction products
was verified through the Vermont Cancer Center DNA Sequencing Facility.
Photomicroscopy--
Vacuolar staining was conducted using
carboxy-DCFDA or FM 4-64 (Molecular Probes, Eugene OR). The cells were
grown to mid-log phase under derepressing conditions (described above)
and incubated for 30 min in fresh media containing 10 µM
carboxy-DCFDA or 20 µg/ml FM 4-64. These cells were then washed with
media, and half of the cells were resuspended in H20 for
carboxy-DCFDA staining or allowed to incubate for 30 min post-washing
before visualization for FM 4-64 staining. GFP images were captured at
mid-log phase after 18-28 h under derepressing conditions (described
above). Digital images were captured and analyzed as described
previously (4).
Immunoblot Analysis--
Total cellular protein was isolated
from cells containing nmt1 promoter-driven GFP-Nrf1p fusion
proteins. The cells were grown in EMMS-Leu Lucifer Yellow Uptake--
One ml of cells grown in minimal
media and under derepressing conditions (when necessary) were harvested
and washed twice with media, resuspended in 500 µl of media
containing 5 mg/ml of lucifer yellow carbonyl hydrazine (Sigma), and
incubated for 60 min at 23 °C. Cells were washed three times with
media and visualized. Lucifer yellow uptake assays were conducted as
described previously (9) with the following changes: 5 ml of cells were grown as above and washed twice in ice-cold media and resuspended in
200 µl of ice-cold media containing 5 mg/ml of lucifer yellow carbonyl hydrazine, and half (100 µl) of each sample was incubated at
32 °C with the other half kept on ice. After 90 min, the cultures were washed three times with ice-cold H2O, treated with
zymolyase for 30 min at 37 °C, then lysed with acid-washed glass
beads for 1 min. 400 µl of media was added to the beads, and the
liquid was transferred to a fresh tube, then centrifuged for 15 min at 10,000 × g. The fluorescence (excitation, 426 nm;
emission, 550 nm) was determined using a fluorimeter and quantified by
comparison to a standard curve of lucifer yellow fluorescence.
Nrf1p Has Three Transmembrane Domains--
GFP-Nrf1p was
previously shown to localize to the plasma membrane, nuclear membrane,
septum, and vacuolar membrane (4). To determine if the three predicted
transmembrane domains in Nrf1p were functional, three amino acids in
the middle of each putative domain were mutated to charged residues
(Fig. 1A). These mutations (nrf1tm1, nrf1tm2, and
nrf1tm3) were constructed in
pREP3X-GFP-A8-nrf1+, a high level
expression vector, and expressed as GFP-Nrf1p fusion proteins. Total
protein was isolated from wild-type ED668 cells containing GFP-Nrf1p,
GFP-Nrf1tm1p, GFP-Nrf1tm2p, or
GFP-Nrf1tm3p, and all were expressed at similar levels
(Fig. 1B). Although high level overexpression of GFP-Nrf1p
was lethal (4), the three transmembrane mutant proteins did not confer
lethality when overexpressed (Fig.
2A). The localization of these
mutant proteins was abnormal with no vacuole localization, variable
levels of diminished plasma and nuclear membrane localization, and a
general increase in the presence of cytosolic GFP aggregates (Fig.
2B). These data suggest that all three of these domains are
necessary for efficient localization of GFP-Nrf1p to plasma, nuclear,
and especially vacuolar membranes, supporting the hypothesis that these
are functional transmembrane domains.
The Second Loop of Nrf1p Was Necessary for Its Localization to the
Vacuole--
Site-directed mutagenesis was performed on the putative
extramembranous loops of Nrf1p to determine which were necessary for function. The "positive inside" observation was followed, which predicts that the nontranslocated loops of a protein are enriched in
positively charged residues as compared with translocated loops (10).
Three charged residues within a stretch of five amino acids in the
predicted nontranslocated (i.e. cytoplasmic) loops were
changed to alanine residues in
pREP3X-GFP-A8-nrf1+ (Fig.
1A). Total protein was isolated, and the level of expression of GFP-Nrf1nt-1p, GFP-Nrf1L2-1p,
GFP-Nrf1L2-2p was comparable with wild-type GFP-Nrf1p
(Fig. 1B). Overexpression of GFP-Nrf1nt-1p was
lethal, and localization of the protein appeared similar to GFP-Nrf1p,
suggesting that these charged residues were not necessary for the
proper localization of GFP-Nrf1p or its lethality when overexpressed.
Overexpression of GFP-Nrf1L2-1p did not confer lethality,
and no fluorescence was localized to the vacuole, although the plasma
membrane and nuclear membrane localization appeared enhanced compared
with overexpression of wild-type GFP-Nrf1p (Fig.
3, A and B). These
data indicate that this portion of the second extramembranous loop was
necessary for efficient localization to the vacuoles and was also
necessary for the protein to cause a lethal defect when overexpressed.
Overexpression of GFP-Nrf1L2-2p also did not confer
lethality, but it localized similarly to wild-type GFP-Nrf1p (Fig. 3,
A and B). These data indicate that the
nrf1L2-2 mutation affects the ability of Nrf1p
to cause a lethal phenotype when overexpressed; however, it does not
appear to affect the localization of the protein in wild-type
cells.
Overexpression of nrf1 Mutations in Scd1p and Nrf1p Affect Endocytosis--
These above-mentioned
localization results suggested that Nrf1p may first be targeted to the
plasma membrane and subsequently localize to the vacuolar membrane
through endocytosis, implicating Scd1p and/or Nrf1 in the endocytosis
pathway. To examine this possibility, lucifer yellow uptake was assayed
in wild-type,
To determined whether endocytosis in wild-type or Scd1p and Nrf1p Effects on Endocytosis Appear to Be Dependent on
Cdc42p--
To examine whether Cdc42p may be involved in endocytosis,
dominant-activated Cdc42G12Vp and dominant-negative
Cdc42T17Np were overexpressed in wild-type cells, and the
ability to uptake lucifer yellow was analyzed. Overexpression of
dominant-activated Cdc42G12Vp produced a 3-5-fold increase
in lucifer yellow uptake (Fig. 6A), with no vacuolar
abnormalities or coalescence observed (data not shown). A modest
increase in lucifer yellow uptake was also observed with overexpression
of dominant-negative Cdc42T17Np (Fig. 6A). These
results suggest that overexpression of Cdc42 mutant proteins can
influence endocytosis. A small decrease in lucifer yellow uptake was
also observed upon overexpression of kinase-inactive
Pak1K415R,K416Rp, suggesting that Pak1p may be
involved in the endocytic process. These data raise the possibility
that the endocytic defects observed in the The Vacuolar Coalescence Observed upon Nrf1p Overexpression Was
Independent of the Mitogen-activated Protein Kinase Pmk1p/Spm1p but Was
Dependent on Ypt7p--
Vacuoles fuse in response to osmotic stress
(i.e. placement in H2O), and this fusion has
been shown to be dependent on the Pmk1p/Spm1p mitogen-activated protein
kinase cascade as well as the conserved Rab-like GTPase Ypt7p, which is
necessary for vacuolar fusion in S. cerevisiae (11, 12). To
determine whether the vacuolar coalescence around the nucleus and
subsequent vacuole fusion observed in cells overexpressing Nrf1p act by
a similar mechanism, Nrf1p was overexpressed in Nrf1p and Scd1p Were Not Necessary for Vacuolar Fusion Induced by
Osmotic Stress--
To determine whether Scd1p or Nrf1p may be
necessary for vacuolar fusion induced by osmotic stress,
Nrf1 proteins that contained mutations in the putative
transmembrane domains designed to perturb the predicted hydophobicity of this domain no longer conferred a lethal phenotype and were mislocalized, with a decrease in membrane localization and an increase
in GFP aggregates, suggesting that these domains were necessary for
efficient targeting to membranes. The nrf1nt-1
mutation did not affect the GFP localization or lethality conferred by
overexpression, whereas mutations in the predicted second
extramembranous loop did, suggesting that this region of the protein
was involved in localization of GFP-Nrf1p to the vacuolar membrane.
Both GFP-nrf1L2-1p and
GFP-nrf1L2-2p no longer conferred lethality
when overexpressed, and GFP-nrf1L2-1p did not
localize to the vacuolar membrane, whereas
GFP-nrf1L2-2p localized to the vacuolar
membrane independent of Scd1p.
Both deletion and overexpression of nrf1 led to similar
defects in endocytosis. This seemingly paradoxical result is
reminiscent of results with Cdc24p in S. cerevisiae in which
both deletion and overexpression of Cdc24p led to a loss-of-function
phenotype (13). One explanation could be that loss of a protein and
excess amounts of a protein both adversely affect the stability and/or function of a multiprotein complex essential for the process in question. Deletion of scd1 also led to a decrease in
endocytosis, whereas overexpression of dominant-activated
Cdc42G12Vp led to a 3-5-fold increase in endocytosis and
rescued the defect in In mammalian cells, Cdc42p localizes to Golgi membranes and has been
shown to regulate targeted secretion to the basolateral membrane in
polarized epithelial cells (17, 18). Microinjection of plasmids
encoding dominant-negative Cdc42T17Np resulted in a
mistargeting of proteins normally destined for the basolateral membrane
as well as a mistargeting of basolateral endocytosed proteins.
Microinjection of activated Cdc42Q61Lp-encoding plasmids
resulted in a complete loss of plasma-membrane polarity (18). Cdc42p
has also been shown to interact with the COPI coatomer complex,
specifically through the In S. pombe, vacuoles are numerous, with greater than 50 individual organelles sometimes present within a cell. Under
hypo-osmotic stress conditions, the vacuoles can fuse into one or two
large organelles (11). This osmotic stress-induced fusion is dependent on the Rab-type GTPase Ypt7p and the Sty1p and Pmk1p mitogen-activated protein kinase cascade pathways. Deletion of the Sty1p or Pmk1p mitogen-activated protein kinases or Ypt7p resulted in a severe defect
in vacuole fusion when S. pombe cells were isolated from rich media and placed in H2O (11). The acidification of
these vacuoles did not appear to be affected, as the
pH-dependent fluorescent vital stain carboxy-DCFDA stained
vacuoles in these cells. In this study, it was shown that the vacuolar
fusion induced by overexpression of Nrf1p depended on Ypt7p but not on
Pmk1p, and osmotically induced vacuolar fusion was not dependent on
Nrf1p or Scd1p, suggesting that there exist at least two distinct
vacuolar fusion mechanisms.
Recently the S. cerevisiae Nrf1p homolog (Vtc1p/Nrf1p) was
copurified with the vacuolar-ATPase subunit (v-ATPase) Vma10p (22). A
There is growing evidence for Cdc42p being involved in vesicle
trafficking in mammalian cells. In this study, overexpression of
constitutively GTP-bound Cdc42p led to an increase in endocytosis in
S. pombe and rescued the endocytosis defect in the
nrf1 and
scd1
mutants revealed defects in endocytosis. In addition, overexpression of
constitutively active Cdc42G12Vp resulted in an increase in
endocytosis and an ability to rescue the endocytic defects in
nrf1 and
scd1 cells. These data are consistent with Nrf1p and Scd1p being necessary for efficient endocytosis, possibly through the regulation of Cdc42p.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cdc42
allele exhibiting a phenotype of small, round cells and activated
alleles conferring a phenotype of large, round cells (2). Scd1p, a
putative guanine-nucleotide exchange factor for S. pombe
Cdc42p, shares amino acid identity with the Dbl family of Rho-type
guanine-nucleotide exchange factors including the Saccharomyces
cerevisiae guanine-nucleotide exchange factor Cdc24p. A
scd1 mutant is viable and exhibits a round cell morphology, suggesting that Scd1p plays a role in directed cell growth
(3).
nrf1
mutant was viable; however, high level expression of Nrf1p was lethal,
resulting in an ellipsoidal morphology and abnormal vacuolar phenotypes including vacuolar coalescence around the nucleus and subsequent fusion
of the vacuoles. In S. pombe, vacuoles are numerous, with greater than 50 individual organelles sometimes present within a cell.
Green fluorescent protein
(GFP)1-Nrf1p and GFP-Cdc42p
colocalized to the plasma membrane, nuclear membrane, septum, and
vacuolar membranes. The localization of GFP-Nrf1p to the vacuolar
membrane and subsequent vacuolar coalescence and fusion depended on
Scd1p (4).
scd1 and
nrf1 mutants had a defect in endocytosis, and this defect
could be reversed by expression of activated Cdc42p. Together, these
data suggest that Cdc42p-dependent signaling pathways play
a role in endocytosis in S. pombe.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S. pombe strains used
Thi liquid media to mid-log
phase, collected, washed with H20, resuspended in 100 µl
of 1× phosphate-buffered saline, and spheroplasted at 37 °C in the
presence of 100 µg/ml zymolyase until a sample showing greater than
80% lysis was observed upon the addition of SDS to 0.1%. The
spheroplasts were then collected and resuspended in lysis buffer (0.3 M sorbitol, 140 mM NaCl, 50 mM Tris
pH 8.0) with protease inhibitors (1:100 dilutions of 5 mg/ml aprotinin,
5 mg/ml leupeptin in water, 6 mg/ml phenylmethylsulfonyl fluoride, and
5 mg/ml pepstatin in methanol). SDS (0.1%) was added, and the samples
were vortexed briefly. Protein samples were diluted 1:2 in SDS lysis
buffer (8) containing 40%
-mercaptoethanol, heated at 100 °C for
5 min, and separated on an 10% SDS-polyacrylamide gel, and protein was
transferred to nitrocellulose paper (BA-S83, 0.02 µm; Schleicher & Schuell). 20 µg of protein was loaded in each lane, as
indicated by the Bradford protein assay method. Mouse anti-GFP
antibody (Roche Molecular Biochemicals) was used at a 1:1000 dilution,
and horseradish peroxidase-conjugated goat anti-mouse IgG secondary
antibody (Sigma) was used at a dilution of 1:3000. Antibody-antigen
complexes were visualized using the Renaissance system
(PerkinElmer Life Sciences).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Site-directed mutagenesis of Nrf1p.
A, diagram of predicted Nrf1p membrane topology showing
sites altered by site-directed mutagenesis (shaded amino acids indicate
the sites of amino acid substitution). B, the indicated
GFP- nrf1 plasmid constructs were transformed into ED668
cells and selected on EMMS-Leu Thi media. Individual
transformants were grown in liquid EMMS-Leu
Thi media at 30 °C for
24 h and harvested. Total cellular protein was extracted, and 20 µg of total protein was loaded into each lane. The
GFP-Nrf1 proteins were resolved using mouse anti-GFP antibodies.
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Fig. 2.
Mutations in the transmembrane domains
altered GFP-Nrf1p localization and lethality. A, the
indicated GFP- nrf1 plasmid constructs were transformed into
ED668 cells and selected on EMMS-Leu Thi media. Individual
transformants were streaked at 30° on EMMS-Leu
Thi media.
B, individual transformants were grown in liquid
EMMS-Leu
Thi media at 30° for 18-24 h and examined microscopically
for GFP fluorescence or FM4-64 staining. The fields shown are collages
of individual photos assembled in Adobe Photoshop 5.0. Scale
bars = 10 µm.
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Fig. 3.
Mutations in the cytoplasmic domains of Nrf1p
differentially affected GFP-Nrf1p localization and lethality.
A and B, the indicated GFP- nrf1
plasmid constructs were analyzed as in Fig. 2. The fields shown are
collages of individual photos assembled in Adobe Photoshop 5.0. Scale bars = 10 µm.
scd1 Cells--
The
localization of Nrf1p to vacuoles and subsequent coalescence of
vacuoles around the nucleus and vacuolar fusion depended on Scd1p (4).
To determine if Scd1p may affect the localization of the GFP-Nrf1
mutant proteins, we expressed them in
scd1 cells. Overexpression of wild-type GFP-Nrf1p or GFP-Nrf1nt-1p was
lethal in
scd1 cells; however, overexpression of the
other GFP-Nrf1 mutant proteins did not affect the viability of this strain (data not shown). GFP-Nrf1nt-1p was localized to the
plasma and nuclear membranes but was not observed in the vacuolar
membranes similarly to wild-type GFP-Nrf1p, suggesting that this
mutation does not affect localization of GFP-Nrf1p in
scd1 cells (Fig. 4). The
three transmembrane mutant proteins GFP-Nrf1tm1p,
GFP-Nrf1tm2p, and GFP-Nrf1tm3p all showed a
similar localization pattern with no vacuole localization and variable
levels of diminished plasma membrane and nuclear membrane localization,
comparable with the localization pattern in wild-type cells (data not
shown). Localization of GFP-Nrf1L2-1p was as predicted,
with localization to the plasma and nuclear membranes only (Fig. 4).
However, GFP-Nrf1L2-2p localization was similar to that
observed in wild-type cells, with localization to the plasma membrane,
nuclear membrane, and the vacuolar membranes, indicating a bypass of
the Scd1p requirement for vacuolar membrane localization (Fig. 4).
These data further implicate the second extramembranous loop of Nrf1p
in the localization of GFP-Nrf1p to the vacuolar membranes and suggest
that there may exist a Scd1p-independent mechanism for Nrf1p
localization.
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Fig. 4.
Mutations in the cytoplasmic domains of Nrf1p
differentially affected GFP-Nrf1p localization in
scd1 cells. The indicated
GFP- nrf1 plasmid constructs were transformed into
scd1 cells and selected on EMMS-Leu
Thi media.
Individual transformants were grown in liquid EMMS-Leu
Thi media at
30 °C for 18-24 h and examined microscopically for GFP fluorescence
or FM 4-64 staining. The fields shown are collages of individual photos
assembled in Adobe Photoshop 5.0. Scale bars = 10 µm.
scd1, and
nrf1 cells as well
as
ypt7 cells, which are deficient in vacuolar fusion
and, hence, endocytosis.
scd1 cells showed a decrease in
endocytosis similar to what was observed with
ypt7 cells,
whereas
nrf1 cells showed a lesser deficiency in uptake of lucifer yellow (Fig. 5A).
These data are consistent with Scd1p and Nrf1p having a positive
function in endocytosis.
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Fig. 5.
Nrf1p and Scd1p are involved in
endocytosis. A, ED668, ypt7,
scd1, and
nrf1 cells were grown in YES
media at 30 °C overnight to mid-log phase, harvested, then assayed
for the ability to uptake lucifer yellow. B, Nrf1p mutations
were inserted in the pREP1-nrf1+ expression
plasmid, transformed into ED668 cells, and selected on EMMS-Leu
Thi
media. Individual transformants were grown in liquid EMMS-Leu
Thi
media at 30 °C for 18-24 h harvested and assayed for the ability to
uptake lucifer yellow.
scd1
cells could be affected by overexpression of Nrf1p or the Nrf1 mutant proteins, the nrf1 mutations described previously were
inserted into pREP1-nrf1, a high level expression plasmid.
As with overexpression of GFP-Nrf1p fusion proteins, overexpression of
the wild-type Nrf1p or Nrf1nt-1p from these plasmids led to
lethality after extended induction periods in both strains, whereas
overexpression of the other mutant proteins did not (data not shown).
Overexpression of wild-type Nrf1p or Nrf1nt-1p caused a
significant decrease in lucifer yellow uptake in wild-type cells under
conditions where the cells were still viable, whereas overexpression of
the other mutant proteins led to at least as much lucifer yellow uptake
as the vector control (Fig. 5B). These data suggest that
overexpression of functional Nrf1p leads to a decrease in endocytosis.
Overexpression of Nrf1p or any of the mutant proteins did not affect
lucifer yellow uptake in the
scd1 strain (data not
shown). Overexpression of Nrf1L2-2p, which localizes to
the vacuole in
scd1 cells, did not increase endocytosis
in these cells, suggesting that targeting of Nrf1p to the vacuole (at
least of this mutant protein) could occur independent of
Scd1p-dependent endocytosis.
scd1 and
nrf1 mutants may be the result of decreased Cdc42p
activity. To test this possibility, dominant-activated Cdc42G12Vp was overexpressed in the
scd1 and
nrf1 strains. Overexpression of Cdc42G12Vp
rescued the endocytosis defect in both the
scd1 and
nrf1 strains (Fig. 6B) but not the defect
observed in cells overexpressing Nrf1p (data not shown), suggesting
that the endocytosis defect observed in these strains was due to a
decrease in Cdc42p activity (see "Discussion").
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Fig. 6.
Overexpression of constitutively activated
Cdc42G12Vp suppressed the
scd1 and
nrf1 endocytosis defects.
A, the indicated cdc42 and pak1
alleles were transformed into ED668 cells and selected on EMMS-Leu
Thi
media. Individual transformants were grown in liquid EMMS-Leu
Thi
media at 30 °C for 18-24 h then harvested and assayed for the
ability to uptake lucifer yellow. B,
pREP1-cdc42G12V was transformed into ED668,
scd1, and
nrf1 cells and selected on
EMMS-Leu
Thi media. Individual transformants were grown in liquid
EMMS-Leu
Thi media at 30 °C for 18-24 h then harvested and assayed
for the ability to uptake lucifer yellow. WT, wild
type.
pmk1 and
ypt7 mutant strains. As previously shown in wild-type
cells, overexpression of Nrf1p was lethal in these strains (data not
shown). The vacuolar morphology was observed by carboxy-DCFDA staining
and confirmed by subsequent localization of GFP-Nrf1p. The
pmk1 strain showed the abnormal vacuolar phenotypes
associated with Nrf1p overexpression, but no vacuolar changes were
observed in the
ypt7 cells (Table
II). GFP-Nrf1p localization appeared
normal in the
pmk1 cells, but the
ypt7
strain showed localization only to the plasma membrane (data not
shown). These data suggested that the localization of Nrf1p to the
vacuole and the subsequent abnormal vacuolar morphology was independent
of Pmk1p but was dependent on Ypt7p.
Nrflp-induced vacuolar abnormalities
scd1 and
nrf1 cells were grown in YES
media, stained with carboxy-DCFDA, and shifted into H20 to
observe the vacuolar morphology (Fig. 7).
Fusion was observed in
scd1 cells as well as
nrf1 cells, suggesting that the vacuolar fusion induced
by osmotic stress is independent of Scd1p and Nrf1p. These data are
consistent with the vacuolar fusion induced by overexpression of Nrf1p
occurring through a separate mechanism.
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Fig. 7.
Nrf1p and Scd1p were not required for
osmotically induced vacuolar fusion. ED668, scd1,
and
nrf1 cells were grown in YES media to mid-log phase,
harvested, then stained with carboxy-DCFDA. Half of the cells were
resuspended in YES, and the other half were resuspended in
H20 and examined microscopically for carboxy-DCFDA
staining. Scale bars = 10 µm.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
scd1 and
nrf1 cells.
These data implicate the Cdc42p-dependent signaling pathway
in the endocytic process in S. pombe. Overexpression of
dominant-negative Cdc42T17Np also led to a modest increase
in endocytosis, suggesting that this allele may not function negatively
in the endocytosis pathway. In mammalian cell lysates, endocytic
vesicles have been shown to move at the ends of actin tails, and the
Cdc42p effector, Wiskott-Aldrich syndrome protein (WASp), has been
shown to be involved in the nucleation of actin and subsequent
propulsion of these vesicles (14-16). Two Rho-type GTPase inhibitors,
ToxB and RhoGDI, interfered with this process, and purification of a
soluble factor capable of rescuing the ToxB defect was identified as a
Cdc42p-RhoGDI complex (15). These data are consistent with the
Cdc42p-dependent signaling pathway being involved in the
trafficking of endocytic vesicles.
COP subunit (19). COPI subunits have been
purified from isolated endosomes, suggesting that COPI is involved in
targeting in the endocytic pathway as well (20, 21). The
cdc42F28L-transforming mutant showed a defect in
COPI recruitment to vesicles; however, a triple
Cdc42F28L,K183S,K184S mutant protein that could no
longer interact with
COP resulted in a lack of transformation
ability (19). This mutant protein contained two altered C-terminal
lysine residues necessary for COPI binding, suggesting that Cdc42p
interaction with COPI was necessary for its ability to transform cells.
Altogether, these data implicate mammalian Cdc42p in both exocytic and
endocytic trafficking, and the data herein implicate S. pombe Cdc42p in the endocytic pathway, suggesting a conserved role
for this GTPase in these processes.
vtc1 mutant showed no morphological defect; however, a
reduction in quinacrine uptake into purified vacuoles suggested that
the vacuoles were not properly acidified, which depends on the
v-ATPase. v-ATPase deletion mutant cells can survive a lack of
acidification by taking up external fluid via endocytosis but are
inviable in media at a pH higher than 7.0 (23). A
vma8
vtc1 double mutant was viable at a higher pH, and
vacuoles isolated from a
vtc1 strain showed a decrease in
the level of v-ATPase units associated with these membranes (22). Vtc1p
appears to be a member of a conserved family of proteins (Vtc1p-4) that
are found in a complex on the vacuolar membrane in S. cerevisiae (22).2 These
data suggest a possible role for Nrf1p/Vtc1p in the proper targeting of
v-ATPase subunits to the vacuole.
scd1 and
nrf1 strains. Interestingly, a
recent study has also implicated Cdc42p in the control of endocytosis
in mammalian dendritic cells (24). These results suggest that a
function for Cdc42p in vesicle trafficking may be conserved throughout
eukaryotes. GFP-Cdc42p also localizes to the vacuole and may be
involved in vacuolar fusion (4). Although endocytosis and vacuolar
fusion may not be distinct processes (for example, deletion of
ypt7 leads to a defect in vacuolar fusion and therefore
shows a defect in lucifer yellow uptake), the fact that Nrf1p and Scd1p
are not required for osmotically induced vacuolar fusion suggests that
the endocytic defect in
scd1 and
nrf1
deletion strains is not a result of a general defect in vacuolar
fusion. Further examination of the functions of Nrf1p, Scd1p, and
Cdc42p in S. pombe will help to uncover whether these
proteins act during vesicle formation, trafficking, and/or vacuolar fusion.
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ACKNOWLEDGEMENTS |
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We thank J. Armstrong, E. Chang, P. Fantes, A. Merla, T. Toda, and A. Mayer for strains and reagents. We also thank G. Ward and members of the Johnson lab for thoughtful discussions and critical reading of this manuscript.
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FOOTNOTES |
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* This research was supported by American Cancer Society Grant RPG-89-012-08 and a predoctoral fellowship from the National Science Foundation-VT EPSCoR (to J. M. M.).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.
To whom correspondence should be addressed: Dept. of Microbiology
and Molecular Genetics, 202B Stafford Hall, University of Vermont,
Burlington, VT 05405. Tel.: 802-656-8203; Fax: 802-656-8749; E-mail:
dijohnso@zoo.uvm.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M007389200
2 A. Mayer, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: GFP, green fluorescent protein; YES, yeast extract and supplements; EMM, Edinburgh minimal media; DCFDA, 2',7'-dichlorofluorescein diacetate; v-ATPase, vacuolar-ATPase subunit; Thi, thiamine.
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