From the Cancer Research Group, DuPont
Pharmaceuticals Company, Glenolden Laboratory, Glenolden, Pennsylvania
19036, the § Applied Biotechnology Group, DuPont
Pharmaceuticals Company, Wilmington, Delaware 19803, and ¶ The
Wistar Institute, Philadelphia, Pennsylvania 19104
Received for publication, February 5, 2001, and in revised form, March 22, 2001
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ABSTRACT |
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The BAR adaptor proteins encoded by the
RVS167 and RVS161 genes from
Saccharomyces cerevisiae form a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress. In this study, we identified a human homolog of
RVS161, termed BIN3 (bridging
integrator-3), and a Schizosaccharomyces pombe
homolog of RVS161, termed hob3+
(homolog of Bin3). In
human tissues, the BIN3 gene was expressed ubiquitously
except for brain. S. pombe cells lacking Hob3p were often
multinucleate and characterized by increased amounts of
calcofluor-stained material and mislocalized F-actin. For example,
while wild-type cells localized F-actin to cell ends during interphase,
hob3 Bin/amphiphysin/Rvs domain
(BAR)1 adaptor proteins,
which include proteins encoded by the human genes
Amphiphysin (AMPH), BIN1, and
BIN2 and the Saccharomyces cerevisiae genes
RVS167 and RVS161, are characterized by a unique
N-terminal region termed the BAR domain. While their exact functions
are largely unknown, BAR adaptor proteins appear to integrate signal
transduction pathways that regulate membrane dynamics, F-actin
cytoskeleton, and nuclear processes, roles that are highlighted in the
nomenclature of two recently identified members of the family
(bridging integrators or BIN proteins).
Both genes encoding BAR adaptor proteins in S. cerevisiae
were initially identified in a genetic screen for mutants that lost viability upon nutrient starvation (1, 2). Subsequent work revealed
that Rvs167p and Rvs161p form a physiological complex that regulates
F-actin localization, cell polarity, bud formation, and endocytosis
(2-7). Rvs161p is also important for karyogamy, the nuclear fusion
process which follows mating (8). A variety of Rvs-interacting proteins
were identified that are consistent with Rvs161p and Rvs167p functions
in F-actin regulation, lipid metabolism, cell cycle integration, and
nuclear processes (9-13). Despite the importance of Rvs161p and
Rvs167p in these diverse functions, the RVS161 and
RVS167 genes are not required for viability.
Three genes encoding BAR adaptor proteins have been described in human
cells. Two of these genes, AMPH and BIN1, encode
structural orthologs of RVS167, whereas the third gene,
BIN2, encodes a structurally unique protein. The expression
patterns of each gene suggest different physiological roles:
BIN1 is widely expressed whereas AMPH and BIN2 are tissue-restricted in their expression. Amphiphysin,
the product of the AMPH gene, was identified by virtue of
its biochemical properties (14) and encodes a neuronal adaptor protein
that regulates synaptic vesicle endocytosis (15). The restricted pattern of AMPH expression argues that the physiological
function of this gene is limited to the specialized processes of
synaptic vesicle recovery. In a similar way, BIN2 expression
is restricted to hematopoietic cells. BIN2 function is undefined but
appears to be nonredundant with other mammalian BAR proteins (16). The BIN1 gene has a complex function(s) suggested by its diverse
patterns of alternate splicing. BIN1 splice isoforms have been
identified by virtue of interaction with the c-Myc oncoprotein,
structural similarity to amphiphysin, interaction with the nuclear
tyrosine kinase c-Abl, and characterization of the BIN1 gene
itself (17-23). Brain-specific isoforms, alternately termed
amphiphysin II or amphiphysin-like isoforms, are exclusively cytosolic
and can influence endocytosis (15). However, only brain isoforms
include regions required for interaction with key components of the
endocytosis machinery (24). Thus, it is unclear whether BIN1 proteins
participate in endocytosis outside the brain. Nuclear functions are
suggested by the ability of muscle-specific and ubiquitous isoforms to
localize to the nucleus and to functionally associate with the c-Myc
and c-Abl proteins (17, 19, 25). In particular, c-Myc-interacting isoforms have tumor suppressor and transcriptional properties that
impact cell differentiation and cell death decisions (17, 25-31).
To further investigate the function of BAR adaptor proteins, we
identified a mammalian homolog of RVS161, termed
Bin3 (bridging integrator-3), and a Schizosaccharomyces
pombe homolog of RVS161, termed hob3+
(homolog of Bin 3).
Analysis of hob3 Cloning--
The S. pombe hob3+ gene was identified
through BLAST (32) searches of the S. pombe genome using the
S. cerevisiae RVS161 gene as query. The
hob3+ gene was cloned by PCR from a stationary-phase, S. pombe single-stranded cDNA library
(Library-In-A-TubeTM, QBiogen), using oligonucleotide
primers derived from the hob3+ locus. Sequences encoding the
human BIN3 protein were similarly identified by TBLASTN (32) searches
of the translated EST data base using the S. cerevisiae
Rvs161p as a query. Human BIN3 cDNA was cloned by PCR
from single-stranded Library-In-A-TubeTM cDNA libraries
(QBiogen). Information obtained from the EST data bases was used to
construct a full-length cDNA clone. Sequence determinations
included the full-length IMAGE EST clones obtained from Research
Genetics (Huntsville AL). Human BIN3 cDNA was subcloned as an untagged or hemagglutinin-tagged insert into
pcDNA3/neo (Invitrogen) and these plasmids were used for PCR to
amplify BIN3 cDNA for insertion into yeast vectors.
cDNAs were digested with NdeI and PspAI or
BamHI and cloned into the S. pombe expression plasmid pREP2 (33). Gene deletions in S. pombe were
performed as described (34) using plasmid pFA6a-kanMX6-hemagglutinin as a template for the construction of a disrupted allele. BAR
protein-encoding cDNAs were cloned between the XbaI and
BamHI or SmaI sites of the 2 µ based budding
yeast expression vector YEp195-ACN (courtesy J. Toyn). Yeast
transformations were performed by standard methods (35, 36).
Oligonucleotide sequences are available upon request.
Strains and Media--
S. pombe strains FY71
(h-, ade6-M216, leu1-32,
ura4-D18) and FY72 (h+, ade6-M210, leu1-32,
ura4-D18) were obtained courtesy of S. Henry, Mellon College.
Strain ELR6 (ade6-M210, leu1-32, ura4-D18,
hob3 Immunofluorescence--
Exponential phase S. pombe
cultures were stained for F-actin as described (41) using AlexaFluor
488-conjugated phalloidin (Molecular Probes). Nuclei were stained with
DAPI. Images were captured on a Nikon Eclipse TE300 microscope fitted
with a Nikon Plan Fluor ×100 objective using a Toshiba 3CCD camera.
Images were manipulated using Image ProPlus version 4.0 software (Media Cybernetics).
Endocytosis--
Exponential phase cultures of S. pombe cells were assayed for uptake of the lipophilic styryl dye
FM4-64 (Molecular Probes) as described (42).
Northern Analysis--
MTNI and MTNII human multiple tissue
Northern blots obtained from CLONTECH (Palo Alto
CA) were hybridized to 32P-labeled probes for
BIN3, BIN1, and AMPH generated by the
random priming method as per the vendor's instructions. The
BIN1 and AMPH probes have been described (14,
27). The BIN3 probe was a 32P-labeled 600-base
pair BamHI-BglII fragment of the human
BIN3 cDNA. Hybridization of a Northern blot of RNA isolated
from a panel of tumor cell lines, cultured and processed as described previously (17, 31), was performed using the BIN3 probe and a BIN3 Encodes a Widely Expressed BAR Adaptor Protein Related to
Rvs161p--
Sequences encoding BIN3, a novel human BAR adaptor
protein, were identified using Rvs161p to search the EST data base with the TBLASTN algorithm. Sequence analysis of full-length cDNA clones identified in this manner revealed that BIN3 was a protein of 253 residues in length and was comprised solely of a BAR domain, like
Rvs161p (Fig. 1, a and
b). The BIN3 BAR domain was 27% identical to Rvs161p but
less than 24% identical to other BAR domains (Table I). Northern analysis of human tissue
RNAs was performed to compare the BIN3 expression pattern to
that of other mammalian BAR adaptor genes. A single mRNA species of
~2.2 kilobase was detected at similar levels in all embryonic and
adult tissues examined, except for brain where BIN3 mRNA
was undetectable (Fig. 2a).
This wide expression pattern of BIN3 was similar to
BIN1, which was widely expressed, but contrasted with
AMPH, which was expressed primarily in brain, and
BIN2, which was expressed primarily in hematopoietic cells.
Since BIN1 expression was frequently decreased in malignant cells, BIN3 expression was determined in a panel of human
tumor cell lines. All cell lines tested expressed BIN3 (Fig.
2b). We concluded that BIN3 was a widely expressed BAR
adaptor protein that was structurally most similar to S. cerevisiae Rvs161p.
S. pombe hob3+ Encodes a BAR Adaptor Protein Related to Rvs161p and
BIN3--
Sequences encoding Hob3p (homolog of
Bin 3) were identified using Rvs161p to search
the S. pombe genome (Fig. 1a). Hob3p was 264 residues in length, 56% identical to S. cerevisiae Rvs161p, and 29% identical to BIN3 throughout its entire sequence (Fig. 1b). In contrast, the Hob3p BAR domain sequences were less
than 26% identical to the BAR domain sequences in BIN1, BIN2, and AMPH (Table I). Like Rvs161p and BIN3, Hob3p was comprised solely of a BAR
domain, without the additional C-terminal sequences found in Rvs167p or
known mammalian BAR adaptor proteins (Fig. 1b). The
similarity of BAR sequences and lack of non-BAR sequences suggest that
Rvs161p, BIN3, and Hob3p comprise a subfamily within the family of BAR
adaptor proteins. The other BAR adaptor protein encoded by the S. pombe genome was identified. The structure and characterization of
this predicted protein, which was most similar to Rvs167p, will be
described elsewhere.2
hob3 hob3 hob3 hob3 F-actin Localization Defects in hob3 hob3+, but Not BIN3, Complements the Osmotic Sensitivity of S. cerevisiae rvs161 While the exact role of the N-terminal fold of the BAR family
proteins is unknown, it is apparent that BAR family proteins are
nonredundant in function. However, the BAR family may be subdivided based on structural considerations. Thus, a subset of BAR family members contain a C-terminal SH3 domain. In some cases, as for the
BIN1-binding c-Abl oncoprotein, the protein partner responsible for
interaction is known (19). Another subset of BAR family members contain
domains known to be involved in binding components of the endocytotic
machinery and vesiculation (14, 22, 50). Other domains, such as the
c-Myc-binding domain of BIN1 (17), are unique within the BAR family.
The proteins described in the present study are characterized by a lack
of identifiable functional domains outside of the BAR N-terminal fold.
In combination with the greater homology exhibited between members of
this subset and their ability to cross-complement, this suggests to us
that they form a bona fide subfamily within the BAR family
of proteins. The inability of other BAR-containing proteins to
complement defects in the expression of these proteins, even in the
case of proteins native to the same organism, supports this notion
(48).2 While it is known that some members of the BAR
family are able to interact, such as the yeast Rvs161p and Rvs167p
proteins (7, 51), BIN1 and BIN2 (16), and amphiphysin and the brain
isoform of BIN1 (22), the possibility of homo- or heterotypic
interactions between other BAR family members remains to be determined.
As the case with RVS161 in budding yeast, the phenotype
caused by hob3+ deletion in fission yeast was linked to
cytoskeletal actin regulation. The presence of multiple
calcofluor-reactive primary septa in hob3 The mislocalization of F-actin patches by hob3 We observed cross-species complementation of the hob3 mutants had F-actin patches distributed randomly
around the cell. In addition, medial F-actin rings were rarely found in
hob3
mutants. Notably, in contrast to S. cerevisiae rvs161
mutants, hob3
mutants showed
no measurable defects in endocytosis or response to osmotic stress, yet
hob3+ complemented the osmosensitivity of a
rvs161
mutant. BIN3 failed to rescue the
osmosensitivity of rvs161
, but the actin localization defects of hob3
mutants were completely rescued by
BIN3 and partially rescued by RVS161. These
findings suggest that hob3+ and BIN3 regulate
F-actin localization, like RVS161, but that other roles for
this gene have diverged somewhat during evolution.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mutants revealed an important role for
Hob3p in regulation of F-actin localization, as was found for Rvs161p.
The F-actin localization defect of hob3
mutants was
completely rescued by human BIN3 and partially rescued by
RVS161, raising the possibility that BIN3 regulates F-actin localization in mammalian cells.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
::kanMX6) is a derivative of diploid
strain KGY246/249 (37), obtained by one-step gene disruption.
Integration of the altered allele by homologous recombination was
verified by Southern blotting using a 32P-labeled PCR
product derived from the 5'-untranslated region of the hob3+
locus, extending from 280 to 986 base pairs from the putative start
codon of hob3+ (38). The probe was labeled with the High
Prime DNA Labeling Kit (Roche Molecular Biochemicals) and
[
-32P]dCTP (PerkinElmer Life Sciences). S. cerevisiae strains BY4741 (MATa, ura3, leu2, his3,
met15) and BY4741-3489 (MATa,
ura3, leu2, his3, met15,
rvs161
::kanR) were obtained from J. Toyn,
Applied Biotechnology Group, DuPont Pharmaceuticals Company. S. pombe strains were grown in YE medium or EMM2 containing
appropriate nutritional supplements when necessary (39). Expression
from pREP2 plasmids was achieved by growing cells to early log phase in
medium containing 0.06 mM thiamine, washing the cells 3 times in thiamine-free medium, and resuspending the cells in the same medium. Budding yeast were grown in YPAD or SC medium lacking the
appropriate nutritional supplements, in some cases with the addition of
6% (w/v) NaCl (40).
-tubulin probe to normalize the blot.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BIN3 and Hob3p are members of the BAR
family. a, alignment of BIN3 with S. pombe Hob3p and S. cerevisiae Rvs161p. Identical
residues in all sequences are contained in black boxes,
while residues conserved between at least 50% of sequences are shown
enclosed by gray boxes. b, cartoon
depicting known BAR family proteins. The N-terminal BAR fold is shown
in blue. Domains that interact with c-Myc are in
gray, while those implicated in endocytosis are in
red. SH3 domains are depicted in yellow.
Relationships between known BAR-encoding polypeptides
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Fig. 2.
Distribution of mammalian BAR mRNAs in
normal tissues and tumors. a, multiple tissue Northern
blots probed for BIN1 mRNA (top panel),
BIN3 mRNA (middle panel), and AMPH mRNA
(lower panel). b, BIN3 mRNA
expression in tumor cell lines (upper panel). The membrane
was stripped and reprobed for -tubulin mRNA as a loading control
(lower panel). WM164 and WM1341D were derived from
metastatic melanoma, while C33A, MS751, SiHa, and HeLa were isolated
from cervical carcinomas. A549 is a lung carcinoma line, while HepG2
originates from hepatocellular carcinoma. The C2C12 cell line is an
undifferentiated mouse myoblast line.
Mutants Have a Cell Division Defect--
We began by
studying S. pombe hob3+ since fission yeast genetics allowed
us to rapidly characterize Hob3p function. Using standard methods,
haploid S. pombe strains were made where the entire coding
region of hob3+ was replaced with the kanMX6
cassette, conferring resistance to G418 (34). Southern analysis
confirmed construction of a strain with the hob3
allele
(Fig. 3a). Examination of
hob3
mutants revealed a fraction of cells that were
longer than hob3+ cells and contained more than two nuclei
(Fig. 3b). In particular, about 9% of hob3
cells from an actively growing culture contained more than two nuclei,
with most of these elongated cells containing four nuclei
(n = 108). In contrast, no cells with more than two
nuclei were observed in a parallel culture of hob3+ cells
(n = 125). Calcofluor staining showed that septal material separated most of the nuclei in hob3
cells (Fig.
3b). Furthermore, the hob3
cells contained
increased amounts of calcofluor-stained material, relative to
hob3+ cells (Fig. 3b). Consistent with their multinucleated phenotype, exponentially growing hob3
cells exhibited a continuum of >4N ploidies when analyzed by flow
cytometry (data not shown). The division of cell populations based on
nuclear content by flow cytometry of hob3
and
hob3+ cells mirrored results obtained by microscopic
observation. Both hob3+ and hob3
null cells
grew and mated with kinetics indistinguishable from wild type cells;
however, hob3
cells tended to grow in clumps of 5-15 cells (data not shown) and stopped dividing at lower cell density than
hob3+ cells (Fig. 3c). Overexpression of
hob3+ had no effect on hob3+ cells (data not
shown). Hence, hob3+ had a role in cell division in fission
yeast at the level of septation, with some proportion of cells failing
to separate following septum formation and accumulating increased
levels of septal material.
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Fig. 3.
Generation of a fission yeast strain
harboring a deletion of the mammalian BIN3 homolog.
a, Southern blot of BamHI-digested
genomic DNA from haploid hob3+ (right lane) and
hob3 (left lane). b,
morphology of the hob3
strain. Exponentially growing
cells were stained with calcofluor and DAPI. c, growth
kinetics and viability of hob3
null cells. Graph of cell
density and viability versus time for exponential phase
cells inoculated into YE medium. Cell density was determined by
counting appropriate dilutions of cells with the aid of a
hemocytometer. Viability was determined by a colony formation assay on
solid YE medium. All measurements were obtained in triplicate.
Mutants Frequently Mislocalize F-actin--
S.
cerevisiae rvs161
mutants were defective in F-actin
localization (3, 6, 43). Since S. pombe mutants with F-actin localization defects disrupt septation, cytokinesis, and cell separation (44-46), F-actin localization in hob3
mutants
was determined using fluorescent phalloidin. In hob3+
S. pombe, F-actin was normally localized to cortical patches
during interphase and medial contractile rings during mitosis (Fig.
4) (41). In contrast, hob3
mutants displayed two significant F-actin localization defects (Fig. 4, Table II). First, F-actin patches were
frequently mislocalized in hob3
cells with one nucleus.
In particular, F-actin patches were found equally distributed along the
entire length of mononuclear cells, with 86% of such mononuclear cells
(n = 88) exhibiting this staining pattern
versus 4% of cells from a hob3+ strain
(n = 115). Second, medial F-actin rings and patches
were rarely observed in hob3
mutants with two nuclei. In
hob3
mutant cells with 2 nuclei, 41% had medial F-actin
patches in both compartments with an F-actin ring, 32% had delocalized
F-actin patches in both compartments, and 27% had medial F-actin
patches in one compartment with delocalized F-actin patches in the
other compartment (n = 22). These findings contrast
with hob3+ cells where 100% of cells containing two nuclei exhibited medial F-actin staining (n = 29). Hence,
Hob3p plays an important role in the localization of F-actin in
interphase and mitotic cells. Consistent with loss of cell polarity and
consequent abnormally shaped cells observed in mutants of the S. pombe F-actin-encoding act1+ gene (47), we observed
such misshapen cells in hob3
cultures (Fig. 4).
Specifically, 13% of cells (n = 116) in Fig. 4 lost their cylindrical appearance and took on a rounded appearance, versus 0% of cells in a matched hob3+ culture
(n = 163). Loss of shape was not limited to mononuclear
cells; 48% of misshapen cells possessed two or more nuclei. F-actin
delocalization was observed in all misshapen cells. We concluded that
hob3+ was necessary for F-actin regulation and completion of
septation, the process of cytokinesis in fission yeast cells.
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Fig. 4.
Deletion of hob3+ results in
F-actin localization and septation defects. Exponentially growing
S. pombe cells were fixed and stained with AlexaFluor
488-phalloidin to visualize polymerized actin (top panels).
DNA was stained with DAPI (bottom panels). hob3+
cells are depicted in the left panels, while
hob3 cells are shown in the right panels. Hazy
shading observed in some areas of the figures are due to the presence
of cells in a different focal plane with respect to the majority of
cells. Note that this effect is more pronounced in the
hob3
strain due to the propensity of these cells to grow
in clumps. Insets, smaller fields were magnified to
highlight differences in F-actin staining. Arrows point to
medial F-actin patches and rings. Asterisks indicate
mislocalized F-actin patches.
Quantification of F-actin distribution in hob3 mutants expressing
BAR-containing polypeptides
Mutants Respond Normally to Nutrient and Osmotic
Stress--
RVS161 was discovered in a screen for mutants that
had reduced viability upon starvation for glucose, nitrogen, or sulfur (1). In these experiments, rvs161
mutants had a 35%
reduction in cell viability after 48 h in N005 low nitrogen
medium. Further analysis revealed that rvs161
mutants
showed dramatic morphologic changes in response to high salt and low
nitrogen media, and more significant reductions in cell growth when
shifted to media with high salt (48). Based on these results, the
response of hob3
mutants to nutrient and osmotic stress
was tested. This analysis revealed that hob3
mutant cells
were relatively insensitive to lack of nitrogen or elevated/decreased
temperature, as assayed by growth on plates (Fig.
5). Furthermore, microscopic inspection of hob3
mutant cells following temperature, osmotic, or
nutrient shift did not reveal detectable differences in cell morphology (data not shown). Based on these results, we concluded that
hob3
mutants, unlike rvs161
mutants,
respond like hob3+ cells to changes in temperature,
osmolarity, and nutrients.
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Fig. 5.
hob3 null cells do
not display reduced viability upon starvation. Effect of
temperature and lack of nitrogen (EMMG) on growth on solid medium.
Exponentially growing cells of each strain from liquid YE cultures were
counted in triplicate. 1 × 104 to 1 × 101 cells of each strain were inoculated onto the indicated
medium in 10-fold dilutions from left to right, and the plates were
allowed to grow at the indicated temperature until colonies formed
(3-12 days).
Mutants Undergo Normal Fluid-phase
Endocytosis--
rvs161
mutants were defective in
fluid-phase and receptor-mediated endocytosis (2, 8). To measure the
rate of fluid-phase endocytosis in hob3
mutants, the
ability of cells to accumulate FM4-64 was quantified. FM4-64 is a
fluorescent lipophilic styryl dye which specifically accumulates in
vacuolar membranes of both budding and fission yeasts (42, 49). When
added to cells, FM4-64 initially stained the plasma membrane (Fig.
6). Within 15 min, FM4-64 internalized at
the cell ends in presumed endocytic vesicles (Fig. 6). During the next
60 min, the number of FM4-64 staining structures decreased to 2-3 per
cell and their size increased. Based on published data, these final
structures were vacuoles. A comparison of hob3+ and
hob3
mutants at several times did not reveal any
detectable differences in the kinetics or morphology of the FM4-64
staining structures. Based on these results, we conclude that
fluid-phase endocytosis was normal in hob3
mutants.
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Fig. 6.
hob3 null cells lack
gross endocytotic defects. FM4-64 uptake assay of exponentially
growing cells. Cells were incubated at room temperature in the presence
of FM4-64 and photographed at the indicated times.
Mutants Are Completely
Rescued by BIN3 and Partially Rescued by RVS161--
The structural
similarities between Hob3p, BIN3, and Rvs161p as well as the functional
similarities between rvs161
mutants and
hob3
mutants, suggested that Hob3p, BIN3, and Rvs161p
share common functions. To test this hypothesis, we determined whether ectopic expression of BIN3 and Rvs161 could
rescue the defects of hob3
mutants. For this purpose,
plasmids were constructed where hob3+, BIN3,
RVS161, and RVS167 were expressed using the thiamine-repressible nmt1 promoter of S. pombe
(33). These plasmids or a control plasmid were then introduced into
hob3
mutants and the fraction of elongated cells and
F-actin staining patterns were quantified (Table II, Fig.
7). As expected, ectopic expression of
hob3+ complemented the cell elongation and F-actin defects in hob3
cells whereas the control vector had no effect.
Interestingly, BIN3 expression also corrected the cell
elongation and F-actin defects of hob3
mutants while
RVS161 expression partially corrected the defects of
hob3
mutants. In particular, hob3
mutant
cells expressing RVS161 were not elongated and contained
easily detectable medial F-actin in mitotic cells. RVS161
expression failed, however, to correct the mislocalization of F-actin
patches in hob3
mutants. Rescue of hob3
mutants by BIN3 and RVS161 was specific for these BAR adaptor proteins since RVS167 expression did not correct
the defects of hob3
mutants. We conclude that
BIN3 and RVS161, but not RVS167, at
least partially rescue the F-actin localization defects of
hob3
mutants arguing that these proteins can perform similar functions.
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Fig. 7.
Ectopic expression of Bin3 and Rvs161p
complements the F-actin defect of a hob3
null mutant. A hob3
null strain was
transformed with pREP2-based vectors expressing BAR family proteins.
Exponentially growing transformants were stained with AlexaFluor
488-phalloidin to visualize polymerized actin. pREP2 is the empty
expression vector. pREP2-hob3+, pREP2-RVS161, pREP2-BIN3,
and pREP2-RVS167 encode the hob3+, RVS161,
BIN3, and RVS167 cDNAs, respectively.
Contractile rings are indicated by arrows. Note, delocalized
cortical actin patches aligned along the length of the cells denoted by
asterisks.
Mutants--
Since both RVS161 and
BIN3 complemented the F-actin localization defects of
hob3
mutants, we tested whether expression of BAR-containing proteins could rescue a S. cerevisiae
rvs161
mutant. Complementation was tested by the ability of a
rvs161
strain to grow on synthetic dropout medium
containing 6% NaCl. As expected, rvs161
cells lacking a
plasmid or containing a control plasmid failed to grow on media with
6% NaCl (Fig. 8). In contrast,
rvs161
mutants expressing RVS161 or
hob3+, but not BIN3 or BIN1, grew similarly to RVS161 cells (Fig. 8). All strains which
received a plasmid grew on synthetic dropout medium lacking 6% NaCl
(data not shown). We conclude that, due to the lesser divergence
between the RVS161 and Hob3p proteins, Hob3p was able to complement the osmolarity defect of rvs161
null cells, but that the
greater extent to which Rvs161p and BIN3 have diverged precluded
complementation by BIN3.
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Fig. 8.
The high salt intolerance of
rvs161 can be complemented by ectopic
expression of the fission yeast homolog hob3+.
RVS161 strain BY4741 and rvs161
strain BY4741-3489 were
transformed with the indicated plasmids. Transformants were streaked
onto synthetic uracil dropout medium containing 6% NaCl and incubated
at 30 °C for 7 days. Designations following YEp195-ACN indicate the
identity of the encoded BAR protein.
DISCUSSION
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ABSTRACT
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mutant cells
coupled with the observation of an actin localization defect is
reminiscent of the phenotypes exhibited by known cell separation and
actin regulatory mutants of S. pombe. Thus, inactivating
mutations of the sep2+, sep12+, spn1+, and
rlc1+ genes of S. pombe result in linear,
multiseptated cells, in most cases with increased deposition of septal
material (52-55). sep2+ was identified in a screen for
mutants with increased resistance to lysing enzymes, while
sep12+ was isolated by application of the diploid enrichment
screen of Chang et al. (56). It is worth noting that a
fraction of sep2 cells contained double septa, which yielded
two daughter cells and an anucleate minicell upon cleavage. Neither
double septa nor anucleate cells were observed in cultures of
hob3
. It was shown that cultures of sep12
mutant cells contained 64% hyphae, a much larger percentage than the typical 10-15% observed in hob3
cultures. In addition,
sep12 cells were sterile, while hob3
mated
with normal kinetics. spn1+ is a member of the S. pombe septin family of proteins. As such, it has a role in
promoting septation of fission yeast. However, a role in actin patch
movement has not been predicted. rlc1+ encodes a myosin
regulatory light chain which associates with the yeast Myo2p and Myo3p
gene products. Although the morphology of hob3
cells
closely resembled that of rlc1 mutant cells, the latter were
found to be cold-sensitive for growth, a condition not seen in
hob3
cultures. Interestingly, Rvs161p, Hob3p, and BIN3
are homologous to unconventional myosins; Rvs161p is 25% identical to
Myo1p, the sole type II unconventional myosin of budding yeast; Hob3p
is 20% identical to Myo2p, one of two myosins in S. pombe, and BIN3 is 24% identical to human type VI unconventional myosin. BLAST analysis of the BIN3 protein assigns unconventional type VI
myosins as the most highly homologous non-BAR polypeptides.
null cells
has been previously observed in mutants of the Arp2/3 complex of S. pombe, as well as in mutants of other, known
actin-interacting proteins such as the products of the cdc3+
and cdc8+ genes, which encode profilin and tropomyosin,
respectively (44, 45, 57, 58). However, these mutants do not display
the linear, multiseptated morphology characteristic of
hob3
cells. In addition, loss of Cdc3p, Cdc8p, or Arp3p
function is lethal, whereas loss of Hob3p is not. Given that the major
defect in these mutants is probably actin-related, and that
perturbations of actin organization generally result in gross
morphological defects throughout the cell cycle, it is not surprising
that the hob3+ gene is not essential, nor do
hob3
cells exhibit the profound morphological
abnormalities observed in more severe cases of loss of actin
organization, such as in the cdc3 mutant (58). Nevertheless,
a defect in F-actin patch movement is apparent in hob3
cells. It remains to be determined whether Hob3p is directly associated
with actin or with actin-binding proteins such as profilin,
tropomyosin, or the Arp2/3 complex.
F-actin defect by the budding yeast homolog Rvs161p, and by the human homolog BIN3. A partial rescue of the F-actin localization defect by
Rvs161p was observed insofar as the majority of dividing cells regained
medial F-actin staining, but failed to correctly localize F-actin
patches during interphase. On the other hand, BIN3 was able to rescue
both loss of medial F-actin and localization of F-actin to cortical
patches during interphase. It was noted that budding yeast Rvs167p,
which is not a member of the subfamily of BAR proteins defined by
Rvs161p, Hob3p, and BIN3, was unable to rescue the F-actin defect of
hob3
cells. An alternative explanation for the partial
complementation observed with Rvs161p and the lack of complementation
seen in the case of Rvs167p could be due to decreased steady-state
levels of these proteins in S. pombe. However, we have
confirmed the presence of either BIN1 or BIN3 polypeptides in
hob3+ and hob3
S. pombe strains
transformed with pREP2-based expression vectors. Furthermore, we were
able to ascertain that the resulting BIN1 polypeptide failed to correct
the F-actin defect observed in hob3
cells (data not
shown). We thus favor the interpretation that BIN3 is the human homolog
of Rvs161p and Hob3p, but that there exists a degree of divergence in
this gene during evolution. For example, BIN3 and Hob3p share important roles with Rvs161p in the control of the actin cytoskeleton, but only
Rvs161p exhibits a role in endocytosis, and only Hob3p exhibits a role
in cell division. Despite the lack of any role in cell division in
budding yeast, RVS161 complemented the defects in this process caused
by hob3
gene deletion as well as BIN3. In support of the
notion of some evolutionary drift in the function of this gene during
evolution, BIN3 was found to exhibit a unique localization in human
cells to mitochondria and Golgi rather than to sites of actin
polymerization as in the case of Rvs161p and Hob3p in budding and
fission yeasts.3 Thus, a
major finding of our study is that while BIN3 is clearly homologous to
yeast Rvs161p and Hob3p at some levels, it is also clear that the
function of this BAR adaptor protein has diverged to some extent and/or
is being utilized differently in cells during evolution. Further
insights into the exact mechanistic role of BIN3 in the cell
division processes will require studies in mouse cells in which the
Bin3 gene has been targeted for homozygous deletion.
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ACKNOWLEDGEMENTS |
---|
We thank P. Scott Donover for excellent technical assistance and A. Muller, G. Farmer, W. Du, and K. Prendergast for critical comments.
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FOOTNOTES |
---|
* This work was supported in part by The Wistar Institute and United States Army Breast and Prostate Cancer Research Programs Grants DAMD17-96-1-6324 and PC970326 (to G. C. P.).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.
The nucleotide sequences reported in this paper have been submitted to the GenBankTM/EBI Data Bank with accession numbers AF2717232 (human BIN3 cDNA), AA418871 (human BIN3 EST), AAF76218 (human Bin3 protein), AF271733 (murine Bin3 cDNA), AF275638 (S. pombe hob3+ cDNA), and AAF86459 (S. pombe Hob3p).
The amino acid sequence alignment in Fig. 1 including the budding yeast protein Rvs161p has been submitted to the Swiss Protein Database under Swiss-Prot accession number 25343.
To whom correspondence should be addressed. Tel.:
610-237-7847; Fax: 610-237-7937; E-mail:
george.c.prendergast@dupontpharma.com.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M101096200
2 E. L. Routhier, C. F. Albright, and G. C. Pendergast, manuscript in preparation.
3 J. B. DuHadaway and G. C. Prendergast, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: BAR, Bin/amphiphysin/Rvs; BIN, bridging integrators; EST, expressed sequence tag; PCR, polymerase chain reaction; YE, yeast extract medium; EMM2, Edinburgh minimal medium; EMM2-N, Edinburgh minimal medium without NH4Cl; EMMG, Edinburgh minimal medium without NH4Cl, with glutamate; YPAD, yeast complete medium with adenine; SC, synthetic complete yeast medium; AMPH, amphiphysin; DAPI, 4',6'-diamidino-2-phenylindole.
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