Human BIN3 Complements the F-actin Localization Defects Caused by Loss of Hob3p, the Fission Yeast Homolog of Rvs161p*

Eric L. RouthierDagger , Timothy C. Burn§, Ilgar Abbaszade§, Matthew Summers, Charles F. AlbrightDagger , and George C. PrendergastDagger ||

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, hob3Delta mutants had F-actin patches distributed randomly around the cell. In addition, medial F-actin rings were rarely found in hob3Delta mutants. Notably, in contrast to S. cerevisiae rvs161Delta mutants, hob3Delta mutants showed no measurable defects in endocytosis or response to osmotic stress, yet hob3+ complemented the osmosensitivity of a rvs161Delta mutant. BIN3 failed to rescue the osmosensitivity of rvs161Delta , but the actin localization defects of hob3Delta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 hob3Delta mutants revealed an important role for Hob3p in regulation of F-actin localization, as was found for Rvs161p. The F-actin localization defect of hob3Delta 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

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, hob3Delta ::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 [alpha -32P]dCTP (PerkinElmer Life Sciences). S. cerevisiae strains BY4741 (MATa, ura3, leu2, his3, met15) and BY4741-3489 (MATa, ura3, leu2, his3, met15, rvs161Delta ::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).

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 beta -tubulin probe to normalize the blot.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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.

                              
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Table I
Relationships between known BAR-encoding polypeptides
The BAR domains of known BAR-containing polypeptides were aligned using the GCG program GAP (59) and the percentage of identical residues were calculated for each pairwise combination. Polypeptides were truncated to 253 N-terminal amino acids for comparison.


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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 beta -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.

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

hob3Delta 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 hob3Delta allele (Fig. 3a). Examination of hob3Delta 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 hob3Delta 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 hob3Delta cells (Fig. 3b). Furthermore, the hob3Delta cells contained increased amounts of calcofluor-stained material, relative to hob3+ cells (Fig. 3b). Consistent with their multinucleated phenotype, exponentially growing hob3Delta 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 hob3Delta and hob3+ cells mirrored results obtained by microscopic observation. Both hob3+ and hob3Delta null cells grew and mated with kinetics indistinguishable from wild type cells; however, hob3Delta 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 hob3Delta (left lane). b, morphology of the hob3Delta strain. Exponentially growing cells were stained with calcofluor and DAPI. c, growth kinetics and viability of hob3Delta 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.

hob3Delta Mutants Frequently Mislocalize F-actin-- S. cerevisiae rvs161Delta 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 hob3Delta 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, hob3Delta mutants displayed two significant F-actin localization defects (Fig. 4, Table II). First, F-actin patches were frequently mislocalized in hob3Delta 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 hob3Delta mutants with two nuclei. In hob3Delta 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 hob3Delta 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 hob3Delta 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 hob3Delta 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.

                              
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Table II
Quantification of F-actin distribution in hob3Delta mutants expressing BAR-containing polypeptides
Cells were grown to exponential phase in the medium indicated, fixed, and stained with AlexaFluor 488-phalloidin and DAPI. Cells were counted and distributed according to their nuclear content and F-actin staining pattern. The number (n) of cells analyzed is indicated in parentheses.

hob3Delta 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, rvs161Delta mutants had a 35% reduction in cell viability after 48 h in N005 low nitrogen medium. Further analysis revealed that rvs161Delta 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 hob3Delta mutants to nutrient and osmotic stress was tested. This analysis revealed that hob3Delta 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 hob3Delta 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 hob3Delta mutants, unlike rvs161Delta mutants, respond like hob3+ cells to changes in temperature, osmolarity, and nutrients.


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Fig. 5.   hob3Delta 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).

hob3Delta Mutants Undergo Normal Fluid-phase Endocytosis-- rvs161Delta mutants were defective in fluid-phase and receptor-mediated endocytosis (2, 8). To measure the rate of fluid-phase endocytosis in hob3Delta 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 hob3Delta 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 hob3Delta mutants.


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Fig. 6.   hob3Delta 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.

F-actin Localization Defects in hob3Delta 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 rvs161Delta mutants and hob3Delta 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 hob3Delta 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 hob3Delta 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 hob3Delta cells whereas the control vector had no effect. Interestingly, BIN3 expression also corrected the cell elongation and F-actin defects of hob3Delta mutants while RVS161 expression partially corrected the defects of hob3Delta mutants. In particular, hob3Delta 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 hob3Delta mutants. Rescue of hob3Delta mutants by BIN3 and RVS161 was specific for these BAR adaptor proteins since RVS167 expression did not correct the defects of hob3Delta mutants. We conclude that BIN3 and RVS161, but not RVS167, at least partially rescue the F-actin localization defects of hob3Delta 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 hob3Delta null mutant. A hob3Delta 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.

hob3+, but Not BIN3, Complements the Osmotic Sensitivity of S. cerevisiae rvs161Delta Mutants-- Since both RVS161 and BIN3 complemented the F-actin localization defects of hob3Delta mutants, we tested whether expression of BAR-containing proteins could rescue a S. cerevisiae rvs161Delta mutant. Complementation was tested by the ability of a rvs161Delta strain to grow on synthetic dropout medium containing 6% NaCl. As expected, rvs161Delta cells lacking a plasmid or containing a control plasmid failed to grow on media with 6% NaCl (Fig. 8). In contrast, rvs161Delta 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 rvs161Delta 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 rvs161Delta can be complemented by ectopic expression of the fission yeast homolog hob3+. RVS161 strain BY4741 and rvs161Delta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 hob3Delta 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 hob3Delta . It was shown that cultures of sep12 mutant cells contained 64% hyphae, a much larger percentage than the typical 10-15% observed in hob3Delta cultures. In addition, sep12 cells were sterile, while hob3Delta 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 hob3Delta cells closely resembled that of rlc1 mutant cells, the latter were found to be cold-sensitive for growth, a condition not seen in hob3Delta 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.

The mislocalization of F-actin patches by hob3Delta 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 hob3Delta 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 hob3Delta 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 hob3Delta 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.

We observed cross-species complementation of the hob3Delta 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 hob3Delta 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 hob3Delta 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 hob3Delta 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 hob3Delta 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.

    ACKNOWLEDGEMENTS

We thank P. Scott Donover for excellent technical assistance and A. Muller, G. Farmer, W. Du, and K. Prendergast for critical comments.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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