Roles of the fission yeast UNC-13/Munc13 protein Ync13 in late stages of cytokinesis.

Cytokinesis is a complicated yet conserved step of the cell-division cycle that requires the coordination of multiple proteins and cellular processes. Here we describe a previously uncharacterized protein, Ync13, and its roles during fission yeast cytokinesis. Ync13 is a member of the UNC-13/Munc13 protein family, whose animal homologues are essential priming factors for soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly during exocytosis in various cell types, but no roles in cytokinesis have been reported. We find that Ync13 binds to lipids in vitro and dynamically localizes to the plasma membrane at cell tips during interphase and at the division site during cytokinesis. Deletion of Ync13 leads to defective septation and exocytosis, uneven distribution of cell-wall enzymes and components of cell-wall integrity pathway along the division site and massive cell lysis during cell separation. Interestingly, loss of Ync13 compromises endocytic site selection at the division plane. Collectively, we find that Ync13 has a novel function as an UNC-13/Munc13 protein in coordinating exocytosis, endocytosis, and cell-wall integrity during fission yeast cytokinesis.

Self-association of the Gal4 inhibitor protein Gal80 is impaired by Gal3: evidence for a new mechanism in the GAL gene switch.

The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to GAL gene UASGAL (upstream activation sequence in GAL gene promoter) sites as a dimer via its N-terminal domain and activates transcription via a C-terminal transcription activation domain (AD). In the absence of galactose, a Gal80 dimer binds to a dimer of Gal4, masking the Gal4AD. Galactose triggers Gal3-Gal80 interaction to rapidly initiate Gal4-mediated transcription activation. Just how Gal3 alters Gal80 to relieve Gal80 inhibition of Gal4 has been unknown, but previous analyses of Gal80 mutants suggested a possible competition between Gal3-Gal80 and Gal80 self-association interactions. Here we assayed Gal80-Gal80 interactions and tested for effects of Gal3. Immunoprecipitation, cross-linking, and denaturing and native PAGE analyses of Gal80 in vitro and fluorescence imaging of Gal80 in live cells show that Gal3-Gal80 interaction occurs concomitantly with a decrease in Gal80 multimers. Consistent with this, we find that newly discovered nuclear clusters of Gal80 dissipate in response to galactose-triggered Gal3-Gal80 interaction. We discuss the effect of Gal3 on the quaternary structure of Gal80 in light of the evidence pointing to multimeric Gal80 as the form required to inhibit Gal4.

Rapid GAL gene switch of Saccharomyces cerevisiae depends on nuclear Gal3, not nucleocytoplasmic trafficking of Gal3 and Gal80.

The yeast transcriptional activator Gal4 localizes to UAS(GAL) sites even in the absence of galactose but cannot activate transcription due to an association with the Gal80 protein. By 4 min after galactose addition, Gal4-activated gene transcription ensues. It is well established that this rapid induction arises through a galactose-triggered association between the Gal80 and Gal3 proteins that decreases the association of Gal80 and Gal4. How this happens mechanistically remains unclear. Strikingly different hypotheses prevail concerning the possible roles of nucleocytoplasmic distribution and trafficking of Gal3 and Gal80 and where in the cell the initial Gal3-Gal80 association occurs. Here we tested two conflicting hypotheses by evaluating the subcellular distribution and dynamics of Gal3 and Gal80 with reference to induction kinetics. We determined that the rates of nucleocytoplasmic trafficking for both Gal80 and Gal3 are slow relative to the rate of induction. We find that depletion of the nuclear pool of Gal3 slows the induction kinetics. Thus, nuclear Gal3 is critical for rapid induction. Fluorescence-recovery-after-photobleaching experiments provided data suggesting that the Gal80-Gal4 complex exhibits kinetic stability in the absence of galactose. Finally, we detect Gal3 at the UAS(GAL) only if Gal80 is covalently linked to the DNA-binding domain. Taken altogether, these new findings lead us to propose that a transient interaction of Gal3 with Gal4-associated Gal80 could explain the rapid response of this system. This notion could also explain earlier observations.

Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge.

Many recombinant proteins carry an oligohistidine (His(X))-tag that allows their purification by immobilized metal affinity chromatography (IMAC). This tag can be exploited for the site-specific attachment of chromophores and fluorophores, using the same metal ion-nitrilotriacetic acid (NTA) coordination chemistry that forms the basis of popular versions of IMAC. Labeling proteins in this way can allow their detection at wavelengths outside of the absorption envelopes of un-modified proteins and nucleic acids. Here we describe use of this technology in tracer sedimentation experiments that can be performed in a standard analytical ultracentrifuge equipped with absorbance or fluorescence optics. Examples include sedimentation velocity in the presence of low molecular weight chromophoric solutes, sedimentation equilibrium in the presence of high concentrations of background protein and selective labeling to simplify the assignment of species in a complex interacting mixture.

Gene activation by dissociation of an inhibitor from a transcriptional activation domain.

Gal4 is a prototypical eukaryotic transcriptional activator whose recruitment function is inhibited in the absence of galactose by the Gal80 protein through masking of its transcriptional activation domain (AD). A long-standing nondissociation model posits that galactose-activated Gal3 interacts with Gal4-bound Gal80 at the promoter, yielding a tripartite Gal3-Gal80-Gal4 complex with altered Gal80-Gal4 conformation to enable Gal4 AD activity. Some recent data challenge this model, whereas other recent data support the model. To address this controversy, we imaged fluorescent-protein-tagged Gal80, Gal4, and Gal3 in live cells containing a novel GAL gene array. We find that Gal80 rapidly dissociates from Gal4 in response to galactose. Importantly, this dissociation is Gal3 dependent and concurrent with Gal4-activated GAL gene expression. When galactose-triggered dissociation is followed by galactose depletion, preexisting Gal80 reassociates with Gal4, indicating that sequestration of Gal80 by Gal3 contributes to the observed Gal80-Gal4 dissociation. Moreover, the ratio of nuclear Gal80 to cytoplasmic Gal80 decreases in response to Gal80-Gal3 interaction. Taken together, these and other results provide strong support for a GAL gene switch model wherein Gal80 rapidly dissociates from Gal4 through a mechanism that involves sequestration of Gal80 by galactose-activated Gal3.

Genetic evidence for sites of interaction between the Gal3 and Gal80 proteins of the Saccharomyces cerevisiae GAL gene switch.

Galactose-activated transcription of the Saccharomyces cerevisiae GAL genes occurs when Gal3 binds the Gal4 inhibitor, Gal80. Noninteracting variants of Gal3 or Gal80 render the GAL genes noninducible. To identify the binding determinants for Gal3's interaction with Gal80 we carried out GAL3-GAL80 intergenic suppression analyses and selected for new GAL3 mutations that impair the Gal3-Gal80 interaction. We show that a GAL3(C)-D368V mutation can suppress the noninducibility due to a GAL80(S-1)-G323R mutation, and a GAL80-M350C mutation can suppress the noninducibility due to a gal3-D111C mutation. A reverse two-hybrid selection for GAL3 mutations that impair the Gal3-Gal80 interaction yielded 12 single-amino-acid substitutions at residues that are predicted to be surface exposed on Gal3. The majority of the affected Gal3 residues localized to a composite surface that includes D111 and a sequence motif containing D368, which has been implicated in interaction with Gal80. The striking colocalization of intergenic suppressor residues and Gal80 nonbinder residues identifies a Gal3 surface that likely interacts with Gal80.

Breast cancer metastasis suppressor 1 (BRMS1) is stabilized by the Hsp90 chaperone.

Breast cancer metastasis suppressor 1 (BRMS1) is a member of the mSin3-HDAC transcription co-repressor complex. However, the proteins associated with BRMS1 have not been fully identified. Yeast two-hybrid screen, immuno-affinity chromatography, and co-immunoprecipitation experiments were performed to identify BRMS1 interacting proteins (BIPs). In addition to known core mSin3 transcriptional complex components RBBP1 and mSDS3, BRMS1 interacted with other proteins including three chaperones: DNAJB6 (MRJ), Hsp90, and Hsp70. Hsp90 is a known target of HDAC6 and reversible acetylation is one of the mechanisms that is implicated in regulation of Hsp90 chaperone complex activity. BRMS1 interacted with class II HDACs, HDAC 4, 5, and 6. We further found that BRMS1 is stabilized by Hsp90, and its turnover is proteasome dependent. The stability of BRMS1 protein may be important in maintaining the functional role of BRMS1 in metastasis suppression.

Intragenic suppression of Gal3C interaction with Gal80 in the Saccharomyces cerevisiae GAL gene switch.

Gal4-mediated activation of GAL gene transcription in Saccharomyces cerevisiae requires the interaction of Gal3 with Gal80, the Gal4 inhibitor protein. While it is known that galactose and ATP activates Gal3 interaction with Gal80, neither the mechanism of activation nor the surface that binds to Gal80 is known. We addressed this through intragenic suppression of GAL3C alleles that cause galactose-independent Gal3-Gal80 interaction. We created a new allele, GAL3SOC, and showed that it suppressed a new GAL3C allele. We tested the effect of GAL3SOC on several newly isolated and existing GAL3C alleles that map throughout the gene. All except one GAL3C allele, D368V, were suppressible by GAL3SOC. GAL3SOC and all GAL3C alleles were localized on a Gal3 homology model that is based on the structure of the highly related Gal1 protein. These results provide evidence for allosterism in the galactose- and ATP-activation of Gal3 binding to Gal80. In addition, because D368V and residues corresponding to Gal80-nonbinder mutations colocalized to a domain that is absent in homologous proteins that do not bind to Gal80, we suggest that D368 is a part of the Gal80-binding surface.

Alpha V integrin prolongs collagenase production through Jun activation binding protein 1.

Robust expression of alphav integrin and matrix metalloproteinase 1 (MMP1) plays an important role in cancer metastasis and wound healing. A patient with an abnormal scar that appeared stretched and thinned out was found to have fibroblasts that overexpressed alphav integrin; therefore, a relationship between alphav integrin expression and MMP1 production was sought. A yeast 2 hybrid screen revealed alphav integrin interacts with jun activation binding domain-1 (JAB1). Mesenchymal-derived cells were transfected with the alphav integrin gene and incorporated into collagen lattices. Transfected cells maximally contracted collagen lattices beginning on day 5, whereas control transfected cells did not contract lattices. Late-phase collagen lattice contraction was inhibited by a pan-MMP inhibitor, BB4. Overexpression of alphav correlated with enhanced MMP1 transcription, as determined by a luciferase assay (P < or = 0.05). Diminution of JAB1 with JAB1 antisense abolished alphav integrin up-regulation of MMP1. We conclude alphav integrin signals through JAB1 to prolong MMP1 production and that this signaling pathway in fibroblasts may lead to abnormal scarring.

Self-association of the amino-terminal domain of the yeast TATA-binding protein.

The amino-terminal domain of yeast TATA-binding protein has been proposed to play a crucial role in the self-association mechanism(s) of the full-length protein. Here we tested the ability of this domain to self-associate under a variety of solution conditions. Escherichia coli two-hybrid assays, in vitro pull-down assays, and in vitro cross-linking provided qualitative evidence for a limited and specific self-association. Sedimentation equilibrium analysis using purified protein was consistent with a monomer-dimer equilibrium with an apparent dissociation constant of approximately 8.4 microM. Higher stoichiometry associations remain possible but could not be detected by any of these methods. These results demonstrate that the minimal structure necessary for amino-terminal domain self-association must be present even in the absence of carboxyl-terminal domain structures. On the basis of these results we propose that amino-terminal domain structures contribute to the oligomerization interface of the full-length yeast TATA-binding protein.

Breast cancer metastasis suppressor 1 (BRMS1) forms complexes with retinoblastoma-binding protein 1 (RBP1) and the mSin3 histone deacetylase complex and represses transcription.

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple human and murine cancer cells without inhibiting tumorigenicity. By yeast two-hybrid and co-immunoprecipitation, BRMS1 interacts with retinoblastoma binding protein 1 and at least seven members of the mSin3 histone deacetylase (HDAC) complex in human breast and melanoma cell lines. BRMS1 co-immunoprecipitates enzymatically active HDAC proteins and represses transcription when recruited to a Gal4 promoter in vivo. BRMS1 exists in large mSin3 complex(es) of approximately 1.4-1.9 MDa, but also forms smaller complexes with HDAC1. Deletion analyses show that the carboxyl-terminal 42 amino acids of BRMS1 are not critical for interaction with much of the mSin3 complex and that BRMS1 appears to have more than one binding point to the complex. These results further show that BRMS1 may participate in transcriptional regulation via interaction with the mSin3.HDAC complex and suggest a novel mechanism by which BRMS1 might suppress cancer metastasis.

Overexpression of integrin alphav promotes human osteosarcoma cell populated collagen lattice contraction and cell migration.

Cells attach and interact with the extracellular matrix (ECM) through heterodimeric alphabeta integrin receptors. Specifically, the promiscuous alphavbeta3 integrin and the alpha2beta1 integrin receptors engage numerous matrix components to influence cell adhesion, cell motility, and matrix organization. However, the role of alphav integrin mediating cell-collagen interactions is not clear. In the in vitro cell populated collagen lattice (PCL), a model of cell-matrix interaction, integrin receptors play a role in lattice contraction. To elucidate alphav integrins' effects on cell-collagen interactions, human osteosarcoma (HOS) cells were transfected with alphav integrin (alphav-pcDNA 3.1+). Control HOS cells were transfected with pcDNA 3.1+ vector alone. HOS-alphav cell PCLs contracted to a greater degree than control HOS cell PCLs (P < or = 0.0001). RT-PCR revealed that HOS-alphav cells express both beta1 and beta3 integrins, indicating that alphav has the potential to form a partnership with either beta1 or beta3 integrin. The alphavbeta3 specific inhibitory antibody LM609 significantly retarded HOS-alphav cell PCL contraction (P < or = 0.001), suggesting that alphavbeta3 promotes enhanced HOS-alphav cell PCL contraction. When plated on plastic, control HOS cells show greater elongation compared to HOS-alphav cells. In addition, HOS-alphav cells migrated faster and to a greater degree than control HOS cells (P < or = 0.0001). The possibility that enhanced HOS-alphav cell migration and HOS-alphav cell PCL contraction was caused by increased myosin ATPase activity was examined. HOS-alphav cells showed less myosin ATPase activity than control HOS cells, by an ATP cell contraction bioassay. The enhancement of HOS-alphav cell migration and lattice contraction appears unrelated to increased myosin ATPase activity.

Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein.

Galactose-inducible genes (GAL genes) in yeast Saccharomyces cerevisiae are efficiently transcribed only when the sequence-specific transcription activator Gal4p is activated. Activation of Gal4p requires the interaction between the Gal4p inhibitory protein Gal80p and the galactokinase paralog, Gal3p. It has been proposed that Gal3p binds to a Gal80p-Gal4p complex in the nucleus to activate Gal4p. Here, we present evidence that the Gal3p-Gal80p interaction occurs in the cytoplasm, and concurrently, Gal80p is removed from Gal4p at the GAL gene promoter. We also show that GAL gene expression can be activated by heterologous protein-protein interaction in the cytoplasm that is independent of galactose and Gal3p function. These results indicate that galactose-triggered Gal3p-Gal80p association in the cytoplasm activates Gal4p in the nucleus.

Activation of prophage eib genes for immunoglobulin-binding proteins by genes from the IbrAB genetic island of Escherichia coli ECOR-9.

Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E. coli reference (ECOR) strain ECOR-9. Each eib gene was transferred to test E. coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes, ibrA and ibrB (designation is derived from "immunoglobulin-binding regulator"), which together activated eib expression in the derived lysogens. The proteins encoded by ibrA and ibrB are very similar to uncharacterized proteins encoded by genes of Salmonella enterica serovar Typhi and E. coli O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing ibrA and ibrB has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the E. coli K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five eib-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of ibrAB revealed that the other eib-positive strains diverge by

Gal80 confers specificity on HAT complex interactions with activators.

Several yeast transcription activators have been shown to interact with and recruit histone acetyltransferase complexes to promoters in chromatin. The promiscuity of activator/HAT interactions suggests that additional factors temporally regulate these interactions in response to signaling pathways. In this study, we demonstrate that the negative regulator, Gal80, blocks interactions between the SAGA and NuA4 HAT complexes and the Gal4 activator. By contrast, Gal80 did not inhibit SAGA and NuA4 interaction with another activator Gcn4. The function of Gal80 prevented Gal4 targeting of SAGA and displaced SAGA targeted by Gal4 to a promoter within a nucleosome array. In the same set of experiments, targeting of SAGA by Gcn4 was unaffected by Gal80. These studies demonstrate that the specificity of HAT/activator interactions can be dictated by cofactors that modulate activation domain function in response to cellular signals.