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Results for “Nuclear Localization Signals”

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PubMed2005

Analysis of the subcellular localization of the proteins Rep, Rep' and Cap of porcine circovirus type 1.

Porcine circovirus type 1 (PCV1) encodes two major ORFs. The cap gene comprises the major structural protein of PCV, the rep gene specifies Rep and Rep', which are both essential for initiating the replication of the viral DNA. Rep corresponds to the full-length protein, whereas Rep' is a truncated splice product that is frame-shifted in its C-terminal sequence. In this study, the cellular localization of PCV1-encoded proteins was investigated by immune fluorescence techniques using antibodies against Rep, Rep' and Cap and by expression of viral proteins fused to green and red fluorescence proteins. Rep and Rep' protein co-localized in the nucleus of infected cells as well as in cells transfected with plasmids expressing Rep and Rep' fused to fluorescence proteins, but no signal was seen in the nucleoli. Rep and Rep' carry three potential nuclear localization signals in their identical N-termini, and the contribution of these motifs to nuclear import was experimentally dissected. In contrast to the rep gene products, the localization of the Cap protein varied. While the Cap protein was restricted to the nucleoli in plasmid-transfected cells and was also localized in the nucleoli at an early stage of PCV1 infection, it was seen in the nucleoplasm and the cytoplasm later in infection, suggesting that a shuttling between distinct cellular compartments occurs.

Amino Acid Sequence↗

PubMed2001

ARM domain-dependent nuclear import of adenomatous polyposis coli protein is stimulated by the B56 alpha subunit of protein phosphatase 2A.

Inactivating mutations in the adenomatous polyposis coli (APC) gene correlate with progression of colon cancer and familial adenomatous polyposis. The APC tumor suppressor contributes to chromosome segregation and turnover of the oncogenic transcriptional activator beta-catenin, and these activities are impaired by truncating cancer mutations. APC was recently identified as a shuttling protein whose subcellular distribution is regulated by two nuclear localization signals (NLSs) and multiple nuclear export signals (NESs). Here, we show that mutant disease-linked truncated forms of APC, most of which lack the two central NLSs and certain NES sequences, retain nuclear-cytoplasmic shuttling activity. Nuclear export of truncated APC is mediated by a dominant N-terminal NES. Nuclear import of NLS-deficient APC mutants is facilitated by the N-terminal ARM domain. Furthermore, co-expression of the ARM-binding protein, B56 alpha, increased the nuclear localization of mutant and wild-type APC. The minimal B56 alpha-responsive sequence mapped to APC amino acids 302-625. B56 alpha is a regulatory subunit of protein phosphatase 2A; however, its ability to shift APC to the nucleus was independent of phosphatase activity. We conclude that APC nuclear import is regulated by the ARM domain through its interaction with B56 alpha and postulate that APC/B56 alpha complexes target the dephosphorylation of specific proteins within the nucleus.

Adenomatous Polyposis Coli Protein↗

PubMed2006

Subcellular translocation signals regulate Geminin activity during embryonic development.

BACKGROUND INFORMATION: Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development. RESULTS: We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem. CONCLUSIONS: We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.

Active Transport, Cell Nucleus↗

PubMed2003

Pse1p mediates the nuclear import of the iron-responsive transcription factor Aft1p in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the iron-responsive transcription factor Aft1p plays a critical role in maintaining iron homeostasis. The activity of Aft1p is induced in response to iron starvation and as a consequence the expression of the iron-regulon is increased. We have shown previously that Aft1p is localized to the cytoplasm under iron-replete conditions but that it is localized to the nucleus under iron-depleted conditions. In this study, we identified the transport receptor that mediates the import of Aft1p into the nucleus, located the nuclear localization signal (NLS) sequences of Aft1p, and examined whether the nuclear import of Aft1p is affected by iron status. In pse1-1 cells, which bear a temperature-sensitive mutation of PSE1, Aft1p was misdirected to the cytoplasm during iron starvation at the restrictive temperature. Aft1p could also directly bind to Pse1p and was dissociated from the complex by Ran-GTP in vitro. These results indicate that Aft1p is imported into the nucleus by Pse1p. Supporting this is that the induction of an Aft1p target gene, FTR1, in response to iron starvation was greatly reduced in pse1-1 cells. Furthermore, we demonstrated that the nuclear localization of a mutant Aft1 protein that contains an NLS derived from SV40 was regulated by iron status regardless of whether Pse1p could interact with Aft1p. This suggests that the interaction between Aft1p and Pse1p is not a critical step that controls the iron-regulated nucleo-cytoplasmic transport of Aft1p.

Active Transport, Cell Nucleus↗

PubMed2005

Cross-species sequence analysis reveals multiple charged residue-rich domains that regulate nuclear/cytoplasmic partitioning and membrane localization of a kinase anchoring protein 12 (SSeCKS/Gravin).

A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated.

A Kinase Anchor Proteins↗

PubMed2006

The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor.

Sperm-specific phospholipase C-zeta (PLCzeta) causes intracellular Ca(2+) oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCzeta, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCzeta mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374-381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg(376), Lys(377), Arg(378), Lys(379), or Lys(381) with glutamate, whereas Ca(2+) oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys(299) and/or Lys(301) in the C terminus of X domain, or Trp(13), Phe(14), or Val(18) in the N terminus of EF1. Ca(2+) oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374-383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296-309 or 1-19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2-4 or C2 domain. Thus, PLCzeta is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCdelta1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.

Active Transport, Cell Nucleus↗

PubMed2003

Nuclear CaMKII inhibits neuronal differentiation of PC12 cells without affecting MAPK or CREB activation.

Ca(2+)/calmodulin-regulated protein kinase II (CaMKII) mediates many cellular events. The four CaMKII isoforms have numerous splice variants, three of which contain nuclear localization signals. Little is known about the role of nuclear localized CaMKII in neuronal development. To study this process, PC12 cells were transfected to produce CaMKII targeted to either the cytoplasm or the nucleus and then treated with nerve growth factor (NGF). NGF triggers a signaling cascade (MAPK) that results in the differentiation of PC12 cells into a neuronal phenotype, marked by neurite outgrowth. The present study found that cells expressing nuclear targeted CaMKII failed to grow neurites, whereas cells expressing cytoplasmic CaMKII readily produced neurites. Inhibition of neuronal differentiation by nuclear CaMKII was independent of MAPK signaling, as sustained Erk phosphorylation was not affected. Phosphorylation of CREB was also unaffected. Thus nuclear CaMKII modifies neuronal differentiation by a mechanism independent of MAPK and CREB activation.

Animals↗

PubMed2004

A putative NES mediates cytoplasmic localization of Apoptin in normal cells.

Apoptin, a protein expressed by chicken anemia virus, is found predominantly in the cytoplasm in normal cells, whereas it localizes in the nucleus in transformed and malignant cells. However, the mechanisms that regulate the different subcellular localization of Apoptin in normal and tumor cells have not been fully clarified. In this work, a putative nuclear export signal (NES) in Apoptin was predicted. It was testified that the putative NES (pNES) of Apoptin was not a functional NES, but actually acted as a cytoplasmic retention signal. Deletion of the pNES led to the nuclear accumulation of Apoptin in normal cells. In addition, when a strong nuclear localization signal was introduced into Apoptin, it exclusively translocated to the nucleus in normal cells. These observations indicated that the cytoplasmic localization of Apoptin in normal cells results from the balance between cytoplasmic retention and nuclear import. On the other hand, the pNES was also proved to be necessary for Apoptin multimerization. Mutants lacking the pNES did not form obviously visible globular aggregates in normal or tumor cells.

Active Transport, Cell Nucleus↗

PubMed2004

LEDGF/p75 determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes.

Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integrase-to-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.

Active Transport, Cell Nucleus↗

PubMed1990

The arginine-rich domain of hepatitis B virus precore and core proteins contains a signal for nuclear transport.

Precore and core proteins are two related co-carboxy-terminal proteins of hepatitis B virus. Precore protein contains the entire sequence of core protein plus an amino-terminal extension of 29 amino acid residues. Both proteins can display a common antigenic determinant known as core antigen (HBcAg). Clinically, HBcAg is detected in the nucleus, cytoplasm, or both of hepatitis B virus-infected hepatocytes. In order to understand the mechanism that regulates nuclear transport of HBcAg, various portions of precore and core proteins were linked to a reporter protein, human alpha-globin, and expressed in mammalian cells. Our results indicate that the precore protein-specific sequence, although important for nuclear transport, does not contain a nuclear localization signal. Instead, a signal for nuclear transport is located near the carboxy termini of precore and core proteins in the arginine-rich domain. This signal is made up of a set of two direct PRRRRSQS repeats and is highly conserved among mammalian hepadnaviruses.

Amino Acid Sequence↗

PubMed2000

Recycling of importin alpha from the nucleus is suppressed by loss of RCC1 function in living mammalian cells.

We previously reported that the nuclear import of substrates containing SV40 T antigen nuclear localization signal (NLS) was suppressed in a temperature-sensitive RCC1 mutant cell line, tsBN2, at nonpermissive temperature. Moreover, it was shown that import into wild type BHK21 cell-derived nuclei gradually decreased in heterokaryons between the tsBN2 and BHK21 cells, although the BHK21 nuclei retained wild type RCC1 and should contain RanGTP (Tachibana et al., 1994). In this study, it was found that in the heterokaryons cultured at non-permissive temperature, endogenous importin alpha was not detected immunocytochemically in the cytoplasm or BHK21 nuclei but only in the tsBN2 nuclei, suggesting that importin alpha cannot be exported from the RCC1-depleted nuclei. In fact, importin alpha microinjected into the nucleus of tsBN2 cells at non-permissive temperature remained in the nucleus. These results strongly support the hypothesis that the recycling of importin alpha from the nucleus requires nuclear RanGTP. Moreover, it was found that cytoplasmic injection of importin alpha restored the import of SV40 T-NLS substrates in the BHK21 nuclei but not the tsBN2 nuclei in the heterokaryons. This indicates that the decrease of importin alpha from the cytoplasm in the heterokaryons leads to a suppression of the efficiency of nuclear import of the T-NLS substrate and provides support for the view that nuclear RanGTP is essential for the nuclear entry of the substrates.

Animals↗

PubMed1997

Cloning of a cDNA encoding a novel importin-alpha homologue, Qip1: discrimination of Qip1 and Rch1 from hSrp1 by their ability to interact with DNA helicase Q1/RecQL.

We isolated two cDNA clones encoding human proteins which interact with DNA helicase Q1/RecQL, a human homologue of Eschelichia coli RecQ protein, by two-hybrid screening. One of these proteins, named Qip1, was a novel protein homologous to the nuclear localization signal (NLS) receptor importin-alpha, and the other was the known protein Rch1, which is also a homologue of importin-alpha. DNA helicase Q1 in human cell lysates was coprecipitated with bacterially expressed Qip1 and Rch1 fused with glutathione-S-transferase with glutathione Sepharose beads, confirming the interaction between these proteins and DNA helicase Q1. Two-hybrid experiments revealed that Qip1 interacted with the NLS of SV40 T antigen similar to Rch1 and hSrp1. In addition, interaction of the putative NLS in DNA helicase Q1 with Qip1 and Rch1 but not with hSrp1 was confirmed by the two-hybrid system.

Adenosine Triphosphatases↗

PubMed1998

Molecular cloning of human er1 cDNA and its differential expression in breast tumours and tumour-derived cell lines.

We recently cloned and characterized a novel immediate-early gene, called er1, from Xenopus embryos whose expression levels were increased during mesoderm induction by fibroblast growth factor (FGF). We describe here the isolation and expression pattern of the human er1 sequence. Human ER1 and Xenopus ER1 proteins display 91% similarity; the amino acid sequence motifs, including the putative DNA-binding SANT domain, the predicted nuclear localization signals (NLS) and the putative SH3 binding domain share 100% identity. er1 mRNA expression was negligible in all 50 normal human tissues analyzed. Examination of nine breast carcinoma-derived cell lines and eight breast tumour tissue samples by reverse transcription-polymerase chain reaction (RT-PCR) revealed that human er1 was consistently expressed in all tumour cell lines and tumour tissue while remaining undetectable in normal breast cell lines and breast tissue. These data suggest that er1 expression is associated with the neoplastic state in human breast carcinoma.

Amino Acid Sequence↗

PubMed2004

Integration of stress responses: modulation of calcineurin signaling in Saccharomyces cerevisiae by protein kinase A.

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase required for Saccharomyces cerevisiae to adapt to a variety of environmental stresses. Once activated, calcineurin dephosphorylates the Zn-finger transcription factor Crz1p/Tcn1p, causing it to accumulate in the nucleus where it activates gene expression. Here we show that cyclic AMP-dependent protein kinase A (PKA) phosphorylates and negatively regulates Crz1p activity by inhibiting its nuclear import. Activation of PKA in vivo decreases Crz1p-dependent transcription. PKA phosphorylates Crz1p in vitro, and we identify specific residues required for this phosphorylation, all of which reside in or adjacent to the nuclear localization signal. Mutation of these residues to alanine results in increased nuclear import of Crz1p and results in higher levels of both basal and Ca2+-induced Crz1p transcriptional activity. PKA regulates the general stress response in yeast and coordinates this response with nutrient availability. In contrast, calcineurin regulates the cellular response to a restricted set of environmental insults. Thus, these studies identify a specific biochemical mechanism through which the activities of multiple stress-activated signaling pathways are integrated in vivo.

Active Transport, Cell Nucleus↗

PubMed2006

Dancing in and out of the nucleus: p120(ctn) and the transcription factor Kaiso.

The catenin p120 (hereafter p120(ctn)) was first identified as a Src kinase substrate and subsequently characterized as an Armadillo catenin member of the cell-cell adhesion cadherin-catenin complex. In the past decade, many studies have revealed roles for p120(ctn) in regulating Rho family GTPase activity and E-cadherin stability and turnover, events that occur predominantly at the plasma membrane or in the cytoplasm. However, the recent discovery of the nuclear BTB/POZ-ZF transcription factor Kaiso as a p120(ctn) binding partner, coupled with the detection of p120(ctn) in the nucleus of some cell lines and tumor tissues, suggested that like the classical beta-catenin, p120(ctn) undergoes nucleocytoplasmic trafficking and regulates gene expression. Indeed, p120(ctn) has a classic nuclear localization signal and does traffic to the nucleus. Moreover, nuclear p120(ctn) regulates Kaiso DNA-binding and transcriptional activity, similar to beta-catenin's modulation of TCF/LEF transcription activity. However unlike beta-catenin, p120(ctn) does not appear to be a transcriptional activator. Hence it remains to be determined whether the sole role of nuclear p120(ctn) is regulation of Kaiso or whether p120(ctn) binds and regulates other transcription factors or nuclear proteins.

Active Transport, Cell Nucleus↗

PubMed2004

Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1.

Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 ((51)RRPR(54)) or pat7 ((48)PRVRRPR(54)) NLS2 abrogated nuclear accumulation, whereas deletion of (48)PRV(50) did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 ((11)RRPRR(15)), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids (485)LSAYLTLFVAL(495). This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm.

Active Transport, Cell Nucleus↗

PubMed2000

Protein-protein interactions that precede the nuclear entry of goat uterine estrogen receptor under cell-free conditions.

Mechanisms associated with a regulated nuclear entry of the goat uterine estrogen receptor (gER), under the influence of estradiol, have been examined using a cell-free system. The gER transport into the nucleus is mediated by a 55-kDa cytosolic protein, p55. Experimental evidence has been provided to demonstrate that p55 binds to the nuclear localization signals (NLS) on the gER. Under hormone-free conditions, a 28-kDa protein, p28, binds to the NLS and prevents the p55 interaction with the NLS. This inhibition is reversed by a 73-kDa protein, p73. It appears that p73 associates with the hormone binding domain (HBD) of the gER under hormone-free conditions. Estradiol binding to the HBD facilitates p73 interaction with p28. This leads to the dissociation of p28 from the NLS, which, in turn, facilitates the binding of p55 to the NLS on the gER. Both p28 and p55 cross-react with monoclonal antibodies against hsp-25 and hsp-70, indicating a possibility that p28 and p55 belong to a superfamily of molecular chaperones. J. Cell. Biochem. 78:650-665, 2000.

Actins↗

PubMed1999

Structure-function analysis of the protein-binding domains of Mac1p, a copper-dependent transcriptional activator of copper uptake in Saccharomyces cerevisiae.

The Mac1 protein in Saccharomyces cerevisiae is essential for the expression of yeast high affinity copper uptake. A positive transcription factor, Mac1p binds via its N-terminal domain to GCTC elements in the promoters of CTR1 and FRE1, encoding a copper permease and metal reductase, respectively. Mac1p-dependent transcriptional activation is negatively regulated by copper. We have mapped the domains in Mac1p responsible for its nuclear localization and for the protein-protein interactions that underlie its transcriptional activity. Immunofluorescence studies indicate that Mac1p contains two nuclear localization signals, one each in the N- and C-terminal halves of the protein. Yeast one-hybrid analysis demonstrates that the copper-dependent transcriptional activity in Mac1p resides primarily in a cysteine-rich element encompassing residues 264-279. Two-hybrid analysis indicates that a copper-independent Mac1p-Mac1p interaction linked to DNA binding is due primarily to a predicted helix in the C-terminal region of the protein encompassing residues 388-406. Point mutations within this putative helix abrogate the Mac1-Mac1 interaction in vivo and formation of a ternary (Mac1p)(2).DNA complex in vitro. When produced in normal abundance, Mac1pI396D and Mac1pF400D helix mutants do not support transcriptional activation in vivo consistent with an essential Mac1p dimerization in transcriptional activation. Lastly, the one- and two-hybrid data indicate that an intramolecular interaction between the DNA-binding and transactivation domains negatively modulates Mac1p activity.

Binding Sites↗