Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity

The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Mig1p are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Mig1p homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an α-helix. Received: 30 March 1998 / Accepted: 18 August 1998     

The archaeo-eukaryotic GINS proteins and the archaeal primase catalytic subunit PriS share a common domain

Primase and GINS are essential factors for chromosomal DNA replication in eukaryotic and archaeal cells. Here we describe a previously undetected relationship between the C-terminal domain of the catalytic subunit (PriS) of archaeal primase and the B-domains of the archaeo-eukaryotic GINS proteins in the form of a conserved structural domain comprising a three-stranded antiparallel β-sheet adjacent to an α-helix and a two-stranded β-sheet or hairpin. The presence of a shared domain in archaeal PriS and GINS proteins, the genes for which are often found adjacent on the chromosome, suggests simple mechanisms for the evolution of these proteins.     

Association of 14-3-3 proteins with the C-terminal autoinhibitory domain of the plant plasma-membrane H+-ATPase generates a fusicoccin-binding complex

The plant plasma-membrane H⁺-ATPase (EC 3.6.1.35) contains a C-terminal autoinhibitory domain whose displacement from the catalytic site is caused by treatment of intact plant tissue with the phytotoxin fusicoccin (FC). The FC-induced activation of the H⁺-ATPase was proposed to involve a direct interaction of 14-3-3 proteins with the H⁺-ATPase. By analysing plasma membranes derived from leaves of Commelina communis L., direct biochemical evidence has now been obtained for a complex between the C-terminus of the H⁺-ATPase and a 14-3-3 dimer. Stabilization of this complex was achieved by FC treatment in vivo or in vitro. Furthermore, the C-terminal domain of the H⁺-ATPase in association with a 14-3-3 dimer is essential for the creation of a functional FC-binding complex. Received: 1 August 1998 / Accepted: 15 September 1998     

Identification and structural characterization of FYVE domain-containing proteins of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

FYVE domains have emerged as membrane-targeting domains highly specific for phosphatidylinositol 3-phosphate (PtdIns(3)P). They are predominantly found in proteins involved in various trafficking pathways. Although FYVE domains may function as individual modules, dimers or in partnership with other proteins, structurally, all FYVE domains share a fold comprising two small characteristic double-stranded β-sheets, and a C-terminal α-helix, which houses eight conserved Zn²⁺ ion-binding cysteines. To date, the structural, biochemical, and biophysical mechanisms for subcellular targeting of FYVE domains for proteins from various model organisms have been worked out but plant FYVE domains remain noticeably under-investigated.     

Close Evolutionary Relatedness of α-Amylases from Archaea and Plants

The amino acid sequences of 22 α-amylases from family 13 of glycosyl hydrolases were analyzed with the aim of revealing the evolutionary relationships between the archaeal α-amylases and their eubacterial and eukaryotic counterparts. Two evolutionary distance trees were constructed: (i) the first one based on the alignment of extracted best-conserved sequence regions (58 residues) comprising β2, β3, β4, β5, β7, and β8 strand segments of the catalytic (α/β)₈-barrel and a short conserved stretch in domain B protruding out of the barrel in the β3 →α3 loop, and (ii) the second one based on the alignment of the substantial continuous part of the (α/β)₈-barrel involving the entire domain B (consensus length: 386 residues). With regard to archaeal α-amylases, both trees compared brought, in fact, the same results; i.e., all family 13 α-amylases from domain Archaea were clustered with barley pI isozymes, which represent all plant α-amylases. The enzymes from Bacillus licheniformis and Escherichia coli, representing liquefying and cytoplasmic α-amylases, respectively, seem to be the further closest relatives to archaeal α-amylases. This evolutionary relatedness clearly reflects the discussed similarities in the amino acid sequences of these α-amylases, especially in the best-conserved sequence regions. Since the results for α-amylases belonging to all three domains (Eucarya, Eubacteria, Archaea) offered by both evolutionary trees are very similar, it is proposed that the investigated conserved sequence regions may indeed constitute the ``sequence fingerprints' of a given α-amylase. Received: 3 June 1998 / Accepted: 20 August 1998     

Expression of an apoplast‐localized BURP‐domain protein from soybean (GmRD22) enhances tolerance towards abiotic stress

The BURP‐domain protein family comprises a diverse group of plant‐specific proteins that share a conserved BURP domain at the C terminus. However, there have been only limited studies on the functions and subcellular localization of these proteins. Members of the RD22‐like subfamily are postulated to associate with stress responses due to the stress‐inducible nature of some RD22‐like genes. In this report, we used different transgenic systems (cells and in planta) to show that the expression of a stress‐inducible RD22‐like protein from soybean (GmRD22) can alleviate salinity and osmotic stress. We also performed detailed microscopic studies using both fusion proteins and immuno‐electron microscopic techniques to demonstrate the apoplast localization of GmRD22, for which the BURP domain is a critical determinant of the subcellular localization. The apoplastic GmRD22 interacts with a cell wall peroxidase and the ectopic expression of GmRD22 in both transgenic Arabidopsis thaliana and transgenic rice resulted in increased lignin production when subjected to salinity stress. It is possible that GmRD22 regulates cell wall peroxidases and hence strengthens cell wall integrity under such stress conditions.     

The divergence and positive selection of the plant‐specific BURP‐containing protein family

BURP domain‐containing proteins belong to a plant‐specific protein family and have diverse roles in plant development and stress responses. However, our understanding about the genetic divergence patterns and evolutionary rates of these proteins remain inadequate. In this study, 15 plant genomes were explored to elucidate the genetic origins, divergence, and functions of these proteins. One hundred and twenty‐five BURP protein‐encoding genes were identified from four main plant lineages, including 13 higher plant species. The absence of BURP family genes in unicellular and multicellular algae suggests that this family (1) appeared when plants shifted from relatively stable aquatic environments to land, where conditions are more variable and stressful, and (2) is critical in the adaptation of plants to adverse environments. Promoter analysis revealed that several responsive elements to plant hormones and external environment stresses are concentrated in the promoter region of BURP protein‐encoding genes. This finding confirms that these genes influence plant stress responses. Several segmentally and tandem‐duplicated gene pairs were identified from eight plant species. Thus, in general, BURP domain‐containing genes have been subject to strong positive selection, even though these genes have conformed to different expansion models in different species. Our study also detected certain critical amino acid sites that may have contributed to functional divergence among groups or subgroups. Unexpectedly, all of the critical amino acid residues of functional divergence and positive selection were exclusively located in the C‐terminal region of the BURP domain. In conclusion, our results contribute novel insights into the genetic divergence patterns and evolutionary rates of BURP proteins.     

The conserved 3′ Angio‐domain defines a superfamily of short interspersed nuclear elements (SINEs) in higher plants

Repetitive sequences are ubiquitous components of eukaryotic genomes affecting genome size and evolution as well as gene regulation. Among them, short interspersed nuclear elements (SINEs) are non‐coding retrotransposons usually shorter than 1000 bp. They contain only few short conserved structural motifs, in particular an internal promoter derived from cellular RNAs and a mostly AT‐rich 3′ tail, whereas the remaining regions are highly variable. SINEs emerge and vanish during evolution, and often diversify into numerous families and subfamilies that are usually specific for only a limited number of species. In contrast, at the 3′ end of multiple plant SINEs we detected the highly conserved ‘Angio‐domain’. This 37 bp segment defines the Angio‐SINE superfamily, which encompasses 24 plant SINE families widely distributed across 13 orders within the plant kingdom. We retrieved 28 433 full‐length Angio‐SINE copies from genome assemblies of 46 plant species, frequently located in genes. Compensatory mutations in and adjacent to the Angio‐domain imply selective restraints maintaining its RNA structure. Angio‐SINE families share segmental sequence similarities, indicating a modular evolution with strong Angio‐domain preservation. We suggest that the conserved domain contributes to the evolutionary success of Angio‐SINEs through either structural interactions between SINE RNA and proteins increasing their transpositional efficiency, or by enhancing their accumulation in genes.     

Bioinformatic analysis of the TonB protein family

TonB is a protein prevalent in a large number of Gram-negative bacteria that is believed to be responsible for the energy transduction component in the import of ferric iron complexes and vitamin B₁₂ across the outer membrane. We have analyzed all the TonB proteins that are currently contained in the Entrez database and have identified nine different clusters based on its conserved 90-residue C-terminal domain amino acid sequence. The vast majority of the proteins contained a single predicted cytoplasmic transmembrane domain; however, nine of the TonB proteins encompass a ∼290 amino acid N-terminal extension homologous to the MecR1 protein, which is composed of three additional predicted transmembrane helices. The periplasmic linker region, which is located between the N-terminal domain and the C-terminal domain, is extremely variable both in length (22–283 amino acids) and in proline content, indicating that a Pro-rich domain is not a required feature for all TonB proteins. The secondary structure of the C-terminal domain is found to be well preserved across all families, with the most variable region being between the second α-helix and the third β-strand of the antiparallel β-sheet. The fourth β-strand found in the solution structure of the Escherichia coli TonB C-terminal domain is not a well conserved feature in TonB proteins in most of the clusters. Interestingly, several of the TonB proteins contained two C-terminal domains in series. This analysis provides a framework for future structure-function studies of TonB, and it draws attention to the unusual features of several TonB proteins. Byron C. H. Chu and R. Sean Peacock contributed equally to this work.     

A single residue mutation abolishes attachment of the CBM26 starch-binding domain from <Emphasis Type="Italic">Lactobacillus amylovorus</Emphasis> α-amylase

Starch is degraded by amylases that frequently have a modular structure composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. The C-terminal domain from the Lactobacillus amylovorus α-amylase has an unusual architecture composed of five tandem starch-binding domains (SBDs). These domains belong to family 26 in the carbohydrate-binding modules (CBM) classification. It has been reported that members of this family have only one site for starch binding, where aromatic amino acids perform the binding function. In SBDs, fold similarities are better conserved than sequences; nevertheless, it is possible to identify in CBM26 members at least two aromatic residues highly conserved. We attempt to explain polysaccharide recognition for the L. amylovorus α–amylase SBD through site-directed mutagenesis of aromatic amino acids. Three amino acids were identified as essential for binding, two tyrosines and one tryptophan. Y18L and Y20L mutations were found to decrease the SBD binding capacity, but unexpectedly, the mutation at W32L led to a total loss of affinity, either with linear or ramified substrates. The critical role of Trp 32 in substrate binding confirms the presence of just one binding site in each α-amylase SBD.     

Genome-scale identification of Soybean BURP domain-containing genes and their expression under stress treatments

Background  

Multiple proteins containing BURP domain have been identified in many different plant species, but not in any other organisms. To date, the molecular function of the BURP domain is still unknown, and no systematic analysis and expression profiling of the gene family in soybean (Glycine max) has been reported.     

Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean.

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.     

The NfeD Protein Family and Its Conserved Gene Neighbours Throughout Prokaryotes: Functional Implications for Stomatin-Like Proteins

NfeD-like proteins are widely distributed throughout prokaryotes and are frequently associated with genes encoding stomatin-like proteins (slipins). Here, we reveal that the NfeD family is ancient and comprises three major groups: NfeD1a, NfeD1b and truncated NfeD1b. Members of each group are associated with one of four conserved gene partners, three of which have eukaryotic homologues that are membrane raft associated, namely stomatin, paraslipin (previously SLP-2) and flotillin. The first NfeD group (NfeD1b), comprises proteins of approximately 460-aa long that have three functional domains: an N-terminal protease, a middle membrane-spanning region and a soluble C-terminal region rich in β-strands. The nfeD1b gene is adjacent to eoslipin in prokaryotic genomes except in Firmicutes and Deinococci, where yqfA replaces eoslipin. Proteins in the second major group (NfeD1a) are homologous to the C-terminus of NfeD1b which forms a β-barrel-like domain, and their genes are associated with paraslipin. Using OrthoMCL clustering, we show that nfeD1b genes have become truncated on many independent occasions giving rise to the third major group. These short NfeD homologues frequently remain associated with their ancestral gene neighbour, resembling NfeD1a in structure, yet are much more related to full-length NfeD1b; we term these “truncated NfeD1b”. These conserved associations suggest that NfeD proteins are dependent on gene partners for their function and that the site of interaction may lie within the C-terminal portion that is common to all NfeD homologues. Although NfeD homologues are confined to prokaryotes, this conserved association could represent an excellent system to study slipin and flotillin proteins.     

The C-terminal region of α′ subunit of soybean β-conglycinin contains two types of vacuolar sorting determinants

In maturing seed cells, proteins that accumulate in the protein storage vacuoles (PSVs) are synthesized on the endoplasmic reticulum (ER) and transported by vesicles to the PSVs. Vacuolar sorting determinants (VSDs) which are usually amino acid sequences of short or moderate length direct the proteins to this pathway. VSDs identified so far are classified into two types: sequence specific VSDs (ssVSDs) and C-terminal VSDs (ctVSDs). We previously demonstrated that VSDs of α′ and β subunits of β-conglycinin, one of major storage proteins of soybean (Glycine max), reside in the C-terminal ten amino acids. Here we show that both types of VSDs coexist within this region of the α′ subunit. Although ctVSDs can function only at the very C-termini of proteins, the C-terminal ten amino acids of α′ subunit directed green fluorescent protein (GFP) to the PSVs even when they were placed at the N-terminus of GFP, indicating that an ssVSD resides in the sequence. By mutation analysis, it was found that the core sequence of the ssVSD is Ser-Ile-Leu (fifth to seventh residues counted from the C-terminus) which is conserved in the α and β subunits and some vicilin-like proteins. On the other hand, the sequence composed of the C-terminal three amino acids (AFY) directed GFP to the PSVs when it was placed at the C-terminus of GFP, though the function as a VSD was disrupted at the N-terminus of GFP, indicating that the AFY sequence is a ctVSD.     

A Phylogenetic Study of SPBP and RAI1: Evolutionary Conservation of Chromatin Binding Modules

Our genome is assembled into and array of highly dynamic nucleosome structures allowing spatial and temporal access to DNA. The nucleosomes are subject to a wide array of post-translational modifications, altering the DNA-histone interaction and serving as docking sites for proteins exhibiting effector or “reader” modules. The nuclear proteins SPBP and RAI1 are composed of several putative “reader” modules which may have ability to recognise a set of histone modification marks. Here we have performed a phylogenetic study of their putative reader modules, the C-terminal ePHD/ADD like domain, a novel nucleosome binding region and an AT-hook motif. Interactions studies in vitro and in yeast cells suggested that despite the extraordinary long loop region in their ePHD/ADD-like chromatin binding domains, the C-terminal region of both proteins seem to adopt a cross-braced topology of zinc finger interactions similar to other structurally determined ePHD/ADD structures. Both their ePHD/ADD-like domain and their novel nucleosome binding domain are highly conserved in vertebrate evolution, and construction of a phylogenetic tree displayed two well supported clusters representing SPBP and RAI1, respectively. Their genome and domain organisation suggest that SPBP and RAI1 have occurred from a gene duplication event. The phylogenetic tree suggests that this duplication has happened early in vertebrate evolution, since only one gene was identified in insects and lancelet. Finally, experimental data confirm that the conserved novel nucleosome binding region of RAI1 has the ability to bind the nucleosome core and histones. However, an adjacent conserved AT-hook motif as identified in SPBP is not present in RAI1, and deletion of the novel nucleosome binding region of RAI1 did not significantly affect its nuclear localisation.     

Feeding truncated heat shock protein 70s protect Artemia franciscana against virulent Vibrio campbellii challenge

The 70 kDa heat shock proteins (Hsp70s) are highly conserved in evolution, leading to striking similarities in structure and composition between eukaryotic Hsp70s and their homologs in prokaryotes. The eukaryotic Hsp70 like the DnaK (Escherichia coli equivalent Hsp70) protein, consist of three functionally distinct domains: an N-terminal 44-kDa ATPase portion, an 18-kDa peptide-binding domain and a C-terminal 10-kDa fragment. Previously, the amino acid sequence of eukaryotic (the brine shrimp Artemia franciscana) Hsp70 and DnaK proteins were shown to share a high degree of homology, particularly in the peptide-binding domain (59.6%, the putative innate immunity-activating portion) compared to the N-terminal ATPase (48.8%) and the C-terminal lid domains (19.4%). Next to this remarkable conservation, these proteins have been shown to generate protective immunity in Artemia against pathogenic Vibrio campbellii. This study, aimed to unravel the Vibrio-protective domain of Hsp70s in vivo, demonstrated that gnotobiotically cultured Artemia fed with recombinant C-terminal fragment (containing the conserved peptide binding domain) of Artemia Hsp70 or DnaK protein were well protected against subsequent Vibrio challenge. In addition, the prophenoloxidase (proPO) system, at both mRNA and protein activity levels, was also markedly induced by these truncated proteins, suggesting epitope(s) responsible for priming the proPO system and presumably other immune-related genes, consequently boosting Artemia survival upon challenge with V. campbellii, might be located within this conserved region of the peptide binding domain.     

Evolution of Chitin-Binding Proteins in Invertebrates

Analysis of a group of invertebrate proteins, including chitinases and peritrophic matrix proteins, reveals the presence of chitin-binding domains that share significant amino acid sequence similarity. The data suggest that these domains evolved from a common ancestor which may be a protein containing a single chitin-binding domain. The duplication and transposition of this chitin-binding domain may have contributed to the functional diversification of chitin-binding proteins. Sequence comparisons indicated that invertebrate and plant chitin binding domains do not share significant amino acid sequence similarity, suggesting that they are not coancestral. However, both the invertebrate and the plant chitin-binding domains are cysteine-rich and have several highly conserved aromatic residues. In plants, cysteines have been elucidated in maintaining protein folding and aromatic amino acids in interacting with saccharides [Wright HT, Sanddrasegaram G, Wright CS (1991) J Mol Evol 33:283–294]. It is likely that these residues perform similar functions in invertebrates. We propose that the invertebrate and the plant chitin-binding domains share similar mechanisms for folding and saccharide binding and that they evolved by convergent evolution. Furthermore, we propose that the disulfide bonds and aromatic residues are hallmarks for saccharide-binding proteins. Received: 2 March 1998 / Accepted: 17 July 1998