Tetratricopeptide Repeat Motifs Research Articles

The intraflagellar transport component IFT88/polaris is a centrosomal protein regulating G1-S transition in non-ciliated cells

Loss of normal primary cilia function in mammals is linked to proliferative diseases, such as polycystic kidney disease, suggesting a regulatory relationship between cilia and cell cycle. The primary cilium expressed by most mammalian cells is nucleated from the elder centriole of the centrosome. The relationship between centrosome and cilia suggests that these structures share functions and components. We now show that IFT88/polaris, a component of the intraflagellar transport, remains associated to the centrosome in a proliferative state. IFT88/polaris is tightly associated with the centrosome throughout the cell cycle in a microtubule- and dynein-independent manner. IFT88/polaris tetratricopeptide repeat motifs are essential for this localization. Overexpression of IFT88/polaris prevents G1-S transition and induces apoptotic cell death. By contrast, IFT88/polaris depletion induced by RNA interference promotes cell-cycle progression to S, G2, and M phases. Finally, we demonstrate that IFT88/polaris interacts with Che-1, an Rb-binding protein that inhibits the Rb growth suppressing function. We propose that IFT88/polaris, a protein essential for ciliogenesis, is also crucial for G1-S transition in non-ciliated cells.

Toc64 - A Preprotein-receptor at the Outer Membrane with Bipartide Function

Protein translocation across membranes is assisted by translocation machineries present in the membrane targeted by the precursor proteins. Translocon subunits can be functionally divided into receptor proteins warranting the specificity of this machine and a translocation channel. At the outer envelope of chloroplasts two sets of receptor proteins regulate protein translocation facing the cytosol or acting in the intermembrane space. One, Toc64 is a receptor of the translocon at the outer envelope of chloroplasts (Toc complex) with dual function. Toc64 recognizes Hsp90 delivered precursor proteins via a cytosolic exposed domain containing three tetratrico-peptide repeat motifs and as demonstrated in here, Toc64 functions also as a major component of a complex facing the intermembrane space. The latter complex is composed of an Hsp70 localized in the intermembrane space, its interaction partner Toc12, a J-domain containing protein and the intermembrane space protein Tic22. We analyzed the intermembrane space domain of Toc64. This domain is involved in preprotein recognition and association with the Toc-complex independent of the cytosolic domain of the Toc64 receptor. Therefore, Toc64 is involved in preprotein translocation across the outer envelope at both sites of the membrane.

Induction of abnormal nuclear shapes in two distinct modes by overexpression of serine/threonine protein phosphatase 5 in Hela cells

Okadaic acid-sensitve serine/threonine protein phosphatase 5 (PP5) is expressed ubiquitously in various tissues and is considered to participate in many cellular processes. PP5 has a catalytic domain in the C-terminal region and three tetratricopeptide repeat (TPR) motifs in the N-terminal region, which are suspected to function as a protein-protein interaction domain. Physiological roles of PP5 are still largely unknown, although several PP5-binding proteins were reported and a few in vivo functions of PP5 were suggested. In the present study, the effects of expression of the full-length wild-type PP5 fused with EGFP (EGFP-PP5(WT)) and its phosphatase-dead mutant EGFP-PP5(H304A) were investigated. Transient expression of either EGFP-PP5(WT) or EGFP-PP5(H304A) in HeLa cells induced deformed nuclei with a 10-fold frequency compared to that of EGFP. Abnormal-shaped nuclei were also substantially increased by induced moderate expression of PP5 in tet-on HeLa cells. Many HeLa cells expressing EGFP-PP5(WT) possessed multi-nuclei separated from each other by nuclear membrane, while expression of EGFP-PP5(H304A) induced deformed nuclei which were multiple-like in shape, but not separated completely and were surrounded by one nuclear membrane. These results suggest that PP5 plays important roles at the M-phase of the cell cycle, especially in separation of chromosomes and formation of nuclear membrane.

Tcc1p, a Novel Protein Containing the Tetratricopeptide Repeat Motif, Interacts with Tup1p To Regulate Morphological Transition and Virulence in Candida albicans

The transcriptional factor CaTup1p represses many genes involved in intracellular processes, including the yeast-hypha transition, in the human fungal pathogen Candida albicans. Using tandem affinity purification technology, we identified a novel protein that interacts with CaTup1p, named Tcc1p (Tup1p complex component). Tcc1p is a C. albicans-specific protein with a 736-amino-acid polypeptide with four tetratricopeptide repeat (TPR) motifs in the N-terminal portion. Tcc1p formed a protein complex with CaTup1p via the TPR domain of Tcc1p, independently of CaSsn6p-CaTup1p The tcc1Delta disruptant showed filamentous growth under conditions inducing the yeast form, as is true of the Catup1Delta mutant. Consistent with this result, the common set of hypha-specific genes was negatively regulated by both TCC1 and CaTUP1. These observations will provide new insights into CaTup1p-dependent transcriptional gene regulation in C. albicans.

The Arabidopsis Tetratricopeptide Repeat-Containing Protein TTL1 Is Required for Osmotic Stress Responses and Abscisic Acid Sensitivity

Mutations in the Arabidopsis (Arabidopsis thaliana) TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 1 (TTL1) cause reduced tolerance to NaCl and osmotic stress that is characterized by reduced root elongation, disorganization of the root meristem, and impaired osmotic responses during germination and seedling development. Expression analyses of genes involved in abscisic acid (ABA) biosynthesis and catabolism suggest that TTL1 is not involved in the regulation of ABA levels but is required for ABA-regulated responses. TTL1 regulates the transcript levels of several dehydration-responsive genes, such as the transcription factor DREB2A, and genes encoding dehydration response proteins, such as ERD1 (early response to dehydration 1), ERD3, and COR15a. The TTL1 gene encodes a novel plant protein with tetratricopeptide repeats and a region with homology to thioredoxin proteins. Based on homology searches, there are four TTL members in the Arabidopsis genome with similar intron-exon structure and conserved amino acid domains. Proteins containing tetratricopeptide repeat motifs act as scaffold-forming multiprotein complexes and are emerging as essential elements for plant hormonal responses (such as gibberellin responses and ethylene biosynthesis). In this report, we identify TTL1 as a positive regulator of ABA signaling during germination and seedling development under stress.

TPR Proteins in Plant Hormone Signaling

There is a large number of proteins in nature containing Tetratrico Peptide Repeats (TPRs). TPR motifs are defined as a protein-protein interaction module involved in regulation of different cellular functions. We have recently identified TTL1 as a protein containing TPR motifs required for abscisic acid responses and osmotic stress tolerance. In recent years several of these proteins have been found to be essential for responses to other hormones such ethylene, cytokinin, gibberelling and auxin in Arabidopsis. Thus, proteins containing TPRs are emerging as essential determinants for signal transduction pathways mediated by most plant hormones.

Crystal structure of yeast mitochondrial outer membrane translocon member Tom70p

A majority of the proteins targeted to the mitochondria are transported through the translocase of the outer membrane (TOM) complex. Tom70 is a major surface receptor for mitochondrial protein precursors in the TOM complex. To investigate how Tom70 receives the mitochondrial protein precursors, we have determined the crystal structure of yeast Tom70p to 3.0 A. Tom70p forms a homodimer in the crystal. Each subunit consists primarily of tetratricopeptide repeat (TPR) motifs, which are organized into a right-handed superhelix. The TPR motifs in the N-terminal domain of Tom70p form a peptide-binding groove for the C-terminal EEVD motif of Hsp70, whereas the C-terminal domain of Tom70p contains a large pocket that may be the binding site for mitochondrial precursors. The crystal structure of Tom70p provides insights into the mechanisms of precursor transport across the mitochondrion's outer membrane.

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG–XRCC3 and FANCG–BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein–protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.

The C-terminal TPR Domain of Tom70 Defines a Family of Mitochondrial Protein Import Receptors Found only in Animals and Fungi

In fungi and animals the translocase in the outer mitochondrial membrane (TOM complex) consists of multiple components including the receptor subunit Tom70. Genome sequence analyses suggest no Tom70 receptor subunit exists in plants or protozoans, raising questions about its ancestry, function and the importance of its activity. Here we characterise the relationships within the Tom70 family of proteins. We find that in both fungi and animals, a conserved domain structure exists within the Tom70 family, with a transmembrane segment followed by 11 tetratricopeptide repeat motifs organised in three distinct domains. The C-terminal domain of Tom70 is highly conserved, and crucial for the import of hydrophobic substrate proteins, including those with and those without N-terminal presequences. Tom70 likely arose after fungi and animals diverged from other eukaryote lineages including plants, and subsequent gene duplication gave rise to a paralogue specific to the Saccharomyces group of yeasts. In animals and in fungi, Tom70 plays a fundamental role in the import of precursor proteins, by assisting relatively hydrophobic regions of substrate proteins into the translocation channel in the outer mitochondrial membrane. Proteins that function equivalently to Tom70 may have arisen independently in plants and protists.

Ap58: A novel in situ outer dynein arm-binding protein

Outer arm dynein is a molecular motor that is positioned at 24 nm intervals on outer doublet microtubules in cilia and flagella. In the present paper, we report identification of a 58 kDa novel protein with a tetratricopeptide repeat (TPR), referred to as ap58 (for 58 kDa axonemal protein) in sea urchin sperm axonemes. Ap58 is extracted along with the outer arm dynein by a high salt solution from axonemes. Sucrose density gradient centrifugation or gel filtration of the extract separates the outer arm dynein core from ap58. Most ap58 sediments to the lower density fraction or elutes in fractions of smaller molecules. However, immunogold localization reveals that ap58 is distributed at ∼25 nm intervals on doublet microtubules, suggesting that in situ it is associated with the outer dynein arm. Thus, ap58 with the TPR motif is a new member of outer dynein arm-binding proteins distinct from the outer dynein arm-docking complex.

Convergent Evolution of Receptors for Protein Import into Mitochondria

Mitochondria evolved from intracellular bacterial symbionts. Establishing mitochondria as organelles required a molecular machine to import proteins across the mitochondrial outer membrane. This machinery, the TOM complex, is composed of at least seven component parts, and its creation and evolution represented a sizeable challenge. Although there is good evidence that a core TOM complex, composed of three subunits, was established in the protomitochondria, we suggest that the receptor component of the TOM complex arose later in the evolution of this machine. We have solved by nuclear magnetic resonance the structure of the presequence binding receptor from the TOM complex of the plant Arabidopsis thaliana. The protein fold suggests that this protein, AtTom20, belongs to the tetratricopeptide repeat (TPR) superfamily, but it is unusual in that it contains insertions lengthening the helices of each TPR motif. Peptide titrations map the presequence binding site to a groove of the concave surface of the receptor. In vitro functional assays and peptide titrations suggest that the plant Tom20 is functionally equivalent to fungal and animal Tom20s. Comparison of the sequence and structure of Tom20 from plants and animals suggests that these two presequence binding receptors evolved from two distinct ancestral genes following the split of the animal and plant lineages. The need to bind equivalent mitochondrial targeting sequences and to make similar interactions within an equivalent protein translocation machine has driven the convergent evolution of two distinct proteins to a common structure and function.

Recombinant O-GlcNAc transferase isoforms: identification of O-GlcNAcase, yes tyrosine kinase, and tau as isoform-specific substrates

O-linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine or threonine residues of a variety of substrate proteins, including nuclear pore proteins, transcription factors, and proteins implicated in diabetes and neurodegenerative disorders. We have identified two nucleocytoplasmic isoforms of OGT (ncOGT and sOGT) and one isoform that localizes to the mitochondria (mOGT). These three isoforms contain identical catalytic regions but differ in the number of tetratricopeptide repeat motifs found at the N-terminus of each enzyme. We expressed each of these OGT isoforms in a soluble form in Escherichia coli and have used them to identify novel targets including the Src-family tyrosine kinase yes and O-GlcNAc-ase. We demonstrate that some substrate proteins, such as Nup62 and casein kinase II, are glycosylated by both ncOGT and mOGT, while others such as O-GlcNAcase and tau are specifically modified by ncOGT. The yes kinase was specifically modified by mOGT. The short isoform of OGT (sOGT) did not glycosylate any of the substrates tested, although it retains a potentially active catalytic domain. Our findings demonstrate the potential utility of recombinant OGT in identifying new targets and illustrate the necessity to examine all active isoforms of the enzyme. The identification of a tyrosine kinase and O-GlcNAcase as OGT targets suggests the potential for OGT participation in numerous signal transduction cascades.

Identification of the functional domains of ANT-1, a novel coactivator of the androgen receptor

Previously, we identified a transcriptional coactivator for the activation function-1 (AF-1) domain of the human androgen receptor (AR) and designated it androgen receptor N-terminal domain transactivating protein-1 (ANT-1). This coactivator, which contains multiple tetratricopeptide repeat (TPR) motifs from amino acid (aa) 294, is identical to a component of U5 small nuclear ribonucleoprotein particles and binds specifically to the AR or glucocorticoid receptor. Here, we identified four distinct functional domains. The AR-AF-1-binding domain, which bound to either aa 180–360 or 360–532 in AR-AF-1, clearly overlapped with TAU-1 and TAU-5. This domain and the subnuclear speckle formation domain in ANT-1 were assigned within the TPR motifs, while the transactivating and nuclear localization signal domains resided within the N-terminal sequence. The existence of these functional domains may further support the idea that ANT-1 can function as an AR-AF-1-specific coactivator while mediating a transcription-splicing coupling.

Mutational Analysis of the Catalytic Domain of O-Linked N-Acetylglucosaminyl Transferase

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.

Structure and ESCRT-III protein interactions of the MIT domain of human VPS4A.

The VPS4 AAA ATPases function both in endosomal vesicle formation and in the budding of many enveloped RNA viruses, including HIV-1. VPS4 proteins act by binding and catalyzing release of the membrane-associated ESCRT-III protein lattice, thereby allowing multiple rounds of protein sorting and vesicle formation. Here, we report the solution structure of the N-terminal VPS4A microtubule interacting and transport (MIT) domain and demonstrate that the VPS4A MIT domain binds the C-terminal half of the ESCRT-III protein, CHMP1B (Kd = 20 +/- 13 microM). The MIT domain forms an asymmetric three-helix bundle that resembles the first three helices in a tetratricopeptide repeat (TPR) motif. Unusual interhelical interactions are mediated by a series of conserved aromatic residues that form coiled-coil interactions between the second two helices and also pack against the conserved alanines that interdigitate between the first two helices. Mutational analyses revealed that a conserved leucine residue (Leu-64) on the third helix that would normally bind the fourth helix in an extended TPR is used to bind CHMP1B, raising the possibility that ESCRT-III proteins may bind by completing the TPR motif.

Redefining the Role of the Endogenous XAP2 and C-terminal hsp70-interacting Protein on the Endogenous Ah Receptors Expressed in Mouse and Rat Cell Lines

Studies using transient expression systems have implicated the XAP2 protein in the control of aryl hydrocarbon receptor (AHR) stability and subcellular location. Thus, studies were performed in cell lines that expressed endogenous rat or mouse Ah(b-1) (C57BL/6) or Ah(b-2) (C3H) AHRs with similar levels of endogenous XAP2. Unliganded rat and mouse Ah(b-2) receptor complexes associated with reduced levels of XAP2 and exhibited dynamic nucleocytoplasmic shuttling in comparison with Ah(b-1) receptors. Rat and mouse Ah(b-2) receptors also exhibited a greater magnitude of ligand-induced degradation than Ah(b-1) receptors. Small interfering RNA reduction of endogenous XAP2 by >80% had minimal impact on the level of Ah(b-2) receptors but resulted in a 25-30% reduction of Ah(b-1) receptors. XAP2 reduction resulted in increased susceptibility of the Ah(b-1) receptor to ligand-induced degradation yet produced higher levels of endogenous CYP1A1 induction. Stable expression of the Ah(b-2) receptor in the C57BL/6 background resulted in a protein with reduced association with XAP2, dynamic nucleocytoplasmic shuttling, and increased levels of ligand-induced degradation. Small interfering RNA reduction of endogenous XAP2 in a C-terminal hsp70-interacting protein knockout mouse cell line, exhibited a 25-30% reduction in the level of endogenous Ah(b-1) AHR and showed high levels of ligand-induced degradation. Thus, endogenous XAP2 exerts a negative function on a small fraction of the endogenous Ah(b-1) receptor complex but appears to have a minimal impact on endogenous rat or Ah(b-2) receptors. This implies that the analysis of the AHR-mediated signaling via rat and mouse Ah(b-2) receptors may better represent the physiology of this signal transduction pathway.

Regulation of mitochondrial fission and apoptosis by the mitochondrial outer membrane protein hFis1

Mitochondrial fission is a highly regulated process mediated by a defined set of protein factors and is involved in the early stage of apoptosis. In mammals, at least two proteins, the dynamin-like protein DLP1/Drp1 and the mitochondrial outer membrane protein hFis1, participate in mitochondrial fission. The cytosolic domain of hFis1 contains six alpha-helices that form two tetratricopeptide repeat (TPR) motifs. Overexpression of hFis1 induces DLP1-mediated fragmentation of mitochondria, suggesting that hFis1 is a limiting factor in mitochondrial fission by recruiting cytosolic DLP1. In the present study, we identified two regions of hFis1 that are necessary for correct fission of mitochondria. We found that the TPR region of hFis1 participates in the interaction with DLP1 or DLP1-containing complex and that the first helix (alpha1) of hFis1 is required for mitochondrial fission presumably by regulating DLP1-hFis1 interaction. Misregulated interaction between DLP1 and hFis1 by alpha1 deletion induced mitochondrial swelling, in part by the mitochondrial permeability transition, but significantly delayed cell death. Our data suggest that hFis1 is a main regulator of mitochondrial fission, controlling the recruitment and assembly of DLP1 during both normal and apoptotic fission processes.

Novel Structure of the N Terminus in Yeast Fis1 Correlates with a Specialized Function in Mitochondrial Fission

Mitochondrial fission is facilitated by a multiprotein complex assembled at the division site. The required components of the fission machinery in Saccharomyces cerevisiae include Dnm1, Fis1, and Mdv1. In the present study, we determined the protein structure of yeast Fis1 using NMR spectroscopy. Although the six alpha-helices, as well as their folding, in the yeast Fis1 structure are similar to those of the tetratricopeptide repeat (TPR) domains of the human Fis1 structure, the two structures differ in their N termini. The N-terminal tail of human Fis1 is flexible and unstructured, whereas a major segment of the longer N terminus of yeast Fis1 is fixed to the concave face formed by the six alpha-helices in the TPR domains. To investigate the role of the fixed N terminus, exogenous Fis1 was expressed in yeast lacking the endogenous protein. Expression of yeast Fis1 protein rescued mitochondrial fission in delta fis1 yeast only when the N-terminal TPR binding segment was left intact. The presence of this segment is also correlated to the recruitment of Mdv1 to mitochondria. The conformation of the N-terminal segment embedded in the TPR pocket indicates an intra-molecular regulation of Fis1 bioactivity. Although the TPR-like helix bundle of Fis1 mediates the interaction with Dnm1 and Mdv1, the N terminus of Fis1 is a prerequisite to recruit Mdv1 to facilitate mitochondrial fission.

Local and long-range stability in tandemly arrayed tetratricopeptide repeats.

The tetratricopeptide repeat (TPR) is a 34-aa alpha-helical motif that occurs in tandem arrays in a variety of different proteins. In natural proteins, the number of TPR motifs ranges from 3 to 16 or more. These arrays function as molecular scaffolds and frequently mediate protein-protein interactions. We have shown that correctly folded TPR domain proteins, exhibiting the typical helix-turn-helix fold, can be designed by arraying tandem repeats of an idealized TPR consensus motif. To date, three designed proteins, CTPR1, CTPR2, and CTPR3 (consensus TPR number of repeats) have been characterized. Their high-resolution crystal structures show that the designed proteins indeed adopt the typical TPR fold, which is specified by the correct positioning of key residues. Here, we present a study of the thermodynamic properties and folding kinetics of this set of designed proteins. Chemical denaturation, monitored by CD and fluorescence, was used to assess the folding and global stability of each protein. NMR-detected amide proton exchange was used to investigate the stability of each construct at a residue-specific level. The results of these studies reveal a stable core, which defines the intrinsic stability of an individual TPR motif. The results also show the relationship between the number of tandem repeats and the overall stability and folding of the protein.

Disruption of mouse XAB2 gene involved in pre-mRNA splicing, transcription and transcription-coupled DNA repair results in preimplantation lethality

The XAB2 protein (XPA-binding protein 2) with 15 tetratricopeptide repeat motifs has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A (XPA) protein in the yeast two-hybrid system. It has been shown that XAB2 interacted with Cockayne syndrome groups A and B (CSA and CSB) proteins and RNA polymerase II, which are known to be involved in transcription-coupled repair (TCR) and transcription, and that the antibodies against XAB2 protein inhibited the recovery of RNA synthesis after UV irradiation and normal RNA synthesis when microinjected into living fibroblasts. These results have indicated that XAB2 is involved in TCR and transcription. In this report, to elucidate the function of XAB2 in vivo, two types of mutations were introduced into the XAB2 gene in mice: a deletion of the region encompassing the promoter and exons 1–4, and a deletion of the C-terminal 162 amino acids. Both types of XAB2-heterozygous mice appeared normal physiologically and behaviorally. However, XAB2-homozygotes were selectively absent among the newborn mice. A detailed analysis of embryos at different stages of development indicated that the XAB2-homozygous mutants could survive until the morula stage, but could not develop to the blastocyst stage. These results indicate that XAB2 has an essential function in mouse embryogenesis.