TPR-containing Proteins Research Articles

Different combinations of the heat-shock cognate protein 70 (hsc70) C-terminal functional groups are utilized to interact with distinct tetratricopeptide repeat-containing proteins.

A group of tetratricopeptide repeat (TPR)-containing proteins has been shown to interact with the C-terminal domain of the 70 kDa heat-shock cognate protein (hsc70). In the present study, the effect of the TPR-containing proteins, including the C-terminus of hsc70-interacting protein (CHIP), TPR1 and human glutamine-rich TPR-containing protein (hSGT), on refolding of luciferase by DnaJ and hsc70 was investigated. These proteins inhibited the restoration of luciferase activity by the chaperones. The inhibitory effect exerted by TPR1 and hSGT depended upon their binding to hsc70. However, the interaction with hsc70 did not appear to be required for the inhibition of luciferase refolding by CHIP. We also demonstrate that the peptide, GPTIEEVD, corresponding to the C-terminal end of hsc70, abolished the association of [(3)H]hsc70 with CHIP, TPR1 and hSGT. This implied that the GPTIEEVD motif of hsc70 was responsible for interacting with these TPR-containing proteins. However, the GGXP-repeats (where X is any aliphatic residue), another C-terminal conserved motif of vertebrate hsc70s, were not essential for interacting with the TPR-containing proteins. On the basis of mutagenesis studies, it was clear that a unique combination of the functional groups in the GPTIEEVD motif were utilized to interact with each TPR-containing protein, suggesting that inhibitors can be designed and used to elucidate the functional role of these interactions.

A Pollen-specific Novel Calmodulin-binding Protein with Tetratricopeptide Repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

The tetratricopeptide repeat: a structural motif mediating protein-protein interactions.

The tetratricopeptide repeat (TPR) motif is a protein-protein interaction module found in multiple copies in a number of functionally different proteins that facilitates specific interactions with a partner protein(s). Three-dimensional structural data have shown that a TPR motif contains two antiparallel alpha-helices such that tandem arrays of TPR motifs generate a right-handed helical structure with an amphipathic channel that might accommodate the complementary region of a target protein. Most TPR-containing proteins are associated with multiprotein complexes, and there is extensive evidence indicating that TPR motifs are important to the functioning of chaperone, cell-cycle, transcription, and protein transport complexes. The TPR motif may represent an ancient protein-protein interaction module that has been recruited by different proteins and adapted for specific functions. BioEssays 1999;21:932-939.

Tpr1, a Schizosaccharomyces pombe protein involved in potassium transport

The Schizosaccharomyces pombe Tpr1 was isolated as suppressor of the Saccharomyces cerevisiae Δ trk1,2 potassium uptake deficient phenotype. Tpr1, for tetratrico peptide repeat, encodes a 1039 amino acid residues protein with several reiterated TPR units displaying significant homology to p150 TSP, a recently identified phosphoprotein of mouse, to S. cerevisiae CTR9 and to related sequences of human, Caenorhabditis elegans, Methanoccocus jannaschii and Arabidopsis thaliana. Expression of Tpr1 restored growth on 0.2 mM K + media, induced K + transport with a K T of 4.6 mM and resumed inward currents of −90 pÅ at −250 mV (pH 7.2) conducting K + and other alkali-metal ions. The tetratrico peptide repeat is a degenerate motif of 34 amino acids that is repeated several times within TPR-containing proteins and has been suggested to mediate protein-protein interactions. The sequence and putative binding properties of Tpr1 suggest the protein unlikely as transporter but involved in the enhancement of K + uptake via conventional carriers.

A novel candidate presenilin-1 interacting protein containing tetratricopeptide repeats.

The yeast two-hybrid system, immunofluorescence and co-immunoprecipitation techniques were used to identify a novel candidate protein with which presenilin-1 (PS-1) interacts. This interacting protein, the gene of which is encoded on chromosome 16, contains two tetratricopeptide repeats (TPR) that are known to mediate interactions between proteins, appears to be primarily localized to the cytoplasm of transfected HEK293 cells, and is expressed in brain. Preliminary yeast two-hybrid data suggests this candidate may interact with both heat shock protein-90 and heat shock protein-70 and thus may be a novel member of TPR-containing proteins which interact with this complex.

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions.

The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3-16 motifs, which form scaffolds to mediate protein-protein interactions and often the assembly of multiprotein complexes. TPR-containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90-binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel alpha-helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel alpha-helices. The uniform angular and spatial arrangement of neighbouring alpha-helices defines a helical structure and creates an amphipathic groove. Multiple-TPR motif proteins would fold into a right-handed super-helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of alpha-helices in the PP5-TPR domain is similar to those within 14-3-3 proteins.

Differential interactions of p23 and the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90 mutants.

Hsp90 is required for the normal function of steroid receptors, but its binding to steroid receptors is mediated by Hsc70 and several hsp-associated accessory proteins. An assortment of Hsp90 mutants were tested for their abilities to interact with each of the following accessories: Hop, Cyp40, FKBP52, FKBP51, and p23. Of the 11 Hsp90 mutants tested, all were defective to some extent in associating with progestin (PR) complexes. In every case, however, reduced PR binding correlated with a defect in binding of one or more accessories. Co-precipitation of mutant Hsp90 forms with individual accessories was used to map Hsp90 sequences required for accessory protein interactions. Mutation of Hsp90's highly conserved C-terminal EEVD to AAVD resulted in diminished interactions with several accessory proteins, most particularly with Hop. Deletion of amino acids 661-677 resulted in loss of Hsp90 dimerization and also caused diminished interactions with all accessory proteins. Binding of p23 mapped most strongly to the N-terminal ATP-binding domain of Hsp90 while binding of TPR proteins mapped to the C-terminal half of Hsp90. These results and others further suggest that the N- and C-terminal regions of Hsp90 maintain important conformational links through intramolecular interactions and/or intermolecular influences in homodimers.

P150TSP, a Conserved Nuclear Phosphoprotein That Contains Multiple Tetratricopeptide Repeats and Binds Specifically to SH2 Domains

Src homology 2 (SH2) domains are structural modules that function in the assembly of multicomponent signaling complexes by binding to specific phosphopeptides. The tetratricopeptide repeat (TPR) is a distinct structural motif that has been suggested to mediate protein-protein interactions. Among SH2-binding phosphoproteins purified from the mouse B cell lymphoma A20, a 150-kDa species was identified and the corresponding complementary DNA (cDNA) was molecularly cloned. This protein encoded by this cDNA, which we have termed p150TSP (for TPR-containing, SH2-binding phosphoprotein), is located predominantly in the nucleus and is highly conserved in evolution. The gene encoding p150TSP (Tsp) was mapped to chromosome 7 of the mouse with gene order: centromere-Tyr-Wnt11-Tsp-Zp2. The amino-terminal two-thirds of p150TSP consist almost entirely of tandemly arranged TPR units, which mediate specific, homotypic protein interactions in transfected cells. The carboxyl-terminal third of p150TSP, which is serine- and glutamic acid-rich, is essential for SH2 binding; this interaction is dependent on serine/threonine phosphorylation but independent of tyrosine phosphorylation. The sequence and binding properties of p150TSP suggest that it may mediate interactions between TPR-containing and SH2-containing proteins.

The Tetratricopeptide Repeat Domain of the PAS10 Protein of Saccharomyces cerevisiae Is Essential for Binding the Peroxisomal Targeting Signal -SKL

The PAS10 gene was found in a two-hybrid screen for the isolation of genes encoding proteins which interact with the C-terminal peroxisomal targeting signal -SKL. The PAS10 protein is known to be involved in import of proteins into peroxisomes and to contain a tetratricopeptide repeat (TPR) domain. All TPR-containing proteins involved in diverse processes like mitosis or RNA-synthesis share the ability to interact with other proteins. Here we show that the PAS10 protein interacts in vivo with the C-terminal peroxisomal targeting signal. The part essential for this interaction contains the complete tetratricopeptide repeat domain.

The crooked neck gene of Drosophila contains a motif found in a family of yeast cell cycle genes.

The crooked neck (crn) gene of Drosophila encodes a protein of 702 amino acids and contains 16 tandemly arranged copies of a 34-amino-acid repeat that is similar to the tetratrico peptide repeat (TPR). Multiple copies of the TPR motif have also been found in a family of yeast genes, including several members that are necessary for cell division. TPR-containing proteins encoded by the yeast genes CDC16, CDC23, and nuc2+ are required for progression through the G2/M transition of the cell cycle. Loss of zygotic expression of crn causes defects in the proliferation of brain neuroblasts and results in the absence of identified neuronal lineages in the central and peripheral nervous systems. The sequence similarity and mutant phenotypes are consistent with a cell cycle requirement for the crn gene product.