Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes

The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.     

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa

Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.     

Structure of mouse major urinary protein genes: different splicing configurations in the 3'-non-coding region.

The multigene family which codes for the mouse major urinary proteins (MUPs) consists of approximately 35 genes. Most of these are members of two different groups, Group 1 and Group 2, which can be distinguished by nucleic acid hybridisation. Here we describe the structure of a Group 1 gene and show that two size classes of MUP mRNA which are found in mouse liver result from different splicing events in the 3''-non-coding region and contain different polyadenylation sites. Short mRNA is approximately 750 nucleotides long, contains six exons, and is the main product of the Group 2 genes. Long mRNA is approximately 880 nucleotides long, contains seven exons and is the main product of the Group 1 genes. Five exons and part of the sixth are common to long and short mRNA and contain the coding region. This codes for an acidic protein of 180 amino acids containing an 18 residue signal peptide. A comparison of the mouse sequence with a homologous rat alpha 2u-globulin sequence shows that the rate of evolutionary divergence of the two proteins has been high. Silent sites have diverged four times more rapidly than replacement sites, showing that there has been selection against change in the protein sequence.     

PL-I of Spisula solidissima, a highly elongated sperm-specific histone H1

The major chromosomal protein of the mature sperm of the surf clam, Spisula solidissima, is a histone H1-related protamine-like (PL-I) protein of low electrophoretic mobility. We report here the complete sequence of two isoforms of its encoding genes. These genes encode a protein of 453 and 454 amino acids, respectively. The predicted mass of the larger isoforms (51,437 Da) was confirmed using electrospray ionization mass spectrometry. The amino-terminal tail of the S. solidissima PL-I is greatly elongated because of the presence of 39 tandem hexapeptide repeats of the motif (K/R)KRSAS with a few semiconservative amino acid substitutions. These repeats are very closely mirrored by their encoding DNA sequence, which indicates that an expansion because of sequence duplication most likely occurred. The C-terminal domain consists of a histone H1-related core with a predicted winged-helix tertiary structure, which is followed by an unstructured lysine-rich tail. This information provides additional molecular support for the classification and underlying evolution of sperm nuclear basic proteins in bivalve molluscs.     

Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell.     

MOLECULAR EVOLUTION OF EUGLENOZOAN PARAXONEMAL ROD GENES par1 AND par2 COINCIDES WITH PHYLOGENETIC RECONSTRUCTION BASED ON SMALL SUBUNIT rDNA DATA1

Emergent flagella of Euglenozoa consist of two prominent structural elements: the axoneme built by microtubules with motor proteins to enable the movement of the flagellum and a highly organized protein structure of unknown function, called the paraxonemal rod (PAR), which consists of two major proteins paralleling the axoneme of euglenid and kinetoplastid emergent flagella. These flagellar structures are considered apomorphic characters of Euglenozoa. We examined the evolution of the genes par1 and par2 encoding the two major proteins, where we could show that these proteins are encoded by two very similar genes found in kinetoplastids and euglenids. The branching pattern indicated a gene duplication before the diversification into euglenids and kinetoplastids. In the clades of the genes, subtrees of euglenid and kinetoplastid monophyla arose. Both genes showed strong genetic diversity with biased GC content at taxon rather than at gene level. We also examined phylogenies inferred from PAR genes that are well in agreement with established small subunit rDNA analyses. Both showed further separation of the euglenid subtree into primary osmotrophs and a phototrophic clade, including secondarily derived osmotrophs.     

Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber.

Spider dragline silk is a unique protein fiber possessing both high tensile strength and high elasticity. A partial cDNA clone for one dragline silk protein (Spidroin 1) was previously isolated. However, the predicted amino acid sequence could not account for the amino acid composition of dragline silk. We have isolated a partial cDNA clone for another dragline silk protein (Spidroin 2), demonstrating that dragline silk is composed of multiple proteins. The amino acid sequence exhibits an entirely different repetitive motif than Spidroin 1. Spidroin 2 is predicted to consist of linked beta-turns in proline-rich regions which alternate with beta-sheet regions composed of polyalanine segments. This structure for Spidroin 2 provides a model for dragline silk structure and function.     

Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi

Background

Trypanosoma cruzi, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility.

Methodology/Principal Findings

Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea.

Conclusions/Significance

Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating.     

Computer modeling studies of the structure of a repressor

Due to advances in molecular biology the DNA sequences of structural genes coding for proteins are often known before a protein is characterized or even isolated. The function of a protein whose amino acid sequence has been deduced from a DNA sequence may not even be known. This has created greater interest in the development of methods to predict the tertiary structures of proteins. The a priori prediction of a protein's structure from its amino acid sequence is not yet possible. However, since proteins with similar amino acid sequences are observed to have similar three-dimensional structures, it is possible to use an analogy with a protein of known structure to draw some conclusions about the structure and properties of an uncharacterized protein. The process of predicting the tertiary structure of a protein relies very much upon computer modeling and analysis of the structure. The prediction of the structure of the bacteriophage 434 cro repressor is used as an example illustrating current procedures.     

A Score of the Ability of a Three-Dimensional Protein Model to Retrieve Its Own Sequence as a Quantitative Measure of Its Quality and Appropriateness

Background

Despite the remarkable progress of bioinformatics, how the primary structure of a protein leads to a three-dimensional fold, and in turn determines its function remains an elusive question. Alignments of sequences with known function can be used to identify proteins with the same or similar function with high success. However, identification of function-related and structure-related amino acid positions is only possible after a detailed study of every protein. Folding pattern diversity seems to be much narrower than sequence diversity, and the amino acid sequences of natural proteins have evolved under a selective pressure comprising structural and functional requirements acting in parallel.

Principal Findings

The approach described in this work begins by generating a large number of amino acid sequences using ROSETTA [Dantas G et al. (2003) J Mol Biol 332:449–460], a program with notable robustness in the assignment of amino acids to a known three-dimensional structure. The resulting sequence-sets showed no conservation of amino acids at active sites, or protein-protein interfaces. Hidden Markov models built from the resulting sequence sets were used to search sequence databases. Surprisingly, the models retrieved from the database sequences belonged to proteins with the same or a very similar function. Given an appropriate cutoff, the rate of false positives was zero. According to our results, this protocol, here referred to as Rd.HMM, detects fine structural details on the folding patterns, that seem to be tightly linked to the fitness of a structural framework for a specific biological function.

Conclusion

Because the sequence of the native protein used to create the Rd.HMM model was always amongst the top hits, the procedure is a reliable tool to score, very accurately, the quality and appropriateness of computer-modeled 3D-structures, without the need for spectroscopy data. However, Rd.HMM is very sensitive to the conformational features of the models'' backbone.     

Assembly of the Paraflagellar Rod and the Flagellum Attachment Zone Complex During the Trypanosoma brucei Cell Cycle

Trypanosomes possess a single flagellum that is attached to their cell body via the flagellum attachment zone (FAZ). The FAZ is composed of two structures: a cytoplasmic filament complex and four microtubules situated next to it. There is a complex transmembrane crosslinking of this FAZ to the paraflagellar rod (PFR) and axoneme within the flagellum. We have partially purified the FAZ complex and have produced monoclonal antibodies both against the FAZ and the paraflagellar rod. The two antibodies against the FAZ (L3B2 and L6B3) recognise the cytoplasmic filament in immunofluorescence and in immunoelectron microscopy. On western blot, they detect a doublet of high molecular weight (M(r) 200,000). Two anti-PFR antibodies (L13D6 and L8C4) recognise the paraflagellar rod in immunofluorescence, but show a difference on Western blot: L13D6 recognises both major PFR proteins, whereas L8C4 is specific for only one of them. Using these new antibodies we have shown that although the growth of both cytoplasmic FAZ filament and external PFR are related, their growth initiates at different time points during the cell cycle and the two structures elongate at distinct rates.     

The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes.     

Accurate prediction of protein folding rates from sequence and sequence‐derived residue flexibility and solvent accessibility

Protein folding rates vary by several orders of magnitude and they depend on the topology of the fold and the size and composition of the sequence. Although recent works show that the rates can be predicted from the sequence, allowing for high‐throughput annotations, they consider only the sequence and its predicted secondary structure. We propose a novel sequence‐based predictor, PFR‐AF, which utilizes solvent accessibility and residue flexibility predicted from the sequence, to improve predictions and provide insights into the folding process. The predictor includes three linear regressions for proteins with two‐state, multistate, and unknown (mixed‐state) folding kinetics. PFR‐AF on average outperforms current methods when tested on three datasets. The proposed approach provides high‐quality predictions in the absence of similarity between the predicted and the training sequences. The PFR‐AF's predictions are characterized by high (between 0.71 and 0.95, depending on the dataset) correlation and the lowest (between 0.75 and 0.9) mean absolute errors with respect to the experimental rates, as measured using out‐of‐sample tests. Our models reveal that for the two‐state chains inclusion of solvent‐exposed Ala may accelerate the folding, while increased content of Ile may reduce the folding speed. We also demonstrate that increased flexibility of coils facilitates faster folding and that proteins with larger content of solvent‐exposed strands may fold at a slower pace. The increased flexibility of the solvent‐exposed residues is shown to elongate folding, which also holds, with a lower correlation, for buried residues. Two case studies are included to support our findings. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The conservation profile of a protein bears the imprint of the molecule that is evolutionarily coupled to the protein

The conservation profile of a protein is a curve of the conservation levels of amino acids along the sequence. Biologists are usually more interested in individual points on the curve (namely, the conserved amino acids) than the overall shape of the curve. Here, we show that the conservation curves of proteins bear the imprints of molecules that are evolutionarily coupled to the proteins. Our method is based on recent studies that a sequence conservation profile is quantitatively linked to its structural packing profile. We find that the conservation profiles of nucleic acid (NA) binding proteins are better correlated with the packing profiles of the protein–NA complexes than those of the proteins alone. This indicates that a nucleic acid binding protein evolves to accommodate the nucleic acid in such a way that the residues involved in binding have their conservation levels closely coupled with the specific nucleotides. Proteins 2015; 83:1407–1413. © 2015 Wiley Periodicals, Inc.