Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits.

The collagen prolyl 4-hydroxylases (P4Hs) are essential for proper extracellular matrix formation in multicellular organisms. The vertebrate enzymes are alpha(2)beta(2) tetramers, in which the beta subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the Caenorhabditis elegans catalytic alpha subunit isoforms PHY-1 and PHY-2 and the beta subunit PDI-2. A mixed PHY-1/PHY-2/(PDI-2)(2) tetramer is the major form, while PHY-1/PDI-2 and PHY-2/PDI-2 dimers are also assembled but less efficiently. Cloning and characterization of the orthologous subunits from the closely related nematode Caenorhabditis briggsae revealed distinct differences in the assembly of active P4H forms in spite of the extremely high amino acid sequence identity (92-97%) between the C. briggsae and C. elegans subunits. In addition to a PHY-1/PHY-2(PDI-2)(2) tetramer and a PHY-1/PDI-2 dimer, an active (PHY-2)(2)(PDI-2)(2) tetramer was formed in C. briggsae instead of a PHY-2/PDI-2 dimer. Site-directed mutagenesis studies and generation of inter-species hybrid polypeptides showed that the N-terminal halves of the Caenorhabditis PHY-2 polypeptides determine their assembly properties. Genetic disruption of C. briggsae phy-1 (Cb-dpy-18) via a Mos1 insertion resulted in a small (short) phenotype that is less severe than the dumpy (short and fat) phenotype of the corresponding C. elegans mutants (Ce-dpy-18). C. briggsae phy-2 RNA interference produced no visible phenotype in the wild type nematodes but produced a severe dumpy phenotype and larval arrest in phy-1 mutants. Genetic complementation of the C. briggsae and C. elegans phy-1 mutants was achieved by injection of a wild type phy-1 gene from either species.     

Egg shell collagen formation in Caenorhabditis elegans involves a novel prolyl 4-hydroxylase expressed in spermatheca and embryos and possessing many unique properties

The collagen prolyl 4-hydroxylases (EC ) play a critical role in the synthesis of all collagens. The enzymes from all vertebrate species studied are alpha(2)beta(2) tetramers, in which the beta subunit is identical to protein disulfide isomerase (PDI). Two isoforms of the catalytic alpha subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. We report here on the cloning and characterization of a third C. elegans alpha subunit isoform, PHY-3. It is much shorter than the previously characterized vertebrate and C. elegans alpha subunits and shows 23-30% amino acid sequence identity to PHY-1 and PHY-2 within the catalytic C-terminal region. Recombinant PHY-3 coexpressed in insect cells with a C. elegans PDI isoform that does not associate with PHY-1 was found to be an active prolyl 4-hydroxylase. The phy-3 gene consists of five exons, and its expression pattern differs distinctly from the hypodermally expressed phy-1 and phy-2 in that it is expressed in embryos, late larval stages, and adult nematodes, expression in the latter being restricted to the spermatheca. Nematodes homozygous for a phy-3 deletion are phenotypically of the wild type and fertile, but the 4-hydroxyproline content of phy-3(-/-) early embryos was reduced by about 90%. PHY-3 is thus likely to be involved in the synthesis of collagens in early embryos, probably of those in the egg shell.     

The exoskeleton collagens in Caenorhabditis elegans are modified by prolyl 4-hydroxylases with unique combinations of subunits

The collagen prolyl 4-hydroxylases (P4Hs, EC ) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are alpha(2)beta(2) tetramers, in which protein disulfide isomerase (PDI) serves as the beta subunit. Two conserved alpha subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single beta subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2)(2) mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2-/- and phy-1-/- (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln(121)-Ala(271) and Asp(1)-Leu(122) in PHY-1 and PHY-2, respectively, are critical for its assembly.     

Protein kinase C modulates light responses in Neurospora by regulating the blue light photoreceptor WC-1

The Neurospora protein kinase C (NPKC) is a regulator of light responsive genes. We have studied the function of NPKC in light response by investigating its biochemical and functional interaction with the blue light photoreceptor white-collar 1 (WC-1), showing that activation of NPKC leads to a significant decrease in WC-1 protein levels. Furthermore, we show that WC-1 and NPKC interact in a light-regulated manner in vivo, and that protein kinase C (PKC) phosphorylates WC-1 in vitro. We designed dominant negative and constitutively active forms of PKC which are able to induce either a large increase of WC-1 protein level or a strong reduction respectively. Moreover, these changes in PKC activity result in an altered light response. As WC-1 is a key component of Neurospora circadian clock and regulates the clock oscillator component FRQ we investigated the effect of NPKC-mutated forms on FRQ levels. We show that changes in PKC activity affect FRQ levels and the robustness of the circadian clock. Together these data identify NPKC as a novel component of the Neurospora light signal transduction pathway that modulates the circadian clock.     

Plasmids supplying the Q-qut-controlled gam function permit growth of lambda red- gam- (Fec-) bacteriophages on recA- hosts

A plasmid, pgam, has been constructed which expresses the phage lambda gene, gam, under the control of the lambda late promoter, p'R, contained in a form of a p'R-qut-t'R1 module. Lambda red- gam-, which normally do not grow on recA- hosts, are able to grow on recA- hosts containing pgam, because their Q function can turn on the gam gene expression. This facilitates cloning with lambda red- gam- vectors in recA- hosts.     

Characterization of two thermostable cyanobacterial phytochromes reveals global movements in the chromophore-binding domain during photoconversion

Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B', that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. (1)H-(15)N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.     

Collagen prolyl 4-hydroxylase tetramers and dimers show identical decreases in Km values for peptide substrates with increasing chain length: mutation of one of the two catalytic sites in the tetramer inactivates the enzyme by more than half

The collagen prolyl 4-hydroxylases (collagen P4Hs, EC 1.14.11.2) play a key role in the synthesis of the extracellular matrix. The vertebrate enzymes are alpha(2)beta(2) tetramers, the beta subunit being identical to protein disulfide isomerase (PDI). The main Caenorhabditis elegans collagen P4H form is an unusual PHY-1/PHY-2/(PDI)(2) mixed tetramer consisting of two types of catalytic alpha subunit, but the PHY-1 and PHY-2 polypeptides also form active PHY/PDI dimers. The lengths of peptide substrates have a major effect on their interaction with the P4H tetramers, the K(m) values decreasing markedly with increasing chain length. This phenomenon has been explained in terms of processive binding of the two catalytic subunits to long peptides. We determined here the K(m) values of a collagen P4H having two catalytic sites, the C. elegans mixed tetramer, and a form having only one such site, the PHY-1/PDI dimer, for peptides of varying lengths. All the K(m) values of the PHY-1/PDI dimer were found to be about 1.5-2.5 times those of the tetramer, but increasing peptide length led to identical decreases in the values of both enzyme forms. The K(m) for a nonhydroxylated collagen fragment with 33 -X-Y-Gly-triplets but only 11 -X-Pro-Gly-triplets was found to correspond to the number of the former rather than the latter. To study the individual roles of the two catalytic sites in a tetramer, we produced mutant PHY-1/PHY-2/(PDI)(2) tetramers in which binding of the Fe(2+) ion or 2-oxoglutarate to one of the two catalytic sites was prevented. The activities of the mutant tetramers decreased to markedly less than 50% of that of the wild type, being about 5-10% and 20-30% with the enzymes having one of the two Fe(2+)-binding sites or 2-oxoglutarate-binding sites inactivated, respectively, while the K(m) values for these cosubstrates or peptide substrates were not affected. Our data thus indicate that although collagen P4Hs do not act on peptide substrates by a processive mechanism, prevention of hydroxylation at one of the two catalytic sites in the tetramer impairs the function of the other catalytic site.     

Tools for Fungal Proteomics: Multifunctional Neurospora Vectors for Gene Replacement, Protein Expression and Protein Purification

The completion of genome-sequencing projects for a number of fungi set the stage for detailed investigations of proteins. We report the generation of versatile expression vectors for detection and isolation of proteins and protein complexes in the filamentous fungus Neurospora crassa. The vectors, which can be adapted for other fungi, contain C- or N-terminal FLAG, HA, Myc, GFP, or HAT–FLAG epitope tags with a flexible poly-glycine linker and include sequences for targeting to the his-3 locus in Neurospora. To introduce mutations at native loci, we also made a series of knock-in vectors containing epitope tags followed by the selectable marker hph (resulting in hygromycin resistance) flanked by two loxP sites. We adapted the Cre/loxP system for Neurospora, allowing the selectable marker hph to be excised by introduction of Cre recombinase into a strain containing a knock-in cassette. Additionally, a protein purification method was developed on the basis of the HAT–FLAG tandem affinity tag system, which was used to purify HETEROCHROMATIN PROTEIN 1 (HP1) and associated proteins from Neurospora. As expected on the basis of yeast two-hybrid and co-immunoprecipitation (Co-IP) experiments, the Neurospora DNA methyltransferase DIM-2 was found in a complex with HP1. Features of the new vectors allowed for verification of an interaction between HP1 and DIM-2 in vivo by Co-IP assays on proteins expressed either from their native loci or from the his-3 locus.     

Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases

Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns. Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E. coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs. This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing. Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation. Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.     

Evidence that the N-terminal region of A1-light chain of myosin interacts directly with the C-terminal region of actin. A proton magnetic resonance study

Earlier 1H-NMR experiments on the myosin subfragment-1 (S1) light chain isoenzymes from rabbit fast muscle, containing either the A1 or the A2 alkali light chains [S1(A1) or S1(A2)], have shown that the 41-residue N-terminal extension of A1, rich in proline, alanine and lysine residues, is freely mobile in solution but that this mobility is constrained in the acto-S1(A1) complex [Prince et al. (1981) Eur. J. Biochem. 121, 213-219]. It is now established that this N-terminal region of the A1-light chain interacts directly with the C-terminal region of actin in the acto-S1(A1) complex. This was shown by covalently labelling the Cys-374 residue of actin with a spin-label and observing the enhanced relaxation this paramagnetic centre induced in the 1H-NMR spectrum of S1(A1). In particular, the signal arising from the -N+(CH3)3 protons of alpha-N-trimethylalanine (Me3Ala) were monitored as this residue is uniquely sited at the N-terminus of the A1 light chain [Henry et al. (1982) FEBS Lett. 144, 11-15]. Experiments using complexes of actin with either the N-terminal 37-residue peptide of A1, S1(A1) or heavy meromyosin indicate that the N-terminal region of A1 is binding in a similar manner to actin in each case, with the N-terminal Me3Ala residue within 1.5 nm of the spin label introduced to Cys-374 of actin. A similar strategy was adopted to show that the Me3Ala residue can also be found close (less than 1.5 nm) to the fast-reacting SH1 thiol group on the S1 heavy chain. These data, together with published work, have been used to suggest a possible organisation for the polypeptide chains in the myosin head.     

Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa

Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.     

Bacterial bilin- and flavin-binding photoreceptors

The growing number of sequenced prokaryotic genomes reveals a wide distribution of open reading frames (ORFs) that putatively encode for red- and blue light sensing photoreceptors. They comprise the bilin-binding phytochromes and the flavin-binding cryptochromes, LOV and BLUF proteins, indicating that about 1/4 of bacteria do possess at least one of these photosensory proteins. The distribution of red- and blue-light sensors among different prokaryotic phyla and classes, and their functional activity as light-switched systems are the subject of this perspective. These photoreceptors were originally found in plants by following the associated physiological responses induced by the respective spectral irradiation. Genome-based approaches now require the assignment of a photochemical/physiological function to the heterologously expressed gene product. Database searches demonstrate in some cases several genes of one category in a certain prokaryot, indicating the presence of more than one type of red- or blue-light sensing properties, but also show a combination of proteins with both spectral sensitivities. Another interesting feature now "comes into light": according to their nature as biological sensors, these photoreceptors are equipped with signalling domains, initiating a cellular response, thereby constituting modular systems switchable by light. It is seen that many of these signalling domains, now found together with light-inducible sensing domains, were already described for other stimuli, e.g., osmo-regulation, oxygen, hydrogen, chemicals, or pH. In some cases, the same type of signalling domain can be found in a red- or a blue-light sensing photoreceptor. Following the characterization of their photochemistry, for several of these bacterial photoreceptors physiological functions are now assigned.     

Relationship between two major immunoreactive forms of arginase in Neurospora crassa.

Two major immunoreactive proteins of Mr 41,700 and 36,100 have been detected in crude mycelial extracts with polyclonal antibodies raised against arginase purified from Neurospora crassa. The latter corresponded to the protein used to obtain the antibodies. Both polypeptides were either missing or present in very low amounts in mutant strains having little or no detectable arginase activity. The relative proportion of the two species was altered in strains containing the nitrogen catabolite regulatory mutation nit-2. Peptide mapping indicated that the two species are very closely related, but several of the peptides which appeared to be identical by staining reacted differently with the antibodies. Both species were produced by in vitro translation of poly(A)+ mRNA, although the larger species was produced to a much smaller extent than was expected from its abundance in vivo. The results suggest the existence of multiple forms of arginase in N. crassa which differ in their response to nitrogen catabolite regulation.     

Intrachain disulphide bridges in immunoglobulin G heavy chains. The Fc fragment

The disulphide bridges of the Fc fragment (C-terminal half of the heavy chain) have been studied in several human immunoglobulins, containing heavy chains of different antigenic types (gamma1, gamma2, gamma3 and gamma4), and in heavy-chain-disease proteins. Two intrachain disulphide bridges were found to be present. The sequences appear to be identical in the Fc fragments of two types of chain studied (gamma1 and gamma3), and very similar to corresponding sequences of the Fc fragment in rabbit. These results suggest that the C-terminal half of the heavy chains is covalently folded (in a similar fashion to the light chains) with a C-terminal loop and an N-terminal loop. The similarity is emphasized by comparison of the sequence and location of the disulphide-bridged peptides of the C-terminal loop of heavy and light chains. The N-terminal loop, on the other hand, appears to be very different in Fc fragments and light chains. The C-terminal loop is the only one present in the F'c fragment.     

Secretory protein translocation in a neurospora crassa in vitro system. Hydrolysis of a nucleoside triphosphate is required for posttranslational translocation

An in vitro translocation system has been reconstituted with subcellular fractions from the cell wall-less mutant of Neurospora crassa (fz;sg;os-1). Prepro alpha factor and invertase, secretory proteins from yeast, were faithfully translocated and glycosylated by Neurospora microsomes when presence cotranslationally in the Neurospora translation system. When presence cotranslationally in the Neurospora translation system, microsomes from canine pancreas(cRM) could also translocate and glycosylate the secretory proteins. However, salt-extracted cRM, which is depleted of canine signal recognition particle, could not. Furthermore, prepro alpha factor and a truncated form of invertase, containing the first 262-amino acid residues of the secretory invertase, were glycosylated by Neurospora microsomes posttranslationally, whereas only the truncated form of invertase was glycosylated by cRM when added posttranslationally. The full length invertase was not glycosylated posttranslationally. Posttranslational glycosylation of prepro alpha factor and of the truncated form of invertase is dependent on the hydrolysis of a nucleoside triphosphate. These data suggest that posttranslational glycosylation of prepro alpha factor occurs via a novel type of recognition mechanism which is either absent or ineffective in cRM.