Dynamics of the assembly of a complex macromolecular structure--the coat of spores of the bacterium Bacillus subtilis

The coat is the outermost layer of spores of many Bacillus species, and plays a key role in these spores' resistance. The Bacillus subtilis spore coat contains > 70 proteins in four distinct layers: the basement layer, inner coat, outer coat and crust. In this issue of Molecular Microbiology, McKenney and Eichenberger study the dynamics of spore coat assembly using GFP-fusions to 41 B. subtilis coat proteins. A key finding in the work is that formation of the spore coat is initiated by the apparently simultaneous assembly of foci of proteins from all four coat layers on the developing spore just as forespore engulfment by the mother cell begins. The expansion of these foci before completion of forespore engulfment then sets up the scaffold to which coat proteins added later in sporulation are added. This study provides new understanding of the mechanism of the assembly of a multi-protein, multi-lamellar structure.     

Jelly coat and induction of the acrosome reaction in echinoid sperm

In a previous study we established that sperm from four species of echinoids differ in their specificity for induction of the acrosome reaction by heterotypic jelly coat and we presented evidence that there were only small compositional differences in the active component of the jelly coat, a polysaccharide composed of fucose sulfate units. In the current report we present additional studies related to the species specificity of jelly coat with respect to Ca²⁺ uptake (or exchange), which occurs concomitantly with the acrosome reaction, and activation of phospholipase activity, which appears to occur subsequent to the acrosome reaction. The specificity of jelly coat in inducing these processes is the same as that previously observed in induction of the acrosome reaction. Binding of jelly coat to sperm has been demonstrated, and has been shown to be species specific. This finding raises the possibility that a receptor for jelly coat exists on the surface of the sperm. Finally, based on chemical and physical-chemical studies, evidence is presented that establishes that, despite compositional similarities, the fucose sulfate polysaccharides from the four species of eggs differ in structure.     

Seven novel KIT mutations in horses with white coat colour phenotypes

White coat colour in horses is inherited as a monogenic autosomal dominant trait showing a variable expression of coat depigmentation. Mutations in the KIT gene have previously been shown to cause white coat colour phenotypes in pigs, mice and humans. We recently also demonstrated that four independent mutations in the equine KIT gene are responsible for the dominant white coat colour phenotype in various horse breeds. We have now analysed additional horse families segregating for white coat colour phenotypes and report seven new KIT mutations in independent Thoroughbred, Icelandic Horse, German Holstein, Quarter Horse and South German Draft Horse families. In four of the seven families, only one single white horse, presumably representing the founder for each of the four respective mutations, was available for genotyping. The newly reported mutations comprise two frameshift mutations (c.1126_1129delGAAC; c.2193delG), two missense mutations (c.856G>A; c.1789G>A) and three splice site mutations (c.338-1G>C; c.2222-1G>A; c.2684+1G>A). White phenotypes in horses show a remarkable allelic heterogeneity. In fact, a higher number of alleles are molecularly characterized at the equine KIT gene than for any other known gene in livestock species.     

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: implications for binding requirements.

The coat protein of bacteriophage MS2 is known to bind specifically to an RNA hairpin formed within the MS2 genome. Structurally this hairpin is built up by an RNA double helix interrupted by one unpaired nucleotide and closed by a four-nucleotide loop. We have performed crystallographic studies of complexes between MS2 coat protein capsids and four RNA hairpin variants in order to evaluate the minimal requirements for tight binding to the coat protein and to obtain more information about the three-dimensional structure of these hairpins. An RNA fragment including the four loop nucleotides and a two-base-pair stem but without the unpaired nucleotide is sufficient for binding to the coat protein shell under the conditions used in this study. In contrast, an RNA fragment containing a stem with the unpaired nucleotide but missing the loop nucleotides does not bind to the protein shell.     

Changes of ultrastructure in spore coat of Bacillus thiaminolyticus during germination and outgrowth.

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L-alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Spore coat protein and enterotoxin synthesis in Clostridium perfringens.

Polyacrylamide gel profiles of Clostridium perfringens spore coat protein revealed four and occasionally five components. Pulse-chase experiments indicated that synthesis of coat protein polypeptide and enterotoxin was an early sporulation event. However, maximum synthesis occurred coincident with the onset of heat resistance.     

Changes of Ultrastructure in Spore Coat of Bacillus thiaminolyticus during Germination and Outgrowth

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L -alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at a prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Complete amino acid sequence of the coat protein of the Pseudomonas, aeruginosa RNA bacteriophage PP7

As part of a project intending to assess the evolutionary kinship between the RNA coliphages and RNA bacteriophages of other bacterial genera, we have sequenced the coat protein of $\text{Pseudomonas}\text{,}\text{aeruginosa}$ RNA phage PP7. Like the coat proteins of coliphages MS2 and Qβ and of the broad host range RNA phage PRR1, PP7 coat protein (127 residues) is highly hydrophobic, and contains a cluster of basic residues between positions 40 to 60. Minimal mutation distance values were calculated for comparison of PP7 coat protein with each MS2, Qβ and PRR1 coat proteins. Application of the Moore-Goodman criterion to those values, shows that these four RNA bacteriophage coat proteins very likely descent from a common ancestor.     

RNA binding properties of the coat protein from bacteriophage GA.

The coat protein of bacteriophage GA, a group II RNA phage, binds to a small RNA hairpin corresponding to its replicase operator. Binding is specific, with a Ka of 71 microM -1. This interaction differs kinetically from the analogous coat protein-RNA hairpin interactions of other RNA phage and also deviates somewhat in its pH and salt dependence. Despite 46 of 129 amino acid differences between the GA and group I phage R17 coat proteins, the binding sites are fairly similar. The essential features of the GA coat protein binding site are a based-paired stem with an unpaired purine and a four nucleotide loop having an A at position -4 and a purine at -7. Unlike the group I phage proteins, the GA coat protein does not distinguish between two alternate positions for the unpaired purine and does not show high specificity for a pyrimidine at position -5 of the loop.     

Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.

Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented.     

In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs.

The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis.     

Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.     

Biological, Serological and Coat Protein Properties of a Strain of Turnip Mosaic Virus Causing a Mosaic Disease of Brassica campestris and B. juncea in India

Biological, serological and coat protein properties of a potyvirus (Poty-Rape) causing a mosaic disease of Brassica campestris and B. juncea in India were investigated. The virus readily infected 4 of the 5 plant species in the family Brassicaceae in which it induced severe systemic mosaic symptoms; it also induced chlorotic and necrotic local lesions in Chenopodium amaranticolor , but failed to infect 4 other species of Chenopodiaceae or 20 species of Amaranthaceae, Apiaceae, Canabinaceae, Compositae, Cucurbitaceae, Euphorbiaceae, Leguminosae and Solanaceae. The virus was transmitted in a non-persistant manner by Myzus persicae, Brevicoryne brassicae and Aphis gossypii. The Average size, of the virus particles in a purified preparation was 740 nm × 12 nm. SDS-PAGE analysis of the viral coat protein showed two major bands of approximately 37 kDa and 31 kDa, a pattern very similar to that of a reference isolate of turnip mosaic virus (TuMV) from the U.S. In Western-blot immunoassay, an antiserum to TuMV reacted with both the coat protein bands of the Poty-Rape islate and the reference TuMV, but not with the coat proteins of four other potyviruses. The high performance liquid chromatographic profile of tryptic peptides from the coat protein of Poty-Rape was found to be very similar to that of the reference TuMV, but differed substantially from those of four other potyviruses. The Poty-Rape isolate is considered to be a distinct strain of, TuMV.     

Physical aspects of COPI vesicle formation

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.     

Specific RNA binding by Q beta coat protein

The interaction between the bacteriophage Q beta coat protein and its specific binding site on Q beta genomic RNA was characterized by using a nitrocellulose filter binding assay. Q beta coat protein bound to a synthetic 29-nucleotide RNA hairpin with an association constant of 400 microM-1 at 4 degrees C, 0.2 M ionic strength, pH 6.0. Complex formation had a broad pH optimum centered around pH 6.0 and was favored by both enthalpy and entropy. The salt dependence of Ka revealed that four to five ion pairs may be formed in the complex although approximately 80% of the free energy of complex formation is contributed by nonelectrostatic interactions. Truncation experiments revealed that coat protein binding required only the presence of a hairpin with an eight base pair stem and a three-base loop. Analysis of the binding properties of hairpin variants showed that the sequence of the stem was not important for coat protein recognition and only one of the three loop residues was essential. A bulged adenosine present in the coat protein binding site was not required for coat protein binding. Q beta coat protein binding specific is therefore primarily achieved by the structure and not by the sequence of the operator.     

Physical aspects of COPI vesicle formation

Abstract

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.     

Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis.

The Bacillus subtilis spore coat is composed of at least 15 polypeptides plus an insoluble protein fraction arranged in three morphological layers. The insoluble fraction accounts for about 30% of the coat protein and is resistant to solubilization by a variety of reagents, implying extensive cross-linking. A dodecapeptide was purified from this fraction by formic acid hydrolysis and reverse-phase high-performance liquid chromatography. This peptide was sequenced, and a gene designated cotX was cloned by reverse genetics. The cotX gene encoding the dodecapeptide at its amino end was clustered with four other genes designated cotV, cotW, cotY, and cotZ. These genes were mapped to 107 degrees between thiB and metA on the B. subtilis chromosome. The deduced amino acid sequences of the cotY and cotZ genes are very similar. Both proteins are cysteine rich, and CotY antigen was present in spore coat extracts as disulfide cross-linked multimers. There was little CotX antigen in the spore coat soluble fraction, and deletion of this gene resulted in a 30% reduction in the spore coat insoluble fraction. Spores produced by strains with deletions of the cotX, cotYZ, or cotXYZ genes were heat and lysozyme resistant but readily clumped and responded more rapidly to germinants than did spores from the wild type. In electron micrographs, there was a less densely staining outer coat in spores produced by the cotX null mutant, and those produced by a strain with a deletion of the cotXYZ genes had an incomplete outer coat. These proteins, as part of the coat insoluble fraction, appear to be localized to the outer coat and influence spore hydrophobicity as well as the accessibility of germinants.     

Getting in Touch with the Clathrin Terminal Domain

The N‐terminal domain (TD) of the clathrin heavy chain is folded into a seven‐bladed β‐propeller that projects inward from the polyhedral outer clathrin coat. As the most membrane‐proximal portion of assembled clathrin, the TD is a major protein–protein interaction node. Contact with the TD β‐propeller occurs through short peptide sequences typically located within intrinsically disordered segments of coat components that usually are elements of the membrane‐apposed, inner ‘adaptor’ coat layer. A huge variation in TD‐binding motifs is known and now four spatially discrete interaction surfaces upon the β‐propeller have been delineated. An important operational feature of the TD interaction sites in vivo is functional redundancy. The recent discovery that ‘pitstop’ chemical inhibitors apparently occupy only one of the four TD interaction surfaces, but potently block clathrin‐mediated endocytosis, warrants careful consideration of the underlying molecular basis for this inhibition.     

Polysomal Localization of R17 Bacteriophage-Specific Protein Synthesis

Synthesis of viral ribonucleic acid (RNA) polymerase, maturation protein, and coat protein in Escherichia coli infected with bacteriophage R17 occurs mainly on polysomes containing four or more ribosomes. The 30S ribosomal subunits through trimer-size polysomes, which are associated with all of the R17-specific proteins and are predominant in the infected cell, synthesize only coat protein. These structures may accumulate as products derived from larger polysomes as a result of failure in the release of nascent polypeptides after termination of chain growth. Appreciable amounts of viral coat protein remain attached to ribosomes and polysomes during R17 bacteriophage replication, supporting the hypothesis of the repressor role of this protein. The time course of synthesis of virus-specific proteins obtained from the polysomes of infected cells demonstrated regulated R17 messenger RNA translation consistent with the idea that coat protein is preferentially synthesized whereas the synthesis of noncoat proteins is suppressed.