Morphogenetic pathway of spore wall assembly in Saccharomyces cerevisiae

The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.     

Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology

Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.     

The functions of cell wall polysaccharides in composition and architecture revealed through mutations

The plant cell wall is a highly organized composite of many different polysaccharides, proteins and aromatic substances. These complex matrices define the shape of each individual cell, and ultimately, they are the determinants of plant morphology. The fine structures of the major angiosperm cell wall polysaccharides have been characterized, but it is not well understood how these polysaccharides are assembled into a metabolically active architecture. Cell wall biogenesis and remodeling may be partitioned into six major stages of development (precursor synthesis, polymerization, secretion, assembly, rearrangement and disassembly), and to date, a handful of mutations have been identified that affect the composition and structure in each of these stages. To greatly augment this collection, we have initiated a program to use Fourier transform infrared spectroscopy as a high through-put screen to identify a broad range of cell-wall mutants of Arabidopsis and maize. We anticipate that such mutants will be useful to probe the impact of the individual components and their metabolism on basic processes of plant growth and development. The structures of dicot and grass walls, the identification of representative cell wall mutants, and the use of a novel spectroscopic screen to identify many more cell wall mutants, are briefly reviewed.     

Cell wall composition and structure of Yarrowia lipolytica transposon mutants affected in calcofluor sensitivity

A collection of transposon-mutagenized strains of Yarrowia lipolytica was screened for wall defects by determination of their sensitivity to calcofluor white. A number of strains were hypersensitive, whereas others were resistant. Different non-allelic mutants displayed increased sensitivity to autolysis and lytic enzymes, independently of whether they were sensitive or resistant to calcofluor white. A thorough analysis of their cell walls revealed minor quantitative alterations, and no significant changes in chitin content. Electrophoretic analysis of wall-bound and excreted proteins proved to be a sensitive method that revealed defects in the cell wall structure of the mutants. Important alterations in the patterns of the wall proteins extracted by SDS or by enzymatic treatments were noticed for the mutants, as compared to the parental strain. Mutants released to the growth medium a larger number of protein species than the parental strain, suggesting impairment in wall assembly of certain polypeptides. Patterns of wall-bound and excreted proteins, as well as alterations in wall chemical composition were not diagnostic of calcofluor white sensitivity or resistance, but were specific for each mutant. Our data show that an increase in either sensitivity or resistance of Y. lipolytica to certain levels of calcofluor is equally indicative of alterations in cell wall structure, independent of chitin levels. This revised version was published online in August 2006 with corrections to the Cover Date.     

The hydroxyproline‐rich glycoprotein domain of the Arabidopsis LRX1 requires Tyr for function but not for insolubilization in the cell wall

Extensins, hydroxyproline‐rich repetitive glycoproteins with Ser–Hyp₄ motifs, are structural proteins in plant cell walls. The leucine‐rich repeat extensin 1 (LRX1) of Arabidopsis thaliana is an extracellular protein with both a leucine‐rich repeat and an extensin domain, and has been demonstrated to be important for cell‐wall formation in root hairs. lrx1 mutants develop defective cell walls, resulting in a strong root hair phenotype. The extensin domain is essential for protein function and is thought to confer insolubilization of LRX1 in the cell wall. Here, in vivo characterization of the LRX1 extensin domain is described. First, a series of LRX1 extensin deletion constructs was produced that led to identification of a much shorter, functional extensin domain. Tyr residues can induce intra‐ and inter‐molecular cross‐links in extensins, and substitution of Tyr in the extensin domain by Phe led to reduced activity of the corresponding LRX1 protein. An additional function of Tyr (or Phe) is provided by the aromatic nature of the side chain. This suggests that these residues might be involved in hydrophobic stacking, possibly as a mechanism of protein assembly. Finally, modified LRX1 proteins lacking Tyr in the extensin domain are still insolubilized in the cell wall, indicating strong interactions of extensins within the cell wall in addition to the well‐described Tyr cross‐links.     

The Function of Ile-X-Ile Motif in the Oligomerization and Chaperone-Like Activity of Small Heat Shock Protein AgsA at Room Temperature

Small heat shock proteins assemble as large oligomers in vitro and exhibit ATP-independent chaperone activities. Ile-X-Ile motif is essential in both the function and oligomer formation. AgsA of Salmonella enterica serovar Typhimurium has been demonstrated to adopt large oligomeric structure and possess strong chaperone activity. Size exclusion chromatography, non-denaturing pore gradient PAGE, and negatively stain electron microscopic analysis of the various C-terminal truncated mutants were performed to investigate the role of Ile-X-Ile motif in the oligomer assembly of AgsA. By measuring the ability to prevent insulin from aggregating induced by TCEP, the chaperone-like activity of AgsA and the C-terminal truncated mutants at room temperature were determined. We found that the truncated mutants with Ile-X-Ile motif partially or fully deleted lost the ability to form large oligomers. Contrast to wild type AgsA which displayed weak chaperone-like activity, those mutants shown significantly enhanced activities at room temperature. In summary, biochemical experiment, activity assay and electron microscopic analysis suggested that Ile-X-Ile motif is essential in oligomer assembly of AgsA and might take the role of an inhibitor for its chaperone-like activity at room temperature.     

Structure-function studies of [2Fe-2S] ferredoxins

The ability to overexpress [2Fe-2S] ferredoxins inEscherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegetative and heterocyst forms ofAnabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in these proteins in their oxidized and reduced states has been studied by¹H,²H,¹³C, and¹⁵N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partnerAnabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).     

Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b ₆/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.Abbreviations ENDOR electron nuclear double resonance - ESEEM electron spin echo envelope modulation - LHC light harvesting complex - PSI Photosystem I - PS II Photosystem II - P₆₈₀ primary electron donor in PS II - P₇₀₀ primary electron donor in PS I     

Mutants with altered muscle structure in Caenorhabditis elegans

In the small nematode, Caenorhabditis elegans, mutants with a disorganized myofilament lattice structure have been identified by polarized light and electron microscopy. Genetic analysis places the mutations in 12 complementation groups which are distributed over the six linkage groups of C. elegans. The phenotypes are described for the mutants from the 9 complementation groups not previously reported on in detail. Most are paralyzed, but some exhibit essentially normal movement; mutants of two loci show changes only in later larval stages and adulthood. Morphological studies show that, in general, all the members of a complementation group show similar changes in muscle structure and that these changes are distinctive for that group. In mutants of several genes, disorganization of the myofilament lattice is general with no one component of the lattice more obviously altered than others. In mutants of other genes specific structures are prominently altered. In one of the instances where thick filaments appear to be abnormal, double mutants combining mutations in this gene (unc-82 IV) with mutations in the gene for a myosin heavy chain (MacLeod et al., 1977a,b) or paramyosin (Waterston et al., 1977) were used to show that the unc-82 gene product probably affects thick filament assembly through its actions on paramyosin. Some possible implications of the morphological features of the mutants as well as the conclusions derived from the genetic studies are discussed.     

Folding simulations and computer redesign of protein A three-helix bundle motifs

In solution, the B domain of protein A from Staphylococcus aureus (B domain) possesses a three-helix bundle structure. This simple motif has been previously reproduced by Kolinski and Skolnick (Proteins 18: 353–366, 1994) using a reduced representation lattice model of proteins with a statistical interaction scheme. In this paper, an improved version of the potential has been used, and the robustness of this result has been tested by folding from the random state a set of three-helix bundle proteins that are highly homologous to the B domain of protein A. Furthermore, an attempt to redesign the B domain native structure to its topological mirror image fold has been made by multiple mutations of the hydrophobic core and the turn region between helices I and II. A sieve method for scanning a large set of mutations to search for this desired property has been proposed. It has been shown that mutations of native B domain hydrophobic core do not introduce significant changes in the protein motif. Mutations in the turn region were also very conservative; nevertheless, a few mutants acquired the desired topological mirror image motif. A set of all atom models of the most probable mutant was reconstructed from the reduced models and refined using a molecular dynamics algorithm in the presence of water. The packing of all atom structures obtained corroborates the lattice model results. We conclude that the change in the handedness of the turn induced by the mutations, augmented by the repacking of hydrophobic core and the additional burial of the second helix N-cap side chain, are responsible for the predicted preferential adoption of the mirror image structure. © 1996 Wiley-Liss, Inc.     

The plant cell wall: Biosynthesis,construction, and functions

The plant cell wall is composed of multiple biopolymers, representing one of the most complex structural networks in nature. Hundreds of genes are involved in building such a natural masterpiece. However, the plant cell wall is the least understood cellular structure in plants. Due to great progress in plant functional genomics,manyachievementshavebeenmadein uncovering cell wall biosynthesis, assembly, and architecture, as well as cell wall regulation and signaling. Such information has significantly advanced our understanding of the roles of the cell wall in many biological and physiological processes and has enhanced our utilization of cell wall materials. The use of cutting-edge technologies such as single-molecule imaging,nuclear magnetic resonance spectroscopy, and atomic force microscopy has provided much insight into the plant cell wall as an intricate nanoscale network, opening up unprecedented possibilities for cell wall research. In this review,we summarize the major advances made in understanding the cell wall in this era of functional genomics, including the latest findings on the biosynthesis, construction, and functions of the cell wall.     

De novo genome assembly of Candida glabrata reveals cell wall protein complement and structure of dispersed tandem repeat arrays

Candida glabratais an opportunistic pathogen in humans, responsible for approximately 20% of disseminated candidiasis. Candida glabrata's ability to adhere to host tissue is mediated by GPI-anchored cell wall proteins (GPI-CWPs); the corresponding genes contain long tandem repeat regions. These repeat regions resulted in assembly errors in the reference genome. Here, we performed a de novo assembly of the C. glabrata type strain CBS138 using long single-molecule real-time reads, with short read sequences (Illumina) for refinement, and constructed telomere-to-telomere assemblies of all 13 chromosomes. Our assembly has excellent agreement overall with the current reference genome, but we made substantial corrections within tandem repeat regions. Specifically, we removed 62 genes of which 45 were scrambled due to misassembly in the reference. We annotated 31 novel ORFs of which 24 ORFs are GPI-CWPs. In addition, we corrected the tandem repeat structure of an additional 21 genes. Our corrections to the genome were substantial, with the length of new genes and tandem repeat corrections amounting to approximately 3.8% of the ORFeome length. As most corrections were within the coding regions of GPI-CWP genes, our genome assembly establishes a high-quality reference set of genes and repeat structures for the functional analysis of these cell surface proteins.     

Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages

The filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram-negative bacterial proteins that are essential components of a widely distributed extracellular protein-export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non-functional, interfered with assembly of plV⁺ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non-functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.     

Functional dissection of the major structural protein of bluetongue virus: identification of key residues within VP7 essential for capsid assembly

A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.     

Cell wall mutants

An ever growing collection of cell wall mutants is yielding new insights into the mechanisms underlying the synthesis and assembly of cell walls in plants. In this review, we will provide an update on the use of genetic tools in plant cell wall research and we will discuss the lessons that can be drawn from the study of the first generation of mutants.     

Cryo electron tomography of native HIV-1 budding sites

The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: comparative study of preparations extracted by the phenol-water and the phenol-chloroform-petroleum ether methods

An R-form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN-111 (O3-:K1-) by the phenol-chloroform-petroleum ether method was compared with that extracted by the phenol-water method in the ability to form a hexagonal assembly. The LPS which was extracted by the phenol-water method and dialyzed against tap water to remove phenol showed ribbon-like structures, and it formed a hexagonal lattice structure with a lattice constant of 14.5 +/- 0.3 nm when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol. The LPS which was extracted by the phenol-chloroform-petroleum ether method and lyophilized consisted of ribbon-like structures and their fragments and it often formed small pieces of a hexagonal lattice, although the LPS before lyophilization did not form such a lattice. When the LPS extracted by the phenol-chloroform-petroleum ether method was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, it formed essentially the same hexagonal lattice structure as that formed by the LPS extracted by the phenol-water method. From these results it is concluded that the ability of the LPS to form a hexagonal lattice structure does not depend upon the method of its extraction from bacterial cells.