Unusual nucleotide sequences at the 5'' end of actin genes in Dictyostelium discoideum.

The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined. The amino acid sequences derived from the nucleotide sequences show strong conservation for six of the seven genes relative to the NH2-terminal region of Physarum actin. The region 5' to the AUG initiating codon is greater than 90% A+T residues in all of the genes.     

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.     

ATP-driven steady-state exchange of monomeric and filamentous actin from Dictyostelium discoideum.

When [35S]actin monomer from the slime mold Dictyostelium discoideum is added in trace amounts to a population of unlabeled Dictyostelium actin molecules assembled to steady state, it rapidly exchanges with the pool of actin filaments. This exchange between monomeric and filamentous actin is dependent on the presence of ATP. In addition, the exchange appears to occur via filament ends, because cytochalasin D, a drug that interacts specifically with actin filament ends to inhibit filament assenbly, inhibits the exchange reaction.     

Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly

Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.

The precise inheritance of genetic information relies on the accurate segregation of chromosomes in mitosis and meiosis. Kinetochores are large protein complexes assembled on centromeres and play a crucial role in chromosome segregation. The kinetochore links the chromosome to microtubule polymers, drives the movement of chromosomes, and ensures correct microtubule–kinetochores attachment (1–3). The kinetochore assembly is thus tightly regulated. Yet, the mechanism by which kinetochores are precisely assembled through the cell cycle remains poorly understood.The kinetochore comprises an outer region and an inner region. The outer kinetochore interacts with microtubules and is assembled on the platform of the inner kinetochore. The inner kinetochore consists of a complex of 14 to 16 subunits known as the constitutive centromere–associated network (CCAN) that is directly built on centromeric chromatin (4–6). In centromeres, the histone H3 variant, CENP-A, replaces the canonical histone H3 to form CENP-A–containing nucleosomes (7–9). Most eukaryotes contain large complex regional centromeres where CENP-A–containing nucleosomes are interspersed with canonical H3–containing nucleosomes (10–12). Regional centromeres are epigenetically specified by CENP-A (12–14). But how CENP-A– and histone H3–containing nucleosomes are balanced in centromeres remains unclear.CENP-T, an integral component of CCAN, is also a histone fold–containing protein. CENP-T provides a platform for the assembly of the Ndc80 complex (Ndc80C), an essential outer kinetochore component, during mitosis (5, 15–18). Ndc80C acts as the interface between microtubules and kinetochores and mediates the microtubule attachments (19, 20). The long N terminus of CENP-T contains a conserved Ndc80 receptor motif. The motif forms an alpha-helix that directly interacts with the Spc24-Spc25 heterodimer in Ndc80C (15, 16). The motif can be phosphorylated by cyclin-dependent kinase 1 (CDK1) to stabilize the interaction between CENP-T and Ndc80C (16, 21–24). However, how CENP-T is regulated through the cell cycle to mediate the assembly of Ndc80C is still not well understood.CENP-T has been shown to interact with three other histone fold–containing proteins, CENP-W, CENP-S, and CENP-X, to form the heterotetrameric nucleosome-like structure in vitro (25, 26). The CENP-T-W-S-X complex directly associates with centromeric DNA. The DNA binding activity of the complex is important for kinetochore formation (5, 25). Interestingly, the complex also directly associates with histone H3, not with CENP-A (5, 27), suggesting that CENP-T particles and the CENP-A nucleosome occupy different positions in centromeres. How the spatial relationship between the CENP-A nucleosome and CENP-T particles in centromeres is regulated remains unclear.The fission yeast Schizosaccharomyces pombe contains large regional centromeres and is considered to be a model system for centromere study. The CENP-A homolog, Cnp1, is enriched in centromere cores, which are surrounded by pericentromeric heterochromatin (28–30). CENP-A^Cnp1 nucleosomes nucleate kinetochore assembly. Mislocalization of CENP-A^Cnp1 results in severe chromosome segregation defects in fission yeast (28, 31–34). Fission yeast also contains the CENP-T homolog, Cnp20, which associates with centromeres throughout the cell cycle. The same as in higher eukaryotes, CENP-T^Cnp20 in S. pombe is essential for viability (35).Recently, Ccp1, a nucleosome assembly protein (NAP) family protein, has been shown to play an important role in antagonizing the loading of CENP-A in fission yeast (36). Ccp1 forms a homodimer and is enriched at centromeres. Ccp1 acts as a key player in balancing CENP-A and histone H3 levels in the region (36). How Ccp1 regulates the CENP-A level in centromeres remains elusive. Interestingly, its centromere localization is cell cycle regulated. Ccp1 is dissociated from centromeres at the onset of mitosis and reassociates with centromeres at the end of mitosis (36, 37). The biological importance of the cell cycle–dependent interaction between Ccp1 and centromeres is unknown.Here using mass spectrometry, we found that Ccp1 interacts directly with CENP-T^Cnp20 in fission yeast. We further identified a conserved Ccp1-interaction motif (CIM) at the N terminus of CENP-T^Cnp20, which is adjacent to the Ndc80 receptor motif. We demonstrated that CIM is important for Ccp1 localization. Furthermore, our data suggested that CDK1-mediated phosphorylation of the CIM motif at the onset of mitosis dissociates Ccp1 from CENP-T^Cnp20, allowing proper positioning of Ndc80C. Ccp1 associates with centromeres during mitosis in the phospho-null mutant of the CIM domain, leading to mislocalization of Ndc80C and severe chromosome segregation defects. Our study uncovers a previously unrecognized mechanism regulating kinetochore organization in regional centromeres.     

Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein

Recently, it was shown that glycoproteins with N-glycans close to the NH₂ terminus can directly enter the calnexin/calreticulin cycle and bypass BiP binding. This should allow efficient secretion of glycoproteins such as factor VIII (FVIII) whose secretion is negatively affected by BiP interaction. Examination of the glycosylation pattern of the NH₂ terminus of FV and FVIII revealed N-glycans at positions 23 and 27 in FV and at position 41 in FVIII. To improve FVIII secretion, a 14-amino-acid-long polypeptide with (G3) or without (G0; control) three N-linked glycosylation consensus sites was inserted upstream of the NH₂ terminus of a B-domain deleted FVIII protein. Expression of G3- and G0-constructs in three different cell lines resulted in the same or even higher expression rate of protein as found for the B-domain deleted FVIII. However, as demonstrated by Western blot analysis, the G3- as well as the G0-protein variants were mainly retained inside the cells in similar amounts. Thus, glycosylation alone does not automatically lead to higher secretion rates, but must be in context to the normal structure of the FVIII protein. M. A. Srour and J. Grupp contributed equally to this work.     

Visualization of actin fibers associated with the cell membrane in amoebae of Dictyostelium discoideum.

Amoebae of Dictyostelium discoideum were attached to a surface coated with polylysine, and the upper portion of the cells was sheared off with a stream of buffer. Scanning and transmission electron microscopy showed that the cytoplasmic surface of the exposed membrane was covered with fibers consisting of actin-containing filaments. The actin was identified by its solubility properties and its ability to interact with muscle myosin.     

CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A alpha chain of fibrinogen.

Fibrinogen and fibrin serve as adhesive substrates for a variety of cells including platelets, endothelial cells, and leukocytes. Previously, we identified the C terminus of the gamma chain of fibrinogen as the region of the fibrinogen molecule that contains a ligand for CD11b/CD18 (complement receptor 3) on phorbol ester-stimulated polymorphonuclear leukocytes. In contrast, we report here that neutrophils stimulated with tumor necrosis factor adhere to fibrinogen-coated surfaces, but not to human serum albumin-coated surfaces, via the integrin CD11c/CD18 (p150/95). Monoclonal antibodies LeuM5 and 3.9, which are directed against the alpha subunit of CD11c/CD18, but not monoclonal antibodies OKM10 and OKM1, which are directed against the alpha subunit of CD11b/CD18, inhibit the adhesion of tumor necrosis factor-stimulated neutrophils to fibrinogen-coated surfaces. To identify the site on fibrinogen recognized by CD11c/CD18, we have examined the adhesion of tumor necrosis factor-stimulated neutrophils to surfaces coated with various fibrinogen fragments. Stimulated neutrophils adhere to surfaces coated with the N-terminal disulfide knot fragment of fibrinogen or fibrinogen fragment E. Moreover, peptides containing the sequence Gly-Pro-Arg (which corresponds to amino acids 17-19 of the N-terminal region of the A alpha chain of fibrinogen), and monoclonal antibody LeuM5, block tumor necrosis factor-stimulated neutrophil adhesion to fibrinogen and to the N-terminal disulfide knot fragment of fibrinogen. Thus, CD11c/CD18 on tumor necrosis factor-stimulated neutrophils functions as a fibrinogen receptor that recognizes the sequence Gly-Pro-Arg in the N-terminal domain of the A alpha chain of fibrinogen.     

Cytolytic and ion channel-forming properties of the N terminus of lymphocyte perforin.

Perforin lyses cells by binding to the target cell membrane, where it polymerizes into large nonspecific pores. It is shown here that the first 34 amino acids of the N-terminal region of either human or murine perforin are soluble in aqueous medium and spontaneously insert into membranes. The N-terminal peptides lyse liposomes and nucleated cells, and they form ion channels in planar bilayers, some of which are comparable to those previously described for perforin. The lytic activity of the N-terminal domains does not require calcium, is independent of the lipid headgroup composition, and can be inhibited by heparin. Tumor cells incubated with the N-terminal peptides undergo the same morphological changes as those induced by native perforin. None of the peptides corresponding to the putative membrane-spanning domains from the central region of perforin is cytolytic. Taken together, these results suggest that the N-terminal region is an important part of the pore-forming domain of perforin.     

beta-Endorphin: synthesis of analogs modified at the carboxyl terminus with increased activites.

Three analogs of human beta-endorphin (beta h-EP) have been synthesized: [Gly31]beta h-EP, [Gly31]beta h-endorphinamide, and [Gly31]beta h-endorphinylglycine. All are more active than beta h-EP in both the guinea pig ileum bioassay and the opiate receptor binding assay. The last two analogs are about twice as active as beta h-EP in an assay for analgesia. Modification at position 31 and extension at the COOH terminus may afford a route toward analogs with even greater biological activity.     

Mutational analysis of the N terminus of the protein of maize transposable element Ac.

Mutations of transposable element Ac were tested for their capability to excise themselves from their location autonomously, to be excised by an active Ac, or to act in trans in the excision of an Ac delta element. Removal of 101 amino acids from the N terminus of the Ac protein does not decrease excision. A cis-acting site between base pairs 186 and 207 is important for excision by the wild-type protein but is not necessary for excision by the truncated protein. Improvement of the sequence context of the first AUG does not have a significant effect. Mutations in a small open reading frame of Ac encoding a 102-amino acid protein do not visibly alter excision frequency.     

Direct observation of better hydration at the N terminus of an alpha-helix with glycine rather than alanine as the N-cap residue.

The structural basis for the stability of N termini of helices has been analyzed by thermodynamic and crystallographic studies of three suitably engineered mutants of the barley chymotrypsin inhibitor 2 with Ser, Gly, or Ala at the N-cap position (residue 31). Each mutant has a well-organized shell of hydration of the terminal NH groups of the helix. The three structures are virtually superimposable (rms separations for all atoms, including the common water molecules, are 0.15-0.17 A) and show neither changes in conformation at the site of substitution nor changes in the crystal packing. The only changes on going from Ser-31 to Ala-31 to Gly-31 are in the position of a water molecule (Wat-116). This is bound to the Ser-O gamma atom in the Ser-31 structure but is in a weak hydrogen bonding position with the NH of residue 34 (O ... N = 3.28 A) in the Ala-31 mutant, partly replacing the strong Ser-31-O gamma ... N34 hydrogen bond (O ... N = 2.65 A). The corresponding water molecule completely replaces the Ser hydroxyl hydrogen bond to N34 on mutation to Gly (2.74 A). The only other change between the three structures is an additional water molecule in the Ala-31 structure (Wat-150) that partly compensates for the weak Wat-116 ... N34 hydrogen bond. Perturbation of solvation by the side chain of Ala is consistent with earlier hypotheses on the importance of exposure of the termini of helices to the aqueous solvent.     

Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion.

Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/D363/E364 are not essential for ATP-driven sliding and force generation.     

A cluster of mutations within a short triplet repeat in the C1 inhibitor gene.

Mutations in the C1 inhibitor gene that result in low functional levels of C1 inhibitor protein cause hereditary angioneurotic edema. This disease is characterized by episodic edema leading to considerable morbidity and death. Among 60 unreported kindred with the disease, four patients were discovered to have mutations clustered within a 12-bp segment of exon 5 from nucleotide 8449 to nucleotide 8460. This short segment of DNA contains three direct repeats of the triplet CAA and is immediately preceded by a similar adenosine-rich sequence (CAAGAACAC). These triplet repeats make this region susceptible to mutation by a slipped mispairing mechanism. There are two other short triplet repeat elements in the coding region for this gene, but they have not become mutated in any kindred examined. This suggests that the apparent enhanced mutation rate in this region of exon 5 may be influenced by DNA structural characteristics.     

Generation of a dominant 8-MDa deletion at the left terminus of vaccinia virus DNA.

Vaccinia virus mutants were obtained in high frequency from mouse Friend erythroleukemia (FEL) cells persistently infected with this virus, which contains a large (122-MDa) DNA. During long-term cell passages viral particles with deletions of the DNA are generated in FEL cells. These mutants have a major 8-MDa deletion starting between 2.2 and 3.2 MDa from the left terminus of the viral genome. More than half of the left end terminal repetition is deleted. These mutants have reduced infectivity compared to wild-type virus. The ease with which vaccinia virus mutants are obtained in FEL cells should provide a suitable system for generating mutants with other poxvirus and permit study of the genetic basis of virulence for this group of viruses.     

From the Cover: Dynamin at actin tails

Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane.     

Epidemiology of desmin and cardiac actin gene mutations in a european population of dilated cardiomyopathy.

AIMS: Although dilated cardiomyopathy is the most frequent form of cardiomyopathy, its aetiology is still poorly understood. In about 20-30% of cases the disease is familial with a large predominance of autosomal dominant transmission. Ten different chromosomal loci have been described for autosomal dominant forms of dilated cardiomyopathy. Only two genes have been associated with pure forms (without myopathy and/or conduction disorders) of the disease, the cardiac actin and the desmin genes. Our aim was to determine the proportion of dilated cardiomyopathy affected individuals carrying a mutation in one of these two genes. METHODS AND RESULTS: We performed (1) a systematic polymerase chain reaction-SSCP-sequencing screening of the coding sequences of cardiac actin on DNA samples from 43 probands of dilated cardiomyopathy families and 43 sporadic cases; (2) a systematic polymerase chain reaction-SSCP-sequencing screening of the coding sequences of desmin combined with a search for the described missense mutation (Ile451Met) by restriction fragment length polymorphism analysis on DNA samples from 41 probands of dilated cardiomyopathy families and 22 sporadic cases. CONCLUSION: None of the patients presents a mutation in any of these two genes. Consequently, the proportion of European dilated cardiomyopathy affected individuals bearing a mutation in (1) the cardiac actin gene is less than 1.2%, (2) the desmin gene is less than 1.6%.     

Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.

Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.     

Dominant negative protein kinase mutations that confer a G1 arrest phenotype.

The CDC28 gene of Saccharomyces cerevisiae encodes a protein kinase that is required for passage through the G1 phase of the cell cycle. We have used an inducible promoter fused to the CDC28 coding sequence to isolate conditionally dominant mutant alleles of CDC28. Overexpression of these dominant alleles causes arrest in the G1 phase of the cell cycle but permits the distinctive asymmetric growth that is characteristic of recessive temperature-sensitive cdc28 mutants. The dominant alleles encode products with no detectable protein kinase activity, and their phenotypic effects can be suppressed by simultaneous overproduction of the wild-type protein. DNA sequence analysis showed that the mutant site in at least one of the dominant alleles is in a residue that is highly conserved among protein kinases. These properties are best understood if the dominant mutation results in the catalytic inactivation of the protein kinase but still allows the binding of another component needed for CDC28 function. By this model, high levels of the mutant protein arrest cell division by denying the wild-type protein access to this other component. Suppressors that may encode this other component have been isolated on high-copy-number plasmids.     

Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity.

Pseudomonas exotoxin (PE), a single-chain polypeptide toxin of 613 amino acids, consists of three functional domains: an amino-terminal receptor-binding domain, a middle translocation domain, and a carboxyl-terminal ADP-ribosylation domain. Deletion of as few as 2 or as many as 11 amino acids from the carboxyl terminus of PE does not affect ADP-ribosylation activity but produces noncytotoxic molecules. Deletions and substitutions between positions 602 and 611 of PE show that the last 5 amino acids of PE are very important for its cytotoxic action. The carboxyl-terminal sequence of PE is Arg-Glu-Asp-Leu-Lys. Mutational analysis indicates that a basic amino acid at 609, acidic amino acids at 610 and 611, and a leucine at 612 are required for full cytotoxic activity. Lysine at 613 can be deleted or replaced with arginine but not with several other amino acids. Mutant toxins are able to bind normally to target Swiss mouse 3T3 cells and are internalized by endocytosis, but apparently they do not penetrate into the cytosol. A PE molecule that ends with Lys-Asp-Glu-Leu, which is a well defined endoplasmic reticulum retention sequence [Munro, S. and Pelham, R. B. (1987) Cell 48, 899-907], is fully cytotoxic, suggesting that a common factor may be involved in intoxication of cells by PE and retention of proteins in the lumen of the endoplasmic reticulum. Sequences similar to those at the carboxyl end of PE are also found at the end of Cholera toxin A chain and Escherichia coli heat-labile toxin A chain.