Nuclear migration, nucleokinesis and lissencephaly.

During the past 20 years, biologists have become used to finding that proteins first identified in simple, genetically manipulable eukaryotic organisms are conserved in higher eukaryotes. This article draws attention to the similarity between NUDF protein, which is required for nuclear migration in the filamentous fungus Aspergillus nidulans, and a mammalian homologue, LIS1, whose malfunction causes lissencephaly, a neuronal migration disease. The authors suggest that there might be an underlying similarity of mechanism between nuclear migration in the fungus and neuronal migration in the brain.

The LIS1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein.

The nudF gene of the filamentous fungus Aspergillus nidulans acts in the cytoplasmic dynein/dynactin pathway and is required for distribution of nuclei. NUDF protein, the product of the nudF gene, displays 42% sequence identity with the human protein LIS1 required for neuronal migration. Haploinsufficiency of the LIS1 gene causes a malformation of the human brain known as lissencephaly. We screened for multicopy suppressors of a mutation in the nudF gene. The product of the nudE gene isolated in the screen, NUDE, is a homologue of the nuclear distribution protein RO11 of Neurospora crassa. The highly conserved NH(2)-terminal coiled-coil domain of the NUDE protein suffices for protein function when overexpressed. A similar coiled-coil domain is present in several putative human proteins and in the mitotic phosphoprotein 43 (MP43) of X. laevis. NUDF protein interacts with the Aspergillus NUDE coiled-coil in a yeast two-hybrid system, while human LIS1 interacts with the human homologue of the NUDE/RO11 coiled-coil and also the Xenopus MP43 coiled-coil. In addition, NUDF coprecipitates with an epitope-tagged NUDE. The fact that NUDF and LIS1 interact with the same protein domain strengthens the notion that these two proteins are functionally related.

The crystal structure of a five-stranded coiled coil in COMP: a prototype ion channel?

Oligomerization by the formation of alpha-helical bundles is common in many proteins. The crystal structure of a parallel pentameric coiled coil, constituting the oligomerization domain in the cartilage oligomeric matrix protein (COMP), was determined at 2.05 angstroms resolution. The same structure probably occurs in two other extracellular matrix proteins, thrombospondins 3 and 4. Complementary hydrophobic interactions and conserved disulfide bridges between the alpha helices result in a thermostable structure with unusual properties. The long hydrophobic axial pore is filled with water molecules but can also accommodate small apolar groups. An "ion trap" is formed inside the pore by a ring of conserved glutamines, which binds chloride and probably other monatomic anions. The oligomerization domain of COMP has marked similarities with proposed models of the pentameric transmembrane ion channels in phospholamban and the acetylcholine receptor.

Crystallization and preliminary crystallographic characterization of bacteriophage T4 baseplate protein encoded by gene 9.

The structural protein, gene product 9 (gp9), of bacteriophage T4 controls baseplate expansion at the first steps of virus attachment onto its host bacterial cell with subsequent tail contraction. Gp9, which has an M(r) of 30.8 kDa and contains 287 amino acids, has been purified from a recombinant Escherichia coli strain and crystallized at 25 degrees C using the hanging drop vapor diffusion method at pH 4.0 with ammonium sulfate as precipitant. The crystals of gp9 belong to the space group R32 with hexagonal cell dimensions a = b = 86.5 A and c = 156.2 A and diffract X-rays to at least 2.7 A. There is one molecule per asymmetric unit.

Fibritin encoded by bacteriophage T4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure.

The bacteriophage T4 late gene wac (whisker's antigen control) encodes a fibrous protein which forms a collar/whiskers complex. Whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions. In this work we show that expression of the cloned wac gene in Escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac T4 particles in vitro. CD spectroscopy of fibritin indicates a 90% alpha-helical content, and scanning calorimetry shows that the protein has several distinct domains. The analysis of the 486 amino acid sequence of fibritin reveals three structural components: a 408 amino acid region that contains 12 putative coiled-coil segments with a canonical heptad (a-b-c-d-e-f-g)n substructure where the "a" and "d" positions are preferentially occupied by apolar residues, and the N and C-terminal domains (47 and 29 amino acid residues, respectively) have no heptad substructure. The distribution of hydrophobic residues within heptads is more similar to a triple than to a double coiled-coil. The alpha-helical segments are separated by short "linker" regions, variable in length, that have a high proportion of glycine and proline residues. Each coiled-coil segment has, on the borders with linker regions, residues that are common to the N and C-terminal caps of the alpha-helices. Full-length and amino-terminally truncated fibritins can be reassembled in vitro after temperature-induced denaturation. Co-assembly of full-length fibritin and the N-terminal deletion mutant, as well as analytical centrifugation, indicates that the protein is a parallel triple-standard alpha-helical coiled-coil. Deletions of various N-terminal portions of fibritin did not block trimerisation but the mutant trimers are unable to bind to T4 particles. The last 18 C-terminal residues of fibritin are required for correct trimerisation of gpwac monomers in vivo. We propose that fibritin might serve as a convenient model for the investigation of folding and assembly mechanisms of alpha-fibrous proteins.

A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis.

Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.

The thrombospondin-like chains of cartilage oligomeric matrix protein are assembled by a five-stranded alpha-helical bundle between residues 20 and 83.

The N-terminal fragment of rat cartilage oligomeric matrix protein (COMP), comprising residues 20-83, was over-expressed in E. coli and purified under non-denaturing conditions. The fragment forms pentamers similar to the assembly domain of the native protein. Its five chains can be covalently linked in vitro by oxidation of cysteines 68 and 71. The fragment adopts a predominantly alpha-helical structure as judged by circular dichroism spectroscopy. On the basis of these findings we propose the model of a five-stranded alpha-helical bundle for the assembly domain of COMP. The studied sequence is conserved in thrombospondins 3 and 4 thus raising the possibility that these proteins are also pentamers.

Bacteriophage T4 as a surface display vector.

We describe a method for construction of hymeric bacteriophage T4 particles displaying foreign polypeptides on their surface. The method is based on our finding that minor T4 fibrous protein fibritin encoded by gene wac (whisker's antigen control) could be lengthened at the C terminus without impairing its folding or binding to the phage particle. The lengthened fibritin gene could easily be transferred into the T4 genome by homologous recombination with a plasmid containing the modified gene wac. The modified gene wac is expressed properly during phage reproduction, and the lengthened fibritin is bound to phage particles. As an example of this type of method, we have obtained the hymeric T4 particles carrying a polypeptide of 53 residues, 45 of which are from the pre-S2 region of hepatitis B virus. The T4 display vector extends currently available display systems.

Crystallization and preliminary crystallographic study of the pentamerizing domain from cartilage oligomeric matrix protein: a five-stranded alpha-helical bundle.

Cartilage oligomeric matrix protein (COMP) is a pentameric glycoprotein of the thrombospondin family found in cartilage and tendon. Self-association of COMP is achieved through the formation of a five-stranded alpha-helical bundle that involves 64 N-terminal residues (from 20 to 83). The complex is further stabilized by the interchain disulfide bonds between cysteines 68 and 71. We have prepared, by expression in Escherichia coli, several peptides of different lengths from the N-terminal region of COMP and studied their amenability to crystallization. Crystals of the best quality were obtained with a peptide spanning COMP residues 28-72. This peptide forms disulfide linked pentamers with 87% of alpha-helical structure. Crystals were grown by the hanging drop vapor diffusion method, using polyethylene glycol 1500 as a precipitant. The crystals belong to space group P2(1) with unit cell dimensions a = 38.47 angstroms, b = 49.47 angstroms, c = 54.98 angstroms, beta = 103.84 degrees and contain one pentamer per asymmetric unit. They diffract strongly to at least 1.8 angstrom resolution.

Isolation of a new set of Aspergillus nidulans mutants defective in nuclear migration.

In the filamentous fungus Aspergillus nidulans, nuclear migration in the germ tube is mediated by cytoplasmic dynein. We have previously reported the characterization of four nud (nuclear distribution) genes, nudA, nudC, nudF and nudG, involved in this process. The nudA and nudG genes respectively encode for the heavy chain and the 8-kDa light chain of cytoplasmic dynein. In this work, we describe an improved method for the isolation of nud mutants that has led to the identification of at least ten additional nud loci. We have cloned one of the genes, nudK, and determined that it encodes the actin-related protein Arp1, which is a component of the dynactin complex. This provides the first evidence that dynactin is involved in nuclear migration in A. nidulans.

Cascade of overlapping late genes in bacteriophage T4.

The DNA sequences of genes 9, 10, 11, 12, and wac, which encode the structural proteins in the bacteriophage T4 base plate, were determined. These genes form a single operon which is transcribed in a clockwise direction from a single late promoter in the TATAAATA region located upstream of gene 9 at position -10. A feature of the operon is an overlap between the termination codon of each upstream gene and the initiation codon of its downstream gene. With the exception of gene 10, the open reading frames encode proteins which have a calculated molecular mass close to that obtained experimentally. The reading frame of gene 10 encodes a polypeptide with a calculated molecular mass of 66.2 kDa, which is at least 22 kDa less than that in the phage particle. Thus the mature protein encoded by gene 10 is possibly a product of the fusion of two adjacent phage genes. The hybrid protein may be formed by a frameshift during the translation of messenger RNA at the end of gene 9 or gene 10.

A proposed structure of bacteriophage T4 gene product 22--a major prohead scaffolding core protein.

Gene 22 of bacteriophage T4 encodes a major prohead scaffolding core protein of 269 amino acid residues. From its nucleotide sequence the gene product (gp) 22 has a predicted Mr of 29.9 and a pI of 4.3. The protein is rich in charged residues (glutamic acid and lysine) and contains low amounts of proline and glycine and no cysteine residues. We suggest that gp22 undergoes limited proteolytic processing which eliminates the short C-terminal piece from the molecule during the early steps of prohead assembly. Most amino acid residues of the gp22 polypeptide chain (80%) have an alpha-helical conformation and form seven peculiar alpha-helices. A model suggesting the spatial organization of gp22 is presented. Three long alpha-helices numbered 1 (1A and 1B), 3, and 5 (5A and 5B) are packed in an antiparallel fashion along the major axis of the road-shaped molecule. Two rather short alpha-helices (2 and 4) are located at the distal and proximal ends of the protein molecule, respectively. Helix number 2, which is a proteolytic fragment of gp22 found in mature T4 heads, is packed with helices 1A and 3, similar to a novel element of supersecondary structure, the alpha alpha-corner. Helix number 4 probably interacts with the gp20 connector of the prohead. The implications of the structure of the gp22 molecule for the assembly of the prohead core are discussed.