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.

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 NH2-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.

No abstract

Nuclear migration and positioning in Aspergillus nidulans depend on microtubules, the microtubule-dependent motor protein dynein, and auxiliary proteins, two of which are ApsA and ApsB. In apsA and apsB mutants nuclei are clustered and show various kinds of nuclear navigation defects, although nuclear migration itself is still possible. We studied the role of several components involved in nuclear migration through in vivo fluorescence microscopy using fluorescent-protein tagging. Because ApsA localizes to the cell cortex and mitotic spindles were immobile in apsA mutants, we suggest that astral microtubule-cortex interactions are necessary for oscillation and movement of mitotic spindles along hyphae, but not for post-mitotic nuclear migration. Mutation of apsA resulted in longer and curved microtubules and displayed synthetic lethality in combination with the conventional kinesin mutation ΔkinA. By contrast, ApsB localized to spindle-pole bodies (the fungal centrosome), to septa and to spots moving rapidly along microtubules. The number of cytoplasmic microtubules was reduced in apsB mutants in comparison to the wild type, indicating that cytoplasmic microtubule nucleation was affected, whereas mitotic spindle formation appeared normal. Mutation of apsB suppressed dynein null mutants, whereas apsA mutation had no effect. We suggest that nuclear positioning defects in the apsA and apsB mutants are due to different effects on microtbule organisation. A model of spindle-pole body led nuclear migration and the roles of dynein and microtubules are discussed.

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.