Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora.
(17/1876)
BACKGROUND: Kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. They have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions. RESULTS: We analyzed organelle transport in kinesin and dynein single mutants, and in a kinesin and dynein double mutant of Neurospora crassa. Remarkably, the simultaneous mutation of kinesin and dynein was not lethal and resulted in an additive phenotype that combined the features of the single mutants. The mutation of kinesin and dynein had opposite effects on the apical and retrograde transport, respectively, of vesicular organelles. In the kinesin mutant, apical movement of submicroscopic, secretory vesicles to the Spitzenkorper - an organelle in the hyphal apex - was defective, whereas the predominantly retrograde movement of microscopic organelles was only slightly reduced. In contrast, the dynein mutant still had a prominent Spitzenkorper, demonstrating that apical transport was intact, but retrograde transport was essentially inhibited completely. A major defect in vacuole formation and dynamics was also evident. In agreement with the observations on apical transport, protein secretion into the medium was markedly inhibited in the kinesin mutant but not in the dynein mutant. CONCLUSIONS: Transport of secretory vesicles is necessary but not sufficient for normal apical extension. A component of retrograde transport, presumably precursors of the vacuole system, is also essential. Our findings provide new information on the role microtubule motors play in cell morphogenesis and suggest that kinesin and cytoplasmic dynein have largely independent functions within separate pathways. (+info)
Gene silencing: RNA makes RNA makes no protein.
(18/1876)
A mutation that disrupts post-transcriptional gene silencing in Neurospora crassa has been found to affect the homologue of a plant-encoded RNA-dependent RNA polymerase. This enzyme may produce a specificity determinant of gene silencing and mediate an epigenetic conversion at the RNA level. (+info)
The TamA protein fused to a DNA-binding domain can recruit AreA, the major nitrogen regulatory protein, to activate gene expression in Aspergillus nidulans.
(19/1876)
The areA gene of Aspergillus nidulans encodes a GATA zinc finger transcription factor that activates the expression of a large number of genes subject to nitrogen metabolite repression. The amount and activity of the AreA protein under different nitrogen conditions is modulated by transcriptional, post-transcriptional, and post-translational controls. One of these controls of AreA activity has been proposed to involve the NmrA protein interacting with the DNA-binding domain and the extreme C terminus of AreA to inhibit DNA binding under nitrogen sufficient conditions. In contrast, mutational evidence suggests that the tamA gene has a positive role together with areA in regulating the expression of genes subject to nitrogen metabolite repression. This gene was identified by the selection of mutants resistant to toxic nitrogen source analogues, and a number of nitrogen metabolic activities have been shown to be reduced in these mutants. To investigate the role of this gene we have used constructs encoding the TamA protein fused to the DNA-binding domain of either the FacB or the AmdR regulatory proteins. These hybrid proteins have been shown to activate expression of the genes of acetate or GABA utilization, respectively, as well as the amdS gene. Strong activation was shown to require the AreA protein but was not dependent on AreA binding to DNA. The homologous areA gene of A. oryzae and nit-2 gene of Neurospora crassa can substitute for A. nidulans areA in this interaction. We have shown that the same C-terminal region of AreA and NIT-2 that is involved in the interaction with NmrA is required for the TamA-AreA interaction. However, it is unlikely that TamA requires the same residues as NmrA within the GATA DNA-binding domain of AreA. (+info)
Effects of disrupting the 21 kDa subunit of complex I from Neurospora crassa.
(20/1876)
We have cloned and inactivated in vivo, by repeat-induced point mutations, the nuclear gene encoding a 21 kDa subunit of complex I from Neurospora crassa. Mitochondria from the nuo21 mutant lack this specific protein but retain other subunits of complex I in approximately normal amounts. In addition, this mutant is able to assemble an almost intact enzyme. The electron transfer activities from NADH to artificial acceptors of mitochondrial membranes from nuo21 differ from those of the wild-type strain, suggesting that the absence of the 21 kDa polypeptide results in conformational changes in complex I. Nevertheless, complex I of nuo21 is able to perform NADH:ubiquinone reductase activity, as judged by the observation that the respiration of mutant mitochondria is sensitive to inhibition by rotenone. We discuss these findings in relation to the involvement of complex I in mitochondrial diseases. (+info)
Primary structure and characterisation of a 64 kDa NADH dehydrogenase from the inner membrane of Neurospora crassa mitochondria.
(21/1876)
A cDNA clone encoding a mitochondrial NADH dehydrogenase from Neurospora crassa was sequenced. The total DNA sequence encompasses 2570 base pairs and contains an open reading frame of 2019 base pairs coding for a precursor polypeptide of 673 amino acid residues. The protein is encoded by a single-copy gene located to the right side of the centromere in linkage group IV of the fungal genome. The N-terminus of the precursor protein has characteristics of a mitochondrial targeting pre-sequence. The protein displays homology with mitochondrial NADH dehydrogenases from yeast. In contrast to these polypeptides, however, analysis of its primary structure revealed that it contains a well-conserved calcium-binding domain. Rabbit antiserum against the protein expressed in an heterologous system recognises a mitochondrial protein of N. crassa with an apparent molecular mass of 64 kDa. Analysis of the fungal mitochondria by swelling, digitonin fractionation and alkaline treatment indicate that the protein is located in the inner membrane of the organelles, possibly facing the matrix side. (+info)
Role of a white collar-1-white collar-2 complex in blue-light signal transduction.
(22/1876)
Mutations in either white collar-1 (wc-1) or white collar-2 (wc-2) lead to a loss of most blue-light-induced phenomena in Neurospora crassa. Sequence analysis and in vitro experiments show that WC-1 and WC-2 are transcription factors regulating the expression of light-induced genes. The WC proteins form homo- and heterodimers in vitro; this interaction could represent a fundamental step in the control of their activity. We demonstrate in vivo that the WC proteins are assembled in a white collar complex (WCC) and that WC-1 undergoes a change in mobility due to light-induced phosphorylation events. The phosphorylation level increases progressively upon light exposure, producing a hyperphosphorylated form that is degraded and apparently replaced in the complex by a newly synthesized WC-1. WC-2 is unmodified and also does not change quantitatively in the time frame examined. Light-dependent phosphorylation of WC-1 also occurs in a wc-2 mutant, suggesting that a functional WC-2 is dispensable for this light-specific event. These results suggest that light-induced phosphorylation and degradation of WC-1 could play a role in the transient expression of blue-light-regulated genes. Our findings suggest a mechanism by which WC-1 and WC-2 mediate light responses in Neurospora. (+info)
The 24-kDa iron-sulphur subunit of complex I is required for enzyme activity.
(23/1876)
We have cloned the nuclear gene encoding the 24-kDa iron-sulphur subunit of complex I from Neurospora crassa. The gene was inactivated in vivo by repeat-induced point-mutations, and mutant strains lacking the 24-kDa protein were isolated. Mutant nuo24 appears to assemble an almost intact complex I only lacking the 24-kDa subunit. However, we also found reduced levels of the NADH-binding, 51-kDa subunit of the enzyme. Surprisingly, the complex I from the nuo24 strain lacks NADH:ferricyanide reductase activity. In agreement with this, the respiration of intact mitochondria or mitochondrial membranes from the mutant strain is insensitive to rotenone inhibition. These results suggest that the nuo24 complex is not functioning in electron transfer and the 24-kDa protein is absolutely required for complex I activity. This phenotype may explain the findings that the 24-kDa iron-sulphur protein is reduced or absent in human mitochondrial diseases. In addition, selected substitutions of cysteine to alanine residues in the 24-kDa protein suggest that binding of the iron-sulphur centre is a requisite for protein assembly. (+info)
Circadian biology: clocks for the real world.
(24/1876)
The circadian system of Neurospora crassa includes a molecular feedback loop that is entrainable by light. A recent study has shown that a second, elusive oscillator interacts with the feedback loop to drive output rhythms. (+info)