MHO1, an Evolutionarily Conserved Gene, Is Synthetic Lethal with PLC1; Mho1p Has a Role in Invasive Growth

The novel protein Memo (Mediator of ErbB2 driven cell motility) was identified in a screen for ErbB2 interacting proteins and found to have an essential function in cell motility. Memo is evolutionarily conserved with homologs found in all branches of life; the human and yeast proteins have a similarity of >50%. In the present study we used the model organism S. cerevisiae to characterize the Memo-homologue Mho1 (Yjr008wp) and to investigate its function in yeast. In a synthetic lethal screen we found MHO1 as a novel synthetic lethal partner of PLC1, which encodes the single phospholipase C in yeast. Double-deleted cells lacking MHO1 and PLC1, proliferate for up to ten generations. Introduction of human Memo into the memoΔplc1Δ strain rescued the synthetic lethal phenotype suggesting that yeast and human proteins have similar functions. Mho1 is present in the cytoplasm and the nucleus of yeast cells; the same distribution of Memo was found in mammalian cells. None of the Memo homologues have a characteristic nuclear localization sequence, however, a conserved nuclear export sequence is found in all. In mammalian cells, blocking nuclear export with Leptomycin B led to nuclear Memo accumulation, suggesting that it is actively exported from the nucleus. In yeast MHO1 expression is induced by stress conditions. Since invasive growth in S. cerevisiea is also stress-induced, we tested Mho1's role in this response. MHO1 deletion had no effect on invasion induced by nutrient deprivation, however, Mho1 overexpression blocked the invasive ability of yeast cells, suggesting that Mho1 might be acting in a dominant negative manner. Taken together, our results show that MHO1 is a novel synthetic lethal interactor with PLC1, and that both gene products are required for proliferation. Moreover, a role for Memo in cell motility/invasion appears to be conserved across species

AgTHR4, a new selection marker for transformation of the filamentous fungus Ashbya gossypii, maps in a four-gene cluster that is conserved between A : gossypii and Saccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones of Ashbya gossypii genomic DNA revealed seven sequences with homology to Saccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of the S. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putative AgTHR4 gene of A. gossypii. It comprises 512 codons, two less than the S. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of the A. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying the AgTHR4 gene and various S. cerevisiae ARS elements. Using these plasmids only very weak complementation of a S. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to the AgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved in A. gossypii and S. cerevisiae

Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network

A conserved G1 regulatory circuit promotes asynchronous behavior of nuclei sharing a common cytoplasm

Synthesis and accumulation of conserved cell cycle regulators such as cyclins are thought to promote G1/S and G2/M transitions in most eukaryotes.1 When cells at different stages of the cell cycle are fused to form heterokaryons, the shared complement of regulators in the cytoplasm induces the nuclei to become synchronized.2 However, multinucleate fungi often display asynchronous nuclear division cycles, even though the nuclei inhabit a shared cytoplasm.3 Similarly, checkpoints can induce nuclear asynchrony in multinucleate cells by arresting only the nucleus that receives damage.4–6 The cell biological basis for nuclear autonomy in a common cytoplasm is not known. Here we show that in the filamentous fungus Ashbya gossypii, sister nuclei born from one mitosis immediately lose synchrony in the subsequent G1 interval. A conserved G1 transcriptional regulatory circuit involving the Rb-analog Whi5p promotes the asynchronous behavior yet Whi5 protein is uniformly distributed among nuclei throughout the cell cycle. The homologous Whi5p circuit in S. cerevisiae employs positive feedback to promote robust and coherent entry into the cell cycle. We propose that positive feedback in this same circuit generates timing variability in a multinucleate cell. These unexpected findings indicate that a regulatory program whose products (mRNA transcripts) are translated in a common cytoplasm can nevertheless promote variability in the individual behavior of sister nuclei