The Small Yeast GTPase Rho5 and Its Dimeric GEF Dck1/Lmo1 Respond to Glucose Starvation.

Rho5 is a small GTPase of Saccharomyces cerevisiae and a homolog of mammalian Rac1. The latter regulates glucose metabolism and actin cytoskeleton dynamics, and its misregulation causes cancer and a variety of other diseases. In yeast, Rho5 has been implicated in different signal transduction pathways, governing cell wall integrity and the responses to high medium osmolarity and oxidative stress. It has also been proposed to affect mitophagy and apoptosis. Here, we demonstrate that Rho5 rapidly relocates from the plasma membrane to mitochondria upon glucose starvation, mediated by its dimeric GDP/GTP exchange factor (GEF) Dck1/Lmo1. A function in response to glucose availability is also suggested by synthetic genetic phenotypes of a rho5 deletion with gpr1, gpa2, and sch9 null mutants. On the other hand, the role of mammalian Rac1 in regulating the action cytoskeleton does not seem to be strongly conserved in S. cerevisiae Rho5. We propose that Rho5 serves as a central hub in integrating various stress conditions, including a crosstalk with the cAMP/PKA (cyclic AMP activating protein kinase A) and Sch9 branches of glucose signaling pathways.

Identification of Dck1 and Lmo1 as upstream regulators of the small GTPase Rho5 in Saccharomyces cerevisiae.

The exact function and regulation of the small GTPase Rho5, a putative homolog of mammalian Rac1, in the yeast Saccharomyces cerevisiae have not yet been elucidated. In a genetic screen initially designed to identify novel regulators of cell wall integrity signaling, we identified the homologs of mammalian DOCK1 (Dck1) and ELMO (Lmo1) as upstream components which regulate Rho5. Deletion mutants in any of the encoding genes (DCK1, LMO1, RHO5) showed hyper-resistance to cell wall stress agents, demonstrating a function in cell wall integrity signaling. Live-cell fluorescence microscopy showed that Dck1, Lmo1 and Rho5 quickly relocate to mitochondria under oxidative stress and cell viability assays indicate a role of Dck1/Lmo1/Rho5 signaling in triggering cell death as a response to hydrogen peroxide treatment. A regulatory role in autophagy/mitophagy is suggested by the colocalization of Rho5 with autophagic markers and the decreased mitochondrial turnover observed in dck1, lmo1 and rho5 deletion mutants. Rho5 activation may thus serve as a central hub for the integration of different signaling pathways.

The small GTP-binding proteins AgRho2 and AgRho5 regulate tip-branching, maintenance of the growth axis and actin-ring-integrity in the filamentous fungus Ashbya gossypii.

GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.

A novel role for the non-catalytic intracellular domain of Neprilysins in muscle physiology.

BACKGROUND INFORMATION: Neprilysins (Neps) are membrane-bound M13 endopeptidases responsible for the activation and/or inactivation of peptide signalling events on cell surfaces. By hydrolysing their respective substrates, mammalian Neps are crucial to the metabolism of numerous bioactive peptides, especially in the nervous, immune, cardiovascular and inflammatory systems. On the basis of their involvement in essential physiological processes, proteins of the Nep family constitute putative therapeutic agents as well as targets in different diseases, including Alzheimer's disease. RESULTS: We here demonstrate that overexpression of Neprilysin 4 (Nep4) in Drosophila melanogaster leads to a severe muscle degeneration phenotype. This phenotype is observed for overexpression of full-length Nep4 in somatic muscles and is accompanied by severely impaired movement of larvae and lethality in late larval development. On the contrary, down-regulation of expression caused only the latter two effects. By expressing several mutated and truncated forms of Nep4 in transgenic animals, we show that the intracellular domain is responsible for the observed phenotypes while catalytic activity of the enzyme was apparently dispensable. A yeast two-hybrid screen identified a yet uncharacterised carbohydrate kinase as a first interaction partner of the intracellular domain of Nep4. CONCLUSIONS: These data demonstrate that the physiological significance of Nep4 is not limited to its function as an active peptidase but that the enzyme's intracellular N-terminus is affecting muscle integrity, independent of the protein's enzymatic activity. To our knowledge, this is the first report of an intracellular Nep domain being involved in muscle integrity.

How do I begin? Sensing extracellular stress to maintain yeast cell wall integrity.

The cell wall integrity (CWI) signalling pathway is necessary to remodel the yeast cell wall during normal morphogenesis and in response to cell surface stress. In the Baker's yeast Saccharomyces cerevisiae, a set of five membrane-spanning sensors, namely Wsc1, Wsc2, Wsc3, Mid2 and Mtl1, detect perturbations in the cell wall and/or the plasma membrane and activate a downstream signal transduction pathway with a central MAP kinase module. As a consequence, the expression of genes whose products are involved in cell wall structure and remodelling is induced. This review summarises our recent results on sensor structure and function, as well as the advances made regarding sensor mechanics.

Single-molecule atomic force microscopy reveals clustering of the yeast plasma-membrane sensor Wsc1.

Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains ("rafts") is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of approximately 200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.

Cyk3 acts in actomyosin ring independent cytokinesis by recruiting Inn1 to the yeast bud neck.

Cytokinesis in yeast can be achieved by plasma membrane ingression, which is dependent on actomyosin ring constriction. Inn1 presumably couples these processes by interaction with both the plasma membrane and the temporary actomyosin ring component Hof1. In addition, an actomyosin ring independent cytokinesis pathway exists in yeast. We here identified Cyk3, a key component of the alternative pathway, as a novel interaction partner of Inn1. The carboxy-terminal proline rich part of Inn1 binds the SH3 domains of either Cyk3 or Hof1. Strains with truncated proteins lacking either of these SH3 domains do not display any severe phenotypes, but are synthetically lethal, demonstrating their crucial role in cytokinesis. Overexpression of CYK3 leads to an actomyosin ring independent recruitment of Inn1 to the bud neck, further supporting the significance of this interaction in vivo. Moreover, overexpression of CYK3 in a myo1 or an iqg1 deletion not only restores viability, but also the recruitment of Inn1 to the bud neck. We propose that Cyk3 is part of an actomyosin ring independent cytokinesis pathway, which acts as a rescue mechanism to recruit Inn1 to the bud neck.