LAS24/KOG1, a component of the TOR complex 1 (TORC1), is needed for resistance to local anesthetic tetracaine and normal distribution of actin cytoskeleton in yeast

Abstract

It is known that some local anesthetics inhibit the growth of budding yeast cells. To investigate the pathway of local anesthetics' action, we isolated and characterized mutants that were hyper-sensitive to tetracaine, and at the same time, temperature-sensitive for growth. They were collectively called las ( l ocal a nesthetic s ensitive) mutants. One of the LAS genes, LAS24, was found to be identical to KOG1, which had been independently discovered as a member of the TOR complex 1 (TORC1). Las24p/Kog1p is a widely conserved TOR binding protein containing the NRC domain, HEAT repeats and WD-40 repeats, but its function remains unknown. Like the tor mutants, the las24 mutants were found to have a defect in cell wall integrity and to show sensitivity to rapamycin. Furthermore, Las24p is required not only in TORC1-mediated (rapamycin-sensitive) pathways such as translation initiation control and phosphorylation of Npr1p and Gln3p, but also for the normal distribution of the actin cytoskeleton, which has been regarded as a TORC2-mediated event. Intriguingly, the temperature-sensitivity of the las24 mutant was suppressed by either activation of Tap42/PPase or by down-regulation of the RAS/cAMP pathway. Suppressors of the temperaturesensitivity of the las24-1 mutant were found not to be effective for suppression of the tetracaine-sensitivity of the same mutant. These observations along with the facts that tetracaine and high temperature differentially affected the las24-1 mutant suggest that Las24p/Kog1p is not a target of tetracaine and that the tetracaine-sensitive step may be one of downstream branches of the TORC1 pathway. Consistent with the broad cellular functions exerted by the TOR pathway, we found that Las24p was associated with membranes and was localized at vacuoles, the plasma membrane and small vesicles.

Structural analysis of compounds with actions similar to local anesthetics and antipsychotic phenothiazines in yeast

“…Recently we found that local anesthetics, antipsychotic phenothiazines and cationic surfactants rapidly shut down both translation initiation and actin polarization in yeast, whereas anionic surfactants shut down only actin polarization without apparent shutdown of translation initiation, similar to the above shutdowns observed in the stresses (b, c) (Araki et al, 2005;Uesono et al, 2008). These shutdown compounds have the amphiphilic structure comprised of hydrophilic and hydrophobic regions, surfactant activity and the ability to lyse yeast cells, and are therefore considered to associate with the lipid bilayer in the cell membranes (Araki et al, 2005;Uesono et al, 2008Uesono et al, , 2009). However, the extents of those shutdowns have not been quantified and the structural details determining shutdown pattern, potency and toxicity remain poorly understood.…”

Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae

“…Several components of the TORC1 and the Tap42-Sit4 complex localize to membranes of the protein secretory pathway (5)(6)(7)(8)(9)(10)(11)(12). Moreover, the EGO-GSE complex, which, in response to amino acids, controls sorting of the general amino acid permease Gap1, and, in combination with TORC1, regulates microautophagy, resides in prevacuolar compartments (29,30).…”

“…A prominent role for endogenous membranes of the protein secretory pathway as a platform for Tor signaling has begun to emerge: (i) different components of the Tor protein complexes, termed TORC1 and TORC2, as well as the Tap42-Sit4 phosphatase complex, have been localized to endosomal and vacuolar compartments (5)(6)(7)(8)(9)(10)(11)(12); (ii) a function for the Golgi Ca 2ϩ /Mn 2ϩ ATPase Pmr1 in negatively regulating TORC1 signaling has been demonstrated (13); and (iii) recent studies have shown genetic interactions between TORC1 and different components of the protein-sorting machinery, including those of the class C Vps complex that functions in docking and fusion of vesicles with the Golgi, endosomes, and vacuoles (refs. 9, 14, and reviewed in ref.…”

TORC2 Signaling Is Antagonized by Protein Phosphatase 2A and the Far Complex in Saccharomyces cerevisiae

“…In contrast, lst8D only mildly reduced the expression of RPL3 and RPS6A, suggesting that lst8D does not lead to severe loss of TORC1 activity. Recent research has demonstrated that the TORC1 components are located on intracellular membranes with a concentration on the vacuolar membrane while TORC2 components appear as punctate spots at the plasma membrane (Wedaman et al 2003;Araki et al 2005;Sturgill et al 2008;Berchtold and Walther 2009;Binda et al 2009). It has been proposed that plasma membrane localization of TORC2 is essential for cell viability (Berchtold and Walther 2009).…”