%0 Journal Article
%T Lack of HXK2 Induces Localization of Active Ras in Mitochondria and Triggers Apoptosis in the Yeast Saccharomyces cerevisiae
%A Loredana Amigoni
%A Enzo Martegani
%A Sonia Colombo
%J Oxidative Medicine and Cellular Longevity
%D 2013
%I Hindawi Publishing Corporation
%R 10.1155/2013/678473
%X We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in the hxk2¦¤ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis in S. cerevisiae. In particular, cells lacking HXK2 and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion of HXK2 causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast. 1. Introduction In Saccharomyces cerevisiae the highly homologous genes RAS1 and RAS2 encode small G-proteins that are activated by the guanine nucleotide exchange factors (GEFs), Cdc25 and Sdc25 [1, 2] and inactivated by the GTPase-activating proteins (GAPs), Ira1 and Ira2 [3]. GEFs and GAPs control the switch of the two small monomeric proteins between the active GTP-bound and the inactive GDP-bound state. The Ras proteins and the GPCR system [4¨C6] constitute two branches that modulate the activity of adenylate cyclase (Cyr1), according to the glucose availability in the environment. In turn Cyr1 [7] activates cAMP-dependent protein kinase (PKA) through cAMP. The amount of this second messenger is also regulated at the level of degradation by the two phosphodiesterases, Pde1 and Pde2. PKA plays a major role in the modulation of metabolism, stress resistance, cell growth, proliferation, morphogenesis, and aging [8]. Recently, our group expressed a probe consisting of a GFP fusion with a trimeric Ras Binding Domain of Raf1 (eGFP-RBD3), which binds Ras-GTP with a much higher affinity than Ras-GDP, to investigate the localization of active Ras in wild-type and in mutant strains in the cAMP/PKA pathway [9]. Our results showed that in W303-1A wild-type cells the probe
%U http://www.hindawi.com/journals/omcl/2013/678473/