Mating was done by replica-plating strain 155 (WT) or its mutants onto YPD plates containing a lawn ofMAT cells (strain 12/8), followed by incubation overnight at 30C. mutant was obtained in conditions where the KlG subunit interacted defectively with the G subunit. The addition of a VEGFA CCAAX motif to the C-end of KlG, partially suppressed the lack of both KlG and KlG subunits. In cells lacking KlG, the KlG subunit cofractionated with KlG in the plasma membrane, but in the KlG KlG strain was located in the cytosol. When the KlG-KlG interaction was affected in the KlG mutant, most KlG fractionated to the cytosol. In contrast to the generic model of G-protein function, the G subunit ofK. lactishas the capacity to attach to the membrane and to activate mating effectors in absence of the G subunit. == INTRODUCTION == Signal transduction mediated by heterotrimeric G proteins coupled to seven transmembrane receptors is an extremely widespread phenomenon in eukaryotic cells. InSaccharomyces cerevisiaethe heterotrimer G(Gpa1p)/G(Ste4p)/G(Ste18p) is required for response to mating pheromones. This G protein is the same inMATaandMAT cells. On pheromone interaction with a cell typespecific receptor, the G protein dissociates into G(GTP) and the G/G dimer, which in turn initiates a cascade of events that results in transcriptional activation of genes required for mating (Elionet al., 1993,Olsonet al., 2000). Mating between haploid cells of the opposite mating types leads to the formation of a diploidMATa/MAT cell. InS. cerevisiaedisruption of the gene encoding the G-protein subunit leads to permanent growth arrest and therefore to lethality (Dietzel and Kurjan, 1987;Miyajimaet al., 1987), whereas inactivation of both, the G and G subunits leads to sterility (Whitewayet al., 1989). Moreover, G subunit overexpression induces growth arrest and mating. On pheromone activation inS. cerevisiae, the liberated G dimer Esaxerenone directly associates with a scaffold protein Ste5p and with a p21-activated kinase (PAK), Ste20p, which is essential for activation of the MAPKKK Ste11p. Activation of Ste11p is also promoted by action of the adaptor protein Ste50p. Ste11p in turn, activates the MAPKK Ste7p. Downstream from Ste7p, Fus3p and Kss1p, two partially redundant MAPKs, induce the activation of transcription factors, Ste12p among others, which regulate the mating process (Breitkreutzet al., 2001). In the budding yeastKluyveromyces lactis, the signal transduction system that mediates mating is triggered by both G (Savin-Tejedaet al., 2001) and G (Kawasakiet al., 2005) subunits of the heterotrimeric G protein. In contrast toS. cerevisiae, inactivation of G inK. lactisdoes not affect cell viability, but produces partial sterility (Savin-Tejedaet al., 2001); and overexpression of G has no effect in mating, but its inactivation produces total sterility (Kawasakiet al., 2005). These features ofK. lactisG protein function, not observed inS. cerevisiae, may reflect a different control mechanism of the process for sexual reproduction. The actual knowledge of the pheromone response pathway inK. lactisshows that activation of G protein by binding of pheromone to G proteincoupled receptor triggers two branches: one is essential for mating and is triggered by the G subunit, and the second is dispensable and is activated by G. These two branches converge in the Esaxerenone MAP module formed by the scaffold KlSte5p, the MAPKKK KlSte11p, the MAPKK KlSte7p, and the MAPK KlFus3p (Kawasakiet al., 2008). In the heterotrimeric G proteincoupled receptor systems, the dimer is a fundamental part of the transduction mechanism. Yeast G and G form a stable dimeric complex similar in Esaxerenone its structural and functional organization to the G-dimer of vertebrates. The G complex is associated with the membrane via isoprenyl modifications of the G subunit and promotes G association with membranes and receptors (Zhang and Casey, 1996). The G dimer can also activate effector proteins on its own.