Polarized secretion is a tightly regulated event generated by conserved, asymmetrically localized multiprotein complexes, and the mechanism(s) underlying its temporal and spatial regulation are only beginning to emerge. Although yeast Iqg1p has been identified as a positional marker linking polarity and exocytosis cues, studies on its mammalian counterpart, IQGAP1, have focused on its role in organizing cytoskeletal architecture, for which the underlying mechanism is unclear. Here, we report that IQGAP1 associates and co-localizes with the exocyst-septin complex, and influences the localization of the exocyst and the organization of septin. We further show that activation of CDC42 GTPase abolishes this association and inhibits secretion in pancreatic β-cells. Whereas the N-terminus of IQGAP1 binds the exocyst-septin complex, enhances secretion and abrogates the inhibition caused by CDC42 or the depletion of IQGAP1, the C-terminus, which binds CDC42, inhibits secretion. Pulse-chase experiments indicate that IQGAP1 influences protein-synthesis rates, thus regulating exocytosis. We propose and discuss a model in which IQGAP1 serves as a conformational switch to regulate exocytosis.

Summary IQGAP1 regulates cell proliferation through a novel CDC42-mTOR pathway

The Rho-type GTPase Cdc42p has been implicated in diverse cellular functions including cell shape, cell motility, and cytokinesis, all of which involve the reorganization of the actin cytoskeleton. Targets of Cdc42p that interface the actin cytoskeleton are likely candidates for mediating cellular activities. In this report, we identify and characterize a yeast homologue for the mammalian IQGAP, a cytoskeletal target for Cdc42p. The yeast IQGAP homologue, designated Iqg1p, displays a two-hybrid interaction with activated Cdc42p and coimmunoprecipitates with actin filaments. Deletion of IQG1 results in a temperature-sensitive lethality and causes aberrant morphologies including elongated and round multinucleated cells. This together with its localization at the mother–bud neck, suggest that Iqg1p promotes budding and cytokinesis. At restrictive temperatures, the vacuoles of the mutant cells enlarge and vesicles accumulate in the bud. Interestingly, Iqg1p shows two-hybrid interactions with the ankyrin repeat–containing protein, Akr1p (Kao, L.-R., J. Peterson, J. Ruiru, L. Bender, and A. Bender. 1996. Mol. Cell. Biol. 16:168–178), which inhibits pheromone signaling and appears to promote cytokinesis and/or trafficking. We also show two-hybrid interactions between Iqg1p and Afr1p, a septin-binding protein involved in projection formation (Konopka, J.B., C. DeMattei, and C. Davis. 1995. Mol. Cell. Biol. 15:723–730). We propose that Iqg1p acts as a scaffold to recruit and localize a protein complex involved in actin-based cellular functions and thus mediates the regulatory effects of Cdc42p on the actin cytoskeleton.

Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids. However, no studies have been done of the biochemical pathways involved. Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sanvwn and Arabidopsis thaEaa and in a cDNA library from Arabidopsis. A cDNA was cloned and sequenced, and its predicted amino acid sequence Is 60.7% identical to that of the cyanobacterial RecA protein. This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts.The observation of chloroplast DNA recombinants in somatic hybrids of higher plants (1), genetic studies of the inheritance of chloroplast markers in several crosses of Chlamydomonas (2-4), the integration of donor DNA by homologous recombination in chloroplasts of transformed Chlamydomonas (5), and extensive comparative analyses of chloroplast genome structure (6-9) indicate that DNA recombination occurs in chloroplasts of both higher plants and green algae. The biochemistry of any recombinational mechanism in chloroplasts is completely unknown, however.It is generally accepted that plastids originated from cyanobacterial progenitors, acquired by an ancestral eukaryotic cell through an endosymbiotic event (10,11). Therefore it seemed probable that any chloroplast recombination system should be related to a eubacterial counterpart. In Escherichia coli and many other prokaryotes, the RecA protein is essential for homologous recombination and for a variety of SOS responses to DNA damage (12)(13)(14)(15)(16)(17)(18)(19)(20)

SummaryDefining the mechanisms that control cell growth and division is crucial to understanding cell homeostasis, which impacts human diseases such as cancer and diabetes. IQGAP1, a widely conserved effector and/or regulator of the GTPase CDC42, is a putative oncoprotein that controls cell proliferation; however, its mechanism in tumorigenesis is unknown. The mechanistic target of rapamycin (mTOR) pathway, the center of cell growth control, is commonly activated in human cancers, but has proved to be an ineffective clinical target because of an incomplete understanding of its mechanisms in cell growth inhibition. Using complementary studies in yeast and mammalian cells, we examined a potential role for IQGAP1 in regulating the negative feedback loop (NFL) of mTOR complex 1 (mTORC1) that controls cell growth. Two-hybrid screens identified the yeast TORC1-specific subunit Tco89p as an Iqg1p-binding partner, sharing roles in rapamycin-sensitive growth, axial-bud-site selection and cytokinesis, thus coupling cell growth and division. Mammalian IQGAP1 binds mTORC1 and Akt1 and in response to epidermal growth factor (EGF), cells expressing the mTORC1-Akt1-binding region (IQGAP1 IR-WW ) contained attenuated phosphorylated ERK1/2 (ERK1/2-P) activity and inactive glycogen synthase kinase 3a/b (GSK3a/b), which control apoptosis. Interestingly, these cells displayed a high level of Akt1 S473-P, but an attenuated level of the mTORC1-dependent kinase S6K1 T389-P and induced mTORC1-Akt1-and EGF-dependent transformed phenotypes. Moreover, IQGAP1 appears to influence cell abscission and its activity is elevated in carcinoma cell lines. These findings support the hypothesis that IQGAP1 acts upstream on the mTORC1-S6K1RAkt1 NFL and downstream of it, to couple cell growth and division, and thus like a rheostat, regulates cell homeostasis, dysregulation of which leads to tumorigenesis or other diseases. These results could have implications for the development of the next generation of anticancer therapeutics.Key words: IQGAP1, Proliferation, Secretion, mTOR IntroductionThe mechanisms that control cell proliferation continue to be central to cell biology research (Tapon et al., 2001;Sturgill and Hall, 2007;Moseley et al., 2009) and to understanding prevalent human diseases such as diabetes and cancer. The evolutionarily conserved serine/threonine protein kinase mechanistic target of rapamycin (mTOR), the center of cell growth control, interfaces nutrient and growth factor signals to regulate cell proliferation Sabatini, 2007, Sengupta et al., 2010). It is believed that mTOR couples cell growth and division by integrating the nutrient and growth factor signals through the phosphoinositide 3-kinase-RAC-a serine/threonine protein kinase-mTOR (PI3K-Akt1-mTOR) pathway to control cell size, a pre-requisite to entry into the cell cycle, but despite much progress, how the two activities are integrated remains unclear (Tapon et al., 2001;Fingar and Blenis, 2004;Sabatini, 2006;Wullschleger et al., 2006;Polak and Hall, 2006;Sturgill and Hal...