Phosphatidylinositol 4-Phosphate Is Required for Translation Initiation in Saccharomyces cerevisiae

Abstract

The small natural product wortmannin inhibits protein synthesis by modulating several phosphatidylinositol (PI) metabolic pathways. A primary target of wortmannin in yeast is the plasma membrane-associated PI 4-kinase (PI4K) Stt4p, which is required for actin cytoskeleton organization. Here we show that wortmannin treatment or inactivation of Stt4p, but not disorganization of the actin cytoskeleton per se, leads to a rapid attenuation of translation initiation. Interestingly, inactivation of Pik1p, a wortmannin-insensitive, functionally distinct PI4K, implicated in the regulation of Golgi functions and secretion, also results in severe translation initiation defects with a marked increase of the phosphorylation of the translation initiation factor eIF2␣. Because wortmannin largely phenocopies the effects of rapamycin (e.g. it triggers nuclear accumulation of Gln3p), it likely also inhibits the PI kinase-related, target of rapamycin (TOR) kinases. Importantly, however, neither inactivation of Stt4p nor Pik1p significantly affects TOR-controlled readouts other than translation initiation, indicating that these PI4Ks do not simply function upstream of TOR. Together, our results reveal the existence of a novel translation initiation control mechanism in yeast that is tightly coupled to the synthesis of distinct PI4P pools.Under severe environmental conditions, conservation of energy and cellular resources, which is key to cellular survival, is achieved in part by inhibition of translation initiation. The down-regulation of translation initiation depends on multiple signaling pathways that sense internal stresses and subsequently signal downstream effectors that inactivate specific components of the translation machinery. The eIF4E-binding proteins (4E-BPs) 2 are specific translation inhibitors that bind to the translation factor eIF4E and prevent the recruitment of the translation machinery to mRNA (1, 2). In mammalian cells, binding of 4E-BPs to eIF4E is reversible: hypophosphorylated 4E-BPs bind to eIF4E, whereas hyperphosphorylated 4E-BPs do not bind to eIF4E. Growth stimuli including serum, hormones, growth factors, and mitogens induce hyperphosphorylation of 4E-BPs, followed by their dissociation from eIF4E. Conversely, various stresses including nutrient deprivation cause dephosphorylation of 4E-BPs, which results in a rapid association of 4E-BPs with eIF4E and consequent inhibition of translation (1). In the yeast Saccharomyces cerevisiae, two mammalian 4E-BP homologs, Caf20p and Eap1p, have been described (3)(4)(5). Although the precise role of these proteins in the regulation of translation initiation is poorly understood, Eap1p may inhibit translation initiation following membrane and/or heat stress (6, 7).Another mechanism of translation inhibition in response to various environmental stresses is mediated through the phosphorylation of the ␣ subunit of translation initiation factor 2 (eIF2␣) (8 -10). eIF2␣ is part of a trimeric GTPase eIF2 complex that, in its GTP-bound form, delivers the charged methiony...

Perturbations in actin dynamics reconfigure protein complexes that modulate GCN2 activity and promote an eIF2 response

“…S3C). These results are in agreement with previous reports that in yeast, latrunculin-B does not result in increased eIF2α phosphorylation and does not affect translation rates (Cameroni et al, 2006;Kandl et al, 2002). Interestingly, however, disassembly of microtubules by nocodazol resulted in a strong phosphorylation of eIF2α in yeast (Fig.…”

Trans-Golgi Network and Endosome Dynamics Connect Ceramide Homeostasis with Regulation of the Unfolded Protein Response and TOR Signaling in Yeast

“…Transcriptional profiles of sec14-1 ts tlg2⌬ mutants indicate TOR signaling is also sensitive to TGN/endosomal function, consistent with growing evidence that central components of the TOR pathway either reside on TGN/endosomal membranes or are otherwise compromised by membrane trafficking defects through that membrane system (Bertram et al, 2000;Chen and Kaiser, 2003;Wedeman et al, 2003;Cameroni et al, 2006;Aronova et al, 2007;Puria et al, 2008;Rohde et al, 2008). Together, these findings define the TGN/ endosomal system as a central hub for control of intracellular homeostatic signaling.…”

“…Moreover, we find that combined Sec14/Tlg2 defects evoke dramatic increases in ceramide mass with consequent compromise of the UPR, in part via Sit4 protein phosphatase activity. These data extend studies linking nutrient sensing with membrane traffic through the TGN/endosomal system (Chen and Kaiser, 2003;Cameroni et al, 2006;Yan et al, 2006;Puria et al, 2008;Rohde et al, 2008) and identify ceramide as a quality control reporter for late secretory pathway function.…”

“…The unusual Gcn4 translation defects (in the face of elevated phospho-eIF2 levels) have an interesting and relevant precedent. These are also observed in yeast with PtdIns-4-phosphate deficits, indicating this phosphoinositide promotes translation initiation in yeast (Cameroni et al, 2006). Given Sec14 potentiates both Stt4-and Pik1-mediated PtdIns-4-phosphate synthesis (Hama et al, 1999;Phillips et al, 1999;Rivas et al, 1999;Schaaf et al, 2008), we interpret the Gcn4 translation defects observed in sec14-1 ts tlg2⌬ double mutants at 37°C to primarily stem from reduced PtdIns-4-phosphate levels.…”

Membrane stress is coupled to a rapid translational control of gene expression in chlorpromazine-treated cells

“…We recently showed that a decrease of phosphatidyl inositol 4-phosphate (PI4-P) levels on Golgi and ER organelles in a pik1 (PI4-kinase) mutant result in a rapid phosphorylation of eIF2 (Cameroni et al 2006). Like sac1 and drs2 mutants that deregulate the net charge of phospholipids on Golgi/endosomal compartments (see Table 1), the pik1 temperature sensitive mutant is highly sensitive to CPZ at permissive temperature (data not shown).…”