A Membrane Transport Defect Leads to a Rapid Attenuation of Translation Initiation in Saccharomyces cerevisiae

Abstract

Transport of lipids and proteins is a highly regulated process, which is required to maintain the integrity of various intracellular organelles in eukaryotic cells. Mutations along the yeast secretory pathway repress transcription of rRNA, tRNA, and ribosomal protein genes. Here, we show that these mutations also lead to a rapid and specific attenuation of translation initiation that occurs prior to the transcriptional inhibition of ribosomal components. Using distinct vesicular transport mutants and chlorpromazine, we have identified the eIF2alpha kinase Gcn2p and the eIF4E binding protein Eap1p as major mediators of the translation attenuation response. Finally, in chlorpromazine-treated cells, this response does not require Wsc1p or the protein kinase Pkc1p, both of which are upstream of the transcriptional repression of ribosomal components. Altogether, our results suggest that yeast cells not only evolved a transcriptional but also a translational control to assure efficient attenuation of protein synthesis when membranes are stressed.

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

“…Interestingly, the eIF4B deletion strain was found to be highly sensitive to rapamycin, an observation that corroborates the Wnding that the mammalian eIF4B is an indirect target of mTOR, which is responsive to amino acid starvation (Raught et al 2004). In this regard, we previously observed that the eIF4B mutant is synthetically lethal with mutants that aVect Golgi function (e.g., vps54 , mon2 (Deloche et al 2004)) and is partially sensitive to CPZ ( Table 1), indicating that the function of eIF4B is required for cell growth during a membrane (Stevens et al 1986) stress. Finally, at 200 M CPZ, the level of ribosomal subunits dramatically dropped, resulting in a total arrest of protein synthesis (Fig.…”

“…8), thereby allowing cells to preserve energy and cellular resources. We have shown previously that this inhibition of translation initiation is not completely abrogated in a mutant strain where eIF2 cannot be phosphorylated (eIF2 -S51A), implying the existence of additional mechanisms of translation inhibition (Deloche et al 2004). Notably, we reported that the eIF4E-binding protein Eap1p functions as a translation initiation inhibitor in cells treated with CPZ and in mutants that block secretory or endocytic pathways (e.g., sec4 and end3).…”

“…At 100 M CPZ, the UPR was slightly lower, a result that might be explained by the inhibitory eVects of CPZ on protein synthesis and/or GCN4 transcription. We previously showed that a CPZ treatment causes a rapid phosphorylation of eIF2 (Deloche et al 2004). In accord with this observation and with the role of eIF2 in the activation of GCN4, we showed that 30 M CPZ leads to a 3.1-fold increase of the -galactosidase activity in cells containing the GCN4-lacZ reporter (Fig.…”

“…Thus, it is possible that CPZ induces eIF2 phosphorylation by interfering with the function of charged lipids such as PI4-P. PI4-Ps are essential for protein transport from the Golgi apparatus to the plasma membrane. However, because some sec mutants do not induce phosphorylation of eIF2 (e.g., sec4; Deloche et al 2004), a block of protein transport to the cell surface does not seem to be directly linked to phosphorylation of eIF2 . Additionally, the activation of UPR does not induce eIF2 phoshorylation (data not shown) and GCN4 mRNA translation (SteVensen and Pedersen 2006), demonstrating that Gcn2p is not stimulated by an ER stress.…”

“…These Wndings demonstrate that cells respond to a block of membrane transport to the cell surface by inducing a speciWc transcriptional program. In this context, we previously showed, using either sec mutants or chlorpromazine (CPZ), that eIF2 phosphorylation is strongly induced upon a membrane stress (Deloche et al 2004). Thus, the expression of Hac1p and Gcn4p may be required to induce an appropriate cellular response that enables cells to cope with a membrane transport defect.…”