Volume 13, Issue 3, Pages 357-366 (February 2004) A Membrane Transport Defect Leads to a Rapid Attenuation of Translation Initiation in Saccharomyces cerevisiae  Olivier Deloche, Jesús de la Cruz, Dieter Kressler, Monique Doère, Patrick Linder  Molecular Cell  Volume 13, Issue 3, Pages 357-366 (February 2004) DOI: 10.1016/S1097-2765(04)00008-5 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Polysome Profile Analysis of Vesicular Transport Mutants (A) Different steps of vesicular transport, which are blocked at 37°C in several ts mutants used in this study. ER, endoplasmic reticulum; PM, plasma membrane. (B) Isogenic wild-type (RH448) and the above-mentioned mutants were grown in YPD at 22°C. Mid-log phase cultures were shifted to 37°C for 15 min. Cells were harvested before and after the shift, and polysomes were analyzed. The positions corresponding to the 40S r subunit, 60S r subunit, 80S monosomes/couples, and polysomal ribosomes are indicated for the profile of wild-type cells at 22°C. Molecular Cell 2004 13, 357-366DOI: (10.1016/S1097-2765(04)00008-5) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Inhibition of Translation in sec4-2 and end3-1 Mutants after a Temperature Shift Up (A) Isogenic wild-type (RH448), sec4-2 (RH1554), and end3-1 (RH1623) cells were grown in YPD at 22°C. Mid-log phase cultures were shifted to 37°C. Cells were harvested before and at the times indicated after the temperature shift, and polysomes were analyzed. (B–D) [35S]methionine incorporation into proteins over time in wild-type, sec4-2, and end3-1 cells. Cells were grown at either 22°C (B) or 37°C (C and D). Aliquots were taken at the indicated times after addition of [35S]methionine and the relative levels of incorporation into proteins were determined. (B) Wild-type strain (circles), sec4-2 strain (triangles), and end3-1 strain (squares) at 22°C. (C and D) Wild-type strain (circles), sec4-2 strain (triangles), and end3-1 strain (squares) at 37°C. (E) [35S]methionine uptake by wild-type, sec4-2, and end3-1 cells. Aliquots were taken immediately (open bars) or 45 min after the addition of [35S]methionine (filled bars). Then, the relative levels of uptake into cells were determined. Values from (B) to (D) belong to a single representative experiment. The experiment was repeated at least three times with similar results (measurements differ less than 5%). Molecular Cell 2004 13, 357-366DOI: (10.1016/S1097-2765(04)00008-5) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Pre-rRNA Processing and Northern Analyses in sec4-2 and end3-1 Mutants (A and B) Isogenic wild-type (RH448), sec4-2 (RH1554), and end3-1 (RH1623) cells were grown at 22°C. Mid-log phase cultures were shifted to 37°C for 15 (A) or 90 min (B). Each culture was pulse labeled with [methyl-3H]methionine for 2 min and then chased with an excess of nonradioactive methionine for 0, 5, and 15 min. Total RNA was prepared and 20,000 c.p.m. from each sample was subjected to electrophoresis and transferred to a nylon membrane. The positions of the different pre-rRNAs and mature rRNAs are indicated. (C) Wild-type, sec4-2, and end3-1 cells were grown in YPD at 22°C. Mid-log phase cultures were shifted to 37°C. Cells were harvested before and at various times after the shift. Total RNA was prepared and 5 μg from each sample was subjected to electrophoresis. Gels were transferred to nylon membranes and hybridized with different probes to detect the steady-state levels of the different RNAs. Molecular Cell 2004 13, 357-366DOI: (10.1016/S1097-2765(04)00008-5) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 Gcn2p Mediates the Translation Initiation Attenuation Induced by Chlorpromazine Treatment (A) Δgcn2 (H1333) and SUI2-S51A (ODY198) mutants and their respective isogenic wild-type (H1402) and (ODY197) strains were grown in YPD at 22°C to mid-log phase. Chlorpromazine (CPZ, final concentration 90 μg/ml) was added to the different cultures. After the indicated times of treatment, cells were harvested and polysomes were analyzed. (B) Wild-type (H1402) cells were grown as above and treated with CPZ or with tunicamycin (Ty, final concentration 40 μg/ml) for 30 min. Whole-cell extracts were prepared and phosphorylation of S51 was compared with the total amount of eIF2α protein. As negative controls, extracts from SUI2-S51A (ODY198) and Δgnc2 (H1333) mutants were used. Molecular Cell 2004 13, 357-366DOI: (10.1016/S1097-2765(04)00008-5) Copyright © 2004 Cell Press Terms and Conditions

Figure 5 Gcn2p Mediates the Translation Initiation Attenuation Response in the end3-1 but Not in the sec4-2 Mutant (A) Wild-type (RH448), sec4-2 (RH1554), and end3-1 (RH1623) strains were grown in YPD at 22°C. Mid-log phase cultures were shifted to 37°C for 15 min. At the different time points after the shift, whole-cell extracts were prepared and phosphorylation of S51 on eIF2α was analyzed as in Figure 4. (B) Wild-type (RH448), sec4-2 (RH1554), end3-1 (RH1623), Δgcn2 (H1333), sec4-2 Δgcn2 (ODY229), and end3-1 Δgcn2 (ODY211) strains were grown and shifted as above. Cells were harvested before and after the shift, and polysomes were analyzed. Molecular Cell 2004 13, 357-366DOI: (10.1016/S1097-2765(04)00008-5) Copyright © 2004 Cell Press Terms and Conditions

Figure 6 Role of Eap1p and the Cell Integrity Pathway for the Translation Initiation Attenuation Response (A) Δcaf20 (SH12-1C) and Δeap1 (YGC034) strains were grown in YPD at 22°C to mid-log phase. Cells were harvested before and 15 min after the addition of CPZ (final concentration, 90 μg/ml). (B) Sec4-2 (RH1554), Δeap1 (YGC034), and sec4-2 Δeap1 (ODY279) strains were grown in YPD at 22°C to mid-log phase and shifted to 37°C for 15 min. Cells were harvested before and after the shift. (C) Wild-type (JK9-3da), Δwsc1 (PA39-1b), and Δpkc1 (DL013-4b) strains were grown in YPD plus 1 M sorbitol at 22°C and treated with CPZ as above. Molecular Cell 2004 13, 357-366DOI: (10.1016/S1097-2765(04)00008-5) Copyright © 2004 Cell Press Terms and Conditions

Figure 7 Model of Signaling Pathways Leading to an Attenuation of Translation Initiation and a Transcriptional Repression of Ribosome Components Block of vesicular transport through the plasma membrane (i.e., sec and end mutants) or drugs that cause a membrane stress (i.e., chlorpromazine) are proposed to activate one or several of these pathways. Arrows indicate activation and bars indicate repression. The number of steps involved in each branch of the pathways is unknown. This model is modified from Li et al. (2000). Molecular Cell 2004 13, 357-366DOI: (10.1016/S1097-2765(04)00008-5) Copyright © 2004 Cell Press Terms and Conditions