Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway

Abstract

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

Figure 1

Protein translocation returns to wild-type efficiency in the absence of functional SRP54 or SRβ over time. (A) Kar2 immunoprecipitation in the SRP54^dn system. Translocation of Kar2 was compared in strains containing either a pGAL-SRP54^wt plasmid (SMY212) or a pGAL-SRP54^dn plasmid (SMY211). Cells were grown to midlog phase in raffinose-containing selective synthetic medium lacking uracil, and then shifted to the comparable galactose-containing media. Cells were labeled with [³⁵S]methionine for 7 min, and harvested at the indicated times. Lysates at each time point were immunoprecipitated with anti-Kar2 and analyzed by SDS-PAGE followed by autoradiography. Lumenal (Kar2) and cytosolic precursor (pre-Kar2) forms are indicated. The amount of precursor protein relative to lumenal protein at each time point was quantified and graphed. The graph and error bars for the SRP54^dn strain reflect the average and SD of nine experiments. (B) DPAP-B immunoprecipitation in the SRP54^dn system. Experiments were carried out and analyzed as described for A. Lumenal (DPAP-B) and cytosolic precursor (pre-DPAP-B) forms are indicated. (C) DPAP-B immunoprecipitation in the SRβ^ts system. Translocation of DPAP-B in a wild-type strain (SMY286, srp102::URA3, pTH123) or a strain containing the srp102(K51I) ts allele of SRP102 (SMY288, srp102::URA3, pSO462). Cells were grown to midlog phase in YPD at 23°C and then shifted to 37°C to induce the SRβ^ts allele. Cells were labeled and immunoprecipitated as described in A.

Figure 2

Adaptation is a reversible, physiological process. Kar2 immunoprecipitation in the SRP54^dn system. Cells (SMY211) were grown to midlog phase in raffinose-containing synthetic medium lacking uracil and shifted to galactose-containing medium. After 16 h, the cells were washed free of galactose and returned to raffinose-containing medium for a period of 24 h. After this recovery period, cells were again shifted to galactose-containing medium and allowed to grow for 12 h. Cells were labeled with [³⁵S]methionine at the indicated time points, and Kar2 was immunoprecipitated as described in Figure 1. Cytosolic precursor forms (pre-Kar2) and lumenal forms (Kar2) are indicated. The relative amount of untranslocated protein at each time point was quantified and graphed as in Figure 1A. Differences in protein levels in lane 1, 4, 5, and 6 are due to experimental handling.

Figure 3

The translocon in adapted cells is identical to that of wild-type cells. Sec63 complex proteins were purified from the following strains: SMY212 (lane 1, no tag); SMY268 bearing pSM131 (lane2, Sec64prA); SMY268 bearing pSM110 (lane 3, Sec63prA); srp102::URA3, SMY266 (lane 4 and 5, Sec63prA) as described in MATERIALS AND METHODS. Cells were grown in raffinose-containing medium to midlog phase and switched to growth in galactose-containing medium lacking methionine. Cultures were maintained in log phase in galactose-containing medium for 24 h (lanes 1–3). Alternatively, cultures were grown to midlog phase in YPD at 23°C (lane 4 and 5) and switched to 37°C for 12 h (lane 5). Cells were steady-state labeled with [³⁵S]methionine for 45 min to 1 h at 30°C (lanes 1–3), 23°C (lane 4), or 37°C (lane 5), and membranes were isolated. Digitonin extracts of membranes were incubated with IgG Sepharose for 3 h at 4°C with rotation. The IgG Sepharose beads were washed and protein complexes were eluted with 100 mM glycine pH 2.0. The eluate was concentrated by TCA precipitation and analyzed by SDS-PAGE. The major protein components of the translocon are indicated.

Figure 4

Heat shock and chaperone gene transcription is induced during adaptation. Strains were grown to midlog phase in either YPD at 23°C (SRβ^ts [SMY288]; versus SRβ^wt [SMY286]), or 30°C (ΔSRP54 [SMY284a]; ΔSRP102 (YTH119); ΔSCR1 (SOY60); all versus W303 rho⁻), or in synthetic raffinose-containing medium at 30°C SRP54^dn (SMY211); versus SRP54^wt [SMY212]). The SRβ^ts strain and the SRβ^wt control strain were then both shifted to 37°C for the times indicated. The SRP54^dn strain and the SRP54^wt control strain were shifted to galactose-containing medium for the times indicated. It was critical to ensure that all strains were diluted as necessary to keep them continuously in log phase growth, and the medium used was derived from the same batch for each experiment. At the indicated time points, cells were centrifuged and snap-frozen in liquid nitrogen. Fluorescently labeled cDNA probes were made as described in MATERIALS AND METHODS. Cy3 (green) labeled probes are SRβ^wt at 23°C (0-h time point) or 37°C (2–12-h time points), W303 rho⁻, and SRP54^wt in raffinose (0-h time point) or galactose (2–31-h time points). Cy5 (red) labeled probes are SRβ^ts, SRP54^dn, ΔSRP54, ΔSRP102, and ΔSCR1. For each time point or deletion, the differentially labeled probe pairs were mixed and hybridized to a microarray, and the relative abundance of each mRNA was measured by intensity of red or green fluorescence. The red/green fluorescence intensity ratio gives a measure of relative expression for each ORF as shown in the color scale. Brightest red color blocks indicate genes most highly induced relative to the control strain, brightest green blocks represent highest repression, black indicates no change in expression, and gray indicates no data. (A) Relative expression levels of selected genes encoding for heat shock proteins and chaperones are depicted with color blocks. (B) Relative expression levels of several heat shock and chaperone genes throughout the SRβ^ts time course. (C) Relative expression levels of several heat shock and chaperone genes throughout the SRP54^dn time course.

Figure 5

Constitutive high expression of heat shock proteins and chaperones is not sufficient to adapt to the loss of SRP⁵⁴. Accumulation of untranslocated Kar2 upon induction of SRP54^dn was measured in a wild-type strain (SMY211; □; n = 4) and in a strain containing a constitutively active allele of HSF1 (SMY226 [HSF1^C; hsf::LEU2, with pHF35]; ▪; n = 2). Cells were grown in galactose-containing medium for 4 h, labeled, and processed for immunoprecipitation with anti-Kar2 as described in Figure 1. The precursor form of Kar2 is plotted as percentage of total Kar2 immunoprecipitated.

Figure 6

Ribosome biogenesis is repressed during adaptation. Strains, conditions, and color codes are exactly as described in Figure 4. (A) Relative expression levels of selected genes involved in ribosome biogenesis are depicted. (B) Relative expression levels of several ribosomal protein genes throughout the SRβ ^ts time course are plotted.

Figure 7

Decreased protein synthesis caused by cycloheximide treatment can suppress translocation defects due to SRP54^dn expression. SRP54^dn cells were grown in galactose for 3 h, and cycloheximide was added to the concentrations indicated for an additional hour. Cells were labeled and processed for immunoprecipitation with anti-Kar2 as described in Figure 1.