A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis

Abstract

The coupling of environmental conditions to cell growth and division is integral to cell fitness. In Saccharomyces cerevisiae, the transcription factor Sfp1 couples nutrient status to cell growth rate by controlling the expression of ribosome biogenesis (Ribi) and ribosomal protein (RP) genes. Sfp1 is localized to the nucleus in rich nutrients, but upon nutrient limitation or target of rapamycin (TOR) pathway inhibition by rapamycin, Sfp1 rapidly exits the nucleus, leading to repression of the Ribi/RP regulons. Through systematic cell-based screens we found that many components of the secretory system influence Sfp1 localization. Notably, the essential Rab escort protein Mrs6 exhibited a nutrient-sensitive interaction with Sfp1. Overexpression of Mrs6 prevented nuclear localization of Sfp1 in rich nutrients, whereas loss of Mrs6 resulted in nuclear Sfp1 localization in poor nutrients. These effects were specific to Sfp1 and independent of the protein kinase C (PKC) pathway, suggesting that Mrs6 lies in a distinct branch of TOR and ribosome biogenesis regulation. Rapamycin-resistant alleles of MRS6 were defective in the cytoplasmic retention of Sfp1, the control of cell size, and in the repression of the Ribi/RP regulons. The Sfp1-Mrs6 interaction is a nexus for growth regulation that links the secretory system and TOR-dependent nutrient signaling to ribosome biogenesis.

Figure 1.

Genome-wide screens for regulators of Sfp1 localization converge on the secretory system. (A) Schematic of cell-based screen for aberrant Sfp1 localization in strains depleted for one of 800 essential genes expressed from the tet promoter or overexpression of 5280 genes from the GAL1 promoter. (B) Elevated PKA activity alters Sfp1 distribution. An sfp1∷SFP1^GFP strain bearing a GAL1-TPK1 plasmid was grown in galactose medium for 2 h prior to shift to glycerol medium for 40 min and visualization of Sfp1^GFP. (C) Increased nuclear export alters Sfp1 distribution. An sfp1∷SFP1^GFP strain bearing a GAL1-MSN5 plasmid was grown in galactose medium for 2 h prior to shift to glycerol medium for 40 min and visualization of Sfp1^GFP. (D) Perturbation of secretory pathway function causes aberrant Sfp1 localization in rich medium. An sfp1∷SFP1^GFP tet-YPT1 strain was grown in 10 μg/mL doxycycline in glucose medium for 16 h and shifted to glycerol medium for 40 min prior to visualization of Sfp1^GFP. (E) Rapid inhibition of secretory pathway flux with Brefeldin A (BFA) causes cytoplasmic relocalization of Sfp1. An sfp1∷SFP1^YFP erg6Δ strain was treated with 100 ng/mL BFA for the indicated times prior to visualization of SFP1^YFP. (F) Strains defective in ER/Golgi trafficking exhibit aberrant Sfp1 localization. Subcellular distribution of Sfp1^YFP was visualized in sfp1∷SFP1^YFP sly1-1 or sfp1∷SFP1^YFP ypt6^169ts strains grown in synthetic complete media at 23°C prior to and following shift for 2 h to 37°C. (G) A log-phase culture of an sfp1∷SFP1^YFP ypt1-1 strain was visualized at a permissive temperature of 30°C.

Figure 2.

Nutrient-sensitive interaction between Sfp1 and the Rab escort protein Mrs6. (A) Identification of an Mrs6–Sfp1 complex. Sfp1^FLAG complexes were immunoprecipitated from glucose or glycerol medium and resolved by SDS-PAGE, and differentially recovered species were identified by mass spectrometry. All peptides identified under each condition are indicated in red. (B) The Sfp1–Mrs6 complex is responsive to carbon source. An sfp1∷SFP1^MYC13 mrs6∷MRS6^3HA strain was grown to log phase in glucose medium and incubated for 30 min in the indicated medium. Sfp1^MYC13 was immunoprecipitated and the presence of Mrs6^3HA was assessed by immunoblot. (C) The Sfp1–Mrs6 complex is responsive to stress conditions. An sfp1∷SFP1^MYC13 mrs6∷MRS6^3HA strain was grown to log phase in glucose medium, exposed to the indicated stresses, and processed as in B. (D) Interactions between Sfp1, Mrs6, and the TORC1 complex. sfp1∷SFP1^MYC13 lst8∷LST8^TAP or mrs6∷MRS6^3HA lst8∷LST8^TAP strains were grown in rich glucose medium and treated for 30 min with 200 ng/mL rapamycin, and Lst8^TAP immune complexes were analyzed for either Sfp1^MYC13 (top panel) or Mrs6^3HA (bottom panel) by immunoblot. (E) Sfp1 and Mrs6 associate with an insoluble membrane fraction. (Left panel) A 100,000g P₁₀₀ fraction was resuspended in lysis buffer and either untreated or incubated with 1 M NaCl and/or 1% Triton X-100, then repelleted at 100,000g and the presence of SFP1^MYC13 and MRS6^3HA in the supernatant (S₁₀₀) and the pellet (P₁₀₀) was assessed by immunoblot.

Figure 3.

Mrs6 controls Sfp1 localization. (A) MRS6 overexpression prevents nuclear accumulation of Sfp1 in glucose medium. An sfp1∷SFP1^YFP strain expressing control vector or GAL1-MRS6^Flag was grown in synthetic raffinose medium, prior to induction with galactose for 120 min. Sfp1^YFP was visualized prior to (left) and after (right) shift into glucose medium. (B) Increased nuclear accumulation of Sfp1 upon Mrs6 depletion in glycerol medium. An sfp1∷SFP1^YFP mrs6∷tet-MRS6 strain was grown in synthetic glucose medium that contained either 0 or 10 μg/mL doxycycline and shifted into glycerol medium prior to visualization of Sfp1^YFP. (C) Mrs6 overexpression causes a reduction in cell size. Size distributions were acquired for log-phase cultures expressing either control vector (black) or GAL1-MRS6 (red) that had been induced for 6 h with galactose. (D) Mrs6 depletion causes an increase in cell size. Size distributions were acquired for log-phase cultures of an mrs6∷tet-MRS6 strain grown in glycerol medium, in the absence (black) or presence (red) of 10 μg/mL doxycycline. As a reference, a wild-type culture was grown in glucose medium without doxycycline (blue). (E) An sfp1Δ mutation bypasses the G2/M delay caused by MRS6 depletion. DNA content was determined for mrs6∷tet-MRS6 (left panel) or sfp1Δ mrs6∷tet-MRS6 (right panel) strains grown in glucose medium in the presence or absence of 10 μg/mL doxycycline. (F) Deletion of SFP1 is epistatic to depletion of MRS6 for cell size. Cell size distributions were acquired for mrs6∷tet-MRS6 and sfp1Δ mrs6∷tet-MRS6 strains grown in glucose medium in the absence (black) or presence (red) of 10 μg/mL doxycycline. (G) The Rab GTPase Ypt1 competes with Sfp1 for Mrs6. An in vivo PCA assay was used to monitor interactions between the indicated protein fusions as judged by growth in the presence of the DHFR inhibitor methotrexate. (H) An sfp1∷SFP1^MYC13 mrs6∷MRS6^3HA strain bearing GAL1-YPT1, GAL1-SEC4, GAL1-RHO1 plasmids or empty vector was grown to early log phase in synthetic raffinose medium and induced with 2% galactose for 2 h prior to harvesting. Sfp1^MYC13 immune complexes were analyzed for the presence of Mrs6^3HA and the indicated Flag-tagged proteins by immunoblot.

Figure 4.

Specificity of Sfp1 regulation by Mrs6. (A) Hyperactivation of the Ras/PKA pathway does not block MRS6-induced Sfp1 relocalization. Sfp1^CFP localization was visualized in wild-type and RAS2^VAL19 cells carrying either GAL1-MRS6 or an empty vector. Cells were grown to log phase in synthetic raffinose medium, induced for 120 min with galactose, and imaged prior to and following glucose addition. (B) The PKC pathway is not required for nutrient- and stress-induced Sfp1 relocalization. Sfp1^YFP localization was visualized in a sfp1∷SFP1^YFP pkc1Δ strain under the indicated conditions. (C) MRS6 does not influence TOR effectors that mediate the NCR response. Localization of Sfp1^GFP, Gap1^GFP, and Gln3^GFP was determined in strains carrying either GAL1-MRS6 or an empty vector grown in raffinose medium following galactose induction for 120 min. (D) MRS6 does not influence the TOR and stress-responsive transcription factor Msn2. Localization of Msn2^GFP and Sfp1^GFP was determined in strains carrying either GAL1-MRS6 or an empty vector grown in galactose medium.

Figure. 5

Isolation and characterization of rapamycin-resistant alleles of MRS6. (A) Nuclear retention of Sfp1 upon rapamycin treatment in the presence of rapamycin-resistant MRS6-R alleles. Localization of Sfp1^GFP in synthetic glucose medium, either untreated or treated with rapamycin (200 ng/mL), and synthetic glycerol medium was assessed in the indicated strains. (B) Location of amino acid substitutions in MRS6-R alleles. Conserved structural Domain I and Domain II of Rab escort proteins and Rab GDI proteins are indicated in green; conserved regions of Rab escort proteins are indicated in purple (Alory and Balch 2003). (C) Growth of wild-type and mutant alleles of MRS6. The indicated strains and, as a control, a rapamycin-resistant TOR1-1 strain were streaked onto glucose medium containing 20 ng/mL rapamycin and imaged after 4 d. (D) Rapamycin-resistant alleles of MRS6 fail to adjust cell size in response to rapamycin. Cell size profiles of indicated strains grown in synthetic glucose medium either with or without rapamycin (200 ng/mL) and, as a control, synthetic glycerol medium.

Figure 6.

MRS6 specifically controls the Ribi regulon. (A) MRS6 is required for rapamycin-induced repression of the Ribi regulon. Expression profiles were determined by microarray analysis for mrs6Δ strains bearing MRS6, MRS6-R1, and MRS6-R2 alleles on a low-copy plasmid treated for 30 and 60 min with 100 ng/mL rapamycin. Values represent the average expression change relative to untreated cells of duplicate samples. Genes exhibiting at least a twofold change in expression are displayed in the clustergram. Genes were classified into the RP and Ribi regulons as described previously (Jorgensen et al. 2004a). Effects on NCR and glycolytic gene clusters are shown to the right. (B) Overexpression of MRS6 causes repression of the Ribi, RP, and glycolytic regulons. A strain bearing a GAL1-MRS6 plasmid was induced in galactose medium for 30, 60, and 120 min and processed as in A. Duplicate experiments are shown. (C) TORC1 regulation of the Sfp1–Mrs6 interaction. Competition between Sfp1 and Rab GTPases for Mrs6 may couple activity of the secretory system to ribosome biogenesis. TORC1 localizes to various elements of the endomembrane system. Membranous structures are shown in light blue, Rab GTPases are shown in orange, TORC1 activators and effectors are shown in steel blue. Not all Rab GTPases are shown; other membrane-associated small GTPases that activate TOR are also not shown. (ER) Endoplasmic reticulum; (PM) plasma membrane; (Endo) endosome; (Rap) rapamycin.