Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast

Abstract

For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2.

Keywords: Chaperone; Cytosolic Protein Quality Control; Fatty Acid Synthase (FAS); Proteasome; Protein Complex; Protein Degradation; Ubiquitylation (Ubiquitination).

FIGURE 1.

Orphan Fas2 is proteolytically susceptible in vitro and its degradation depends on the 26 S proteasome in vivo. A, in vitro trypsin sensitivity assay. Native cell extracts from wild type (FAS1 FAS2) and Δfas1 cells expressing endogenous Fas2 were prepared as described under “Experimental Procedures” and treated with (+) or without (−) trypsin. Samples were taken at the indicated time points, and proteins were precipitated, separated, and analyzed by SDS-PAGE/Western blotting using Fas antibody, which detects Fas1 and Fas2 subunits. Endogenously expressed PGK was used as a control. B, in vivo degradation of orphan Fas2 is dependent on the 26 S proteasome. Cycloheximide-chase experiments of endogenously expressed Fas2 were performed as described under “Experimental Procedures” in the proteasomal mutant cim3-1 Δfas1 and in the respective control strain (CIM3 Δfas1). Samples were taken at the indicated time points and subjected to SDS-PAGE and Western blot analysis. Immunoblots were analyzed with Fas and PGK antibody. PGK served as loading control. The data represent the means of two independent experiments. Error bars indicate the standard deviation of the mean.

FIGURE 2.

Orphan Fas2 is predominantly organized in Fas-assembly intermediates. The Cdc48 machinery is important for disaggregation of ubiquitinated Fas2 intermediates for subsequent proteasomal degradation. A, glycerol step gradient (10–50%) centrifugation was done with native cell extracts prepared from wild type (FAS1 FAS2) and Δfas1 cells, expressing endogenous Fas2. Fractions were collected, starting from the top of the gradient (fraction 1). Proteins were precipitated with TCA and subjected to SDS-PAGE followed by immunoblotting. Endogenously expressed proteins served as molecular mass markers: fatty acid synthase dodecamer (∼2.6 MDa), the Cdc48 hexamer (540 kDa), and glucose-6-phosphate dehydrogenase (G6PDH) tetramer (240 kDa). Detection occurred via the respective antibodies (anti-Fas, anti-Cdc48, and anti-glucose-6-phosphate dehydrogenase). B, the AAA-ATPase Cdc48 is necessary for the degradation of orphan Fas2. Cycloheximide-chase experiments were performed as described under “Experimental Procedures.” Both CDC48 Δfas1 and cdc48^T413R Δfas1 strains were shifted to 37 °C before addition of cycloheximide. Samples were taken at the indicated time points and subjected to SDS-PAGE and Western blot analysis. PGK served as loading control. Immunoblots were done with Fas and PGK antibody. PGK served as loading control. The data represent the means of two independent experiments. Error bars indicate the standard deviation of the mean. C, the Cdc48 machinery acts downstream of the ubiquitination process of Fas2. A Ni-NTA-agarose based pulldown assay was performed using a yeast strain possessing a thermosensitive cdc48 allele and expressing histidine-tagged ubiquitin. Cells were shifted to 37 °C for 1 h before harvesting. Equivalent amount of cells grown at permissive temperature served as control. SDS-PAGE and Western blot were performed after elution of ubiquitinated Fas2 from the Ni-NTA-agarose beads. Immunodetection was done with Fas antibody.

FIGURE 3.

The role of the E3 RING ligase Ubr1, as well as the E2 enzymes Ubc2 and Ubc4, in the quality control process of orphan Fas2. A, pulse-chase experiments of endogenously expressed Fas2 were performed in Δfas1 and Δubr1 Δfas1 deletion strains. Cells were taken at the indicated time points and lysed, and proteins were immunoprecipitated with Fas antibody. After separation by SDS-PAGE, degradation kinetics of orphan Fas2 were detected and quantified using a PhosphorImager system and ImageQuant software, respectively. The data represent the mean values of four independent experiments. Error bars indicate the standard deviation of the mean. B, pulse-chase experiments of endogenously expressed Fas2 were performed in Δubr1 Δfas1 cells, overexpressing either N-terminally Flag-tagged Ubr1 (Flag-Ubr1) or a RING mutant of Ubr1 (Flag-Ubr1^C1220S) from a high copy plasmid. Experimental procedures were as described for A. The data represent the mean values of three independent experiments. Error bars indicate the standard deviation of the mean. C, Fas2 is stable when complexed with Fas1. Cycloheximide-chase experiments were performed using yeast strains endogenously expressing TAP-tagged Fas2 in the wild type or in the FAS1 deletion background, respectively. Fas2-TAP protein was detected at indicated time points after cycloheximide addition using TAP antibody. PGK served as loading control. D, orphan Fas2 is localized to the cytosol. Wild type and Δfas1 cells expressing endogenous EGFP-tagged Fas2 or untagged Fas2 (negative control) were prepared for fluorescence microscopy as described under “Experimental Procedures.” The nucleus was stained with Hoechst 33342. Cells were analyzed by laser-scanning microscopy. E, ubiquitination of Fas2 in absence of Fas1 is dependent on Ubr1. A Ni-NTA-agarose-based pulldown assay was performed using yeast strains expressing histidine-tagged ubiquitin. Eluate and input samples were subjected to SDS-PAGE and immunoblotting using Fas antibody. F, Fas2 in its wild type environment is not ubiquitinated. The ubiquitination assay was performed as described above, using a wild type strain expressing both Fas1 and Fas2 subunits and the FAS1 deletion strain. G, Ubr1 and the RING mutant (Ubr1^C1220S) are physically associated with orphan Fas2 in vivo. Chromosomally expressed Fas2-TAP was purified by one-step TAP purification (as described under “Experimental Procedures”) from the Δubr1 Δfas1 FAS2-TAP mutant strain harboring a high copy plasmid, encoding either N-terminally Flag-tagged Ubr1 (Flag-Ubr1) or a RING mutant of Ubr1 (Flag-Ubr1^C1220S). Cells transformed with empty vector and strains in which Fas2 was not tagged with TAP served as negative controls. 0.5% of supernatant prior to incubation with IgG beads was loaded as input control. Fas2-TAP was pulled down by incubation of the supernatant with IgG beads. Elution of IgG-bound Fas2-TAP was done by TEV protease-mediated cleavage (TEV-C). Eluate and input were subjected to SDS-PAGE followed by immunoblotting. Fas2-TAP and Flag-Ubr1 were detected by Fas and Flag antibody, respectively. PGK detected by PGK antibody served as a negative interaction control. H, pulse-chase experiments of endogenously expressed Fas2 were performed in Δfas1, Δubc2 Δfas1, Δubc4 Δfas1, and Δubc2 Δubc4 Δfas1 cells. Cells were further processed as described in A. The data represent the mean values of three independent experiments. Error bars indicate the standard deviation of the mean. Note: gray vertical line indicates pulse-chase data combined from two different SDS gels.

FIGURE 4.

Degradation and solubility of orphan Fas2 is dependent on the Hsp70 chaperone Ssa1 but shows little change in the absence of the Hsp40 chaperone Ydj1. A, cycloheximide-chase experiments of chromosomally expressed Fas2 were performed as described for temperature-sensitive strains under “Experimental Procedures” with the SSA mutants: SSA1 Δssa2 Δssa3 Δssa4 and ssa1-45 Δssa2 Δssa3 Δssa4, both deleted in FAS1. The Δfas1 strain harboring all four SSA wild type genes served as an additional control. Samples were taken at the indicated time points and subjected to Western blot analysis. Immunoblots were analyzed with Fas and PGK antibody, whereby PGK served as loading control. The data represent the means of four independent experiments. Error bars indicate the standard deviation of the mean. B, the in vivo interaction of orphan Fas2 and Ubr1 does not require a direct Ssa chaperone function. Endogenously expressed Fas2-TAP was purified by one-step TAP purification as described under “Experimental Procedures” from a ssa1-45 Δssa2 Δssa3 Δssa4 Δubr1 Δfas1 FAS2-TAP mutant strain containing either a high copy plasmid, expressing C-terminally HA-tagged Ubr1 (Ubr1-HA), or empty vector. Cells were grown at 25 °C and shifted to 37 °C 1 h before harvesting. Cells transformed with empty vector served as negative control. 0.5% of the lysate and the pellet fraction were loaded as input and pellet fractions, respectively. Fas2-TAP was pulled down by incubation of the supernatant with IgG beads (TAP-IP). Elution of IgG-bound Fas2-TAP was achieved by boiling the beads directly in urea sample buffer. All fractions were subjected to SDS-PAGE followed by immunoblotting. Fas2-TAP and Ubr1-HA were detected by Fas and HA antibody, respectively. PGK detected by PGK antibody served as negative interaction control. C, for the solubility assay of orphan Fas2, 20 A of cells of the yeast strains containing either a wild type SSA1 allele or a temperature-sensitive ssa1-45 allele, both deleted in SSA2, SSA3, and SSA4, were harvested prior to or after a temperature shift to 37 °C for 1 h. The cells were lysed, and the lysates were fractionated into total (T), supernatant (S), and pellet (P) fraction followed by TCA precipitation, solubilization, and SDS-PAGE/immunoblotting. The amount of Fas2 in the different fractions was visualized by using Fas antibody. PGK served as soluble reference protein. D, a Ni-NTA-based pulldown assay for detecting Fas2 ubiquitination was performed using the Δssa2Δssa3Δssa4 deletion strain harboring either the wild type SSA1 gene or the temperature-sensitive ssa1-45 allele. Samples for input and pulldown were taken after 1-h temperature shift from 25 to 37 °C. E, pulse-chase experiments of endogenously expressed Fas2 were performed in YDJ1 Δfas1 and ydj1-151 Δfas1 cells, according to the protocol for temperature-sensitive strains (see “Experimental Procedures”). Samples were taken at the indicated time points and lysed, and proteins were immunoprecipitated with Fas antibody. After separation by SDS-PAGE, degradation kinetics of orphan Fas2 were detected and quantified using a PhosphorImager system and ImageQuant software, respectively. The data represent the mean values of three independent experiments. Error bars indicate the standard deviation of the mean.

FIGURE 5.

Model of the ubiquitin dependent degradation process of orphan protein Fas2, which has lost its partner Fas1. In the absence of Fas1, Fas2 is still organized in an intermediate complex that is recognized by the ubiquitin ligase Ubr1, ubiquitinated with the help of the E2 enzymes Ubc2 and Ubc4, and afterwards disassembled by the Cdc48 machinery for final proteasomal degradation. Degradation of orphan Fas2 requires the presence of the Hsp70 chaperone Ssa1, which keeps Fas2 in a soluble state for efficient ubiquitination and degradation by the proteasome. Its function in the ubiquitination process by attaching Fas2 to Ubr1 via an yet unknown partner is also possible.