Acetylation of N-terminus and two internal amino acids is dispensable for degradation of a protein that aberrantly engages the endoplasmic reticulum translocon

Yeast and bacterial methods

Yeast strains and plasmids used in this study are presented in Tables 1 and 2, respectively. Standard methods were used for manipulation of yeast strains (Guthrie & Fink, 2004). Yeast cells were grown in synthetic defined medium (Guthrie & Fink, 2004). Galactose was included in growth medium instead of glucose to induce expression of Deg1-Sec62-N153D (driven by the GAL1/10 promoter (Johnston & Davis, 1984)) for protein purification. Plasmids were manipulated using PCR-based site-directed mutagenesis using primers presented in Table 2. Mutagenesis was confirmed by digestion with restriction enzymes and plasmid sequencing.

Yeast cell harvest and immunoprecipitation

Overnight cultures of yeast cells grown in plasmid-selective medium containing glucose were diluted to an optical density at 600 nm (OD₆₀₀) of 0.2 in plasmid-selective medium containing galactose. Cultures were incubated, shaking, at 30 °C until the OD₆₀₀ reached 1.5. Approximately 15,000 OD₆₀₀ units (10 L) of logarithmically growing yeast cells were harvested by centrifugation in 1 L batches. Each batch of yeast cells was pelleted by centrifugation for 5 min at 3,800× g, snap-frozen with liquid nitrogen, and stored at −80 °C until lysis. Yeast cell pellets were thawed on ice and suspended in an equal volume of Triton X lysis buffer (500 mM NaCl, 50 mM HEPES pH 7.5, 5 mM EDTA, 1% Triton X-100) supplemented with protease inhibitors (1 tablet cOmplete Mini Protease Inhibitors; Roche Diagnostics, Indianapolis, IN, USA) per 5 mL buffer, 10 µg/mL pepstatin A) and phosphatase inhibitors (1 mM sodium fluoride, 0.5 mM sodium pyrophosphate). Volumes of glass beads approximately equal to the volumes of yeast cell pellets were added, and cell suspensions were vortexed 12 × 30 s at 4 °C, with two min on ice between rounds of vortexing. Lysed cells were centrifuged at 8,700× g for 10 min at 4 °C. The supernatants were subsequently centrifuged at 20,000× g for 10 min at 4 °C. The supernatants were preserved. Protein concentration was determined using the Bradford Assay.

Protein from cells expressing Deg1-Sec62-N153D (which possesses a FLAG affinity tag) was subjected to anti-FLAG immunoprecipitation. Briefly, 250 mg of protein extract was incubated, rotating, for 2.5 h at 4 °C in the presence of 600 µL of 50% αFLAG M2-conjugated agarose affinity gel (Sigma-Aldrich, St. Louis, MO) equilibrated with Triton X lysis buffer. The affinity gel was washed ten times with ice-cold immunoprecipitation wash buffer (500 mM NaCl, 50 mM HEPES pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate). The Deg1-Sec62-N153D protein was eluted from the affinity gel by incubation with 75 mM 3xFLAG peptide (GenScript, Piscataway, NJ, USA) in a total volume of 2 mL of immunoprecipitation wash buffer, rocking, at 4 °C for 30 min. The affinity gel was pelleted by centrifugation at 6,900 × g, and the supernatant with eluted protein was transferred to fresh tubes and preserved. To reduce disulfide linkages and alkylate cysteine side chains prior to mass spectrometry, sequential addition of and incubation with dithiothreitol (5 mM final concentration, 56 °C, 30 min) and iodoacetamide (7 mM final concentration, room temperature, in a light-proof container) was performed.

Purified, reduced, and alkylated Deg1-Sec62-N153D protein was concentrated by trichloroacetic acid (TCA) precipitation. TCA was added to a final concentration of 20%. The sample was vortexed, incubated at 4 °C for 20 min, and centrifuged at 20,000 × g at 4 °C for 20 min. The pellet was washed once with 4 °C 10% TCA and once with ice-cold 100% high-performance liquid chromatography-grade acetone. The pellet was air-dried at room temperature for 10 min and fully dried in a vacuum concentrator for 30 min. The pellet was resuspended in 2X Laemmli Sample Buffer, heated at 95 °C for 5 min, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gel was stained in GelCode Blue (Thermo Scientific, Waltham, MA, USA), and bands corresponding to Deg1-Sec62-N153D were excised with a fresh razor blade.

Mass spectrometry

Similar methods for mass spectrometry data acquisition and analysis have been described (Hughes Hallett, Luo & Capaldi, 2015). The gel slice containing Deg1-Sec62-N153D was washed 15 min each with water, 50% (v/v) acetonitrile, 100% (v/v) acetonitrile, 100 mM ammonium bicarbonate, and 100 mM ammonium bicarbonate in 50% (v/v) acetonitrile. The solution was removed, and the gel slice was dried by vacuum centrifugation. Protein in the dried gel slice was reduced by immersion in 10 mM dithiothreitol/100 mM ammonium bicarbonate at 56 °C for 45 min. The solution was removed and discarded. Protein in the gel slice was alkylated by immersion in 55 mM iodoacetamide/100 mM ammonium bicarbonate in the dark at ambient temperature for 30 min. The solution was removed and discarded. The gel slice was washed with 100 mM ammonium bicarbonate for 10 min on a shaker. An equal amount of acetonitrile was added, and the slice was incubated, shaking, for 10 min. The solution was discarded and the gel slice was dried by vacuum centrifugation for 45 min. The gel slice was cooled on ice. The slice was immersed in an ice-cold solution of 12.5 ng/µL trypsin (Promega, Madison, WI) in 100 mM ammonium bicarbonate. After 45 min, the trypsin solution was discarded, and the gel slice was immersed in 50 mM ammonium bicarbonate for 16 h at 37 °C on a shaker. The sample was sedimented by microcentrifugation, and the supernatant was collected and retained. Peptides were extracted from the gel slice by immersing the slice in 0.1% (v/v) trifluoroacetic acid (TFA) and mixing at ambient temperature for 30 min. An equal volume of acetonitrile was added, and the sample was mixed for an additional 30 min. The sample was sedimented by microcentrifugation, and the supernatant from this extraction was pooled with the post-digestion supernatant. This pool was divided into 4 tubes and concentrated by vacuum centrifugation. One tube was further digested with chymotrypsin (Roche Diagnostics, Indianapolis, IN, USA) by resuspending the tryptically digested peptides in a solution of 100 mM Tris–HCl/10 mM calcium chloride (pH 8) containing 0.25 µg of chymotrypsin. Digestion was performed at room temperature with mixing overnight. The chymotrypsin was quenched by adding TFA to a final concentration of 0.5% (v/v). One of the trypsin digests and the trypsin/chymotrypsin digest were desalted using ZipTip C₁₈ (Millipore, Billerica, MA, USA) and eluted with 70% (v/v) acetonitrile in 0.1% (v/v) TFA. The desalted material was concentrated to dryness in a centrifugal evaporator.

The proteolytically digested samples were suspended in 20 µL of 2% acetonitrile (v/v) in 0.1% (v/v) formic acid, and 18 µL was analyzed by liquid chromatography/electrospray ionization tandem MS (LC/ESI MS/MS) with a Thermo Scientific Easy-nLC II (Thermo Scientific) coupled to an Orbitrap Elite ETD (Thermo Scientific) mass spectrometer using a trap-column configuration as described (Licklider et al., 2002). Inline desalting was accomplished using a reversed-phase trap column (100 µm × 20 mm) packed with Magic C₁₈AQ (5 µm, 200 Å resin; Michrom Bioresources, Auburn, CA) followed by peptide separations on a reversed-phase column (75 µm × 250 mm) packed with Magic C₁₈AQ (5 µm, 100 Å resin) directly mounted on the electrospray ion source. A 45-min gradient from 2% to 35% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 400 nL/min was used for chromatographic separations. A spray voltage of 2,500 V was applied to the electrospray tip, and the Orbitrap Elite instrument was operated in the data-dependent mode, switching automatically between MS survey scans in the Orbitrap (automatic gain control (AGC) target value 1,000,000, resolution 120,000, and injection time 250 ms) with collision induced dissociation (CID) MS/MS spectra acquisition in the linear ion trap (AGC target value of 10,000 and injection time 100 ms), higher-energy collision induced dissociation (HCD) MS/MS spectra acquisition in the Orbitrap (AGC target value of 50,000, 15,000 resolution, and injection time 250 ms), and electron transfer dissociation (ETD) MS/MS spectra acquisition in the Orbitrap (AGC target value of 50,000, 15,000 resolution, and injection time 250 ms). The three most intense precursor ions from the Fourier transform full scan were each consecutively selected for fragmentation in the linear ion trap by CID with a normalized collision energy of 35%, fragmentation in the HCD cell with normalized collision energy of 35%, and fragmentation by ETD with 100 ms activation time. Selected ions were dynamically excluded for 30 s.

Data analysis was performed using Proteome Discoverer 1.4 (Thermo Scientific). The data were searched against the Saccharomyces Genome Database (downloaded 02/03/2011; http://www.yeastgenome.org) that was appended with protein sequences of common contaminants and the sequence for the Deg1-Sec62-N153D protein. Searches were conducted as no-enzyme search. The precursor ion tolerance was set to 10 ppm, and the fragment ion tolerance was set to 0.6 Da. Variable modifications included oxidation on methionine (+15.995 Da), carbamidomethyl (+57.021 Da) on cysteine, and acetylation on lysine and any N-terminus (+42.011 Da). All search results were run through the Percolator algorithm for false discovery rate evaluation of the identified peptides.

Cycloheximide chase, cell lysis, and endoglycosidase H treatment

Cycloheximide chase analysis was performed as described (Buchanan et al., 2016). Briefly, mid-logarithmic growth-phase yeast cells were concentrated to 2.5 OD₆₀₀ units/mL in fresh plasmid-selective media, and cycloheximide was added to a final concentration of 250 µg/mL. 950 µL aliquots (∼2.4 OD₆₀₀ units) of cells were harvested at the indicated time points following cycloheximide administration and added to 50 µL of ice-cold 20X stop mix (200 mM sodium azide, 5 mg/mL bovine serum albumin) and placed on ice until the end of the chase. Proteins were liberated by the post-alkaline protein extraction method (Kushnirov, 2000; Watts et al., 2015). To assay protein N-linked glycosylation, protein extracts derived from 0.375 OD₆₀₀ units of cells were supplemented with potassium acetate, pH 5.6 (80 mM final concentration), and incubated at 37 °C for 2 h in the absence or presence of endoglycosidase H (Endo H; Roche Diagnostics, Indianapolis, IN, USA).

Western blotting

Proteins were separated by SDS-PAGE and transferred to Immobilon polyvinylidene fluoride membranes (Millipore, Billerica, NJ, USA). Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS; 50 mM Tris, pH 7.6, 136 mM NaCl) for 60 min at room temperature or overnight at 4 °C. All antibody incubations were performed for 60 min at room temperature in 1% skim milk in TBS with 0.1% Tween 20 (TBS/T). To detect CPY*-HA, membranes were incubated in the presence of monoclonal mouse anti-HA antibodies (Clone 16B12; Covance, Princeton, NJ, USA) at 1:1,000. To detect Pgk1, membranes were incubated in the presence of monoclonal mouse anti-yeast Pgk1 antibodies (Clone 22C5D8; Life Technologies, Carlsbad, CA, USA) at 1:20,000. Anti-HA and Anti-Pgk1 primary antibody incubations were followed by incubation in the presence of Alexa-Fluor 680-conjugated Rabbit anti-Mouse IgG (H + L) secondary antibodies (Life Technologies, Carlsbad, CA, USA) at 1:40,000. The Deg1-Sec62 protein and its derivatives possess two tandem C-terminal Staphylococcus aureus Protein A tags (Hjelm, Sjodahl & Sjoquist, 1975) and were detected directly with Alexa-Fluor 680-conjugated Rabbit anti-Mouse IgG (H + L) secondary antibodies at 1:40,000. Membranes were imaged using an Odyssey CLx Imaging System (LI-COR, Lincoln, NE, USA).

A model unstable translocon-associated protein is acetylated

We performed MS to gain further insight into the nature of Deg1-Sec62 PTM. We and others have previously mapped the major N-linked glycosylation site to Asn153 (Rubenstein et al., 2012; Scott & Schekman, 2008). In our previous work, mutation of Asn153 to a non-glycosylatable aspartic acid residue did not detectably alter translocon engagement, additional PTM, or Hrd1-dependent degradation (Rubenstein et al., 2012). Therefore, to simplify PTM analysis, we immunoprecipitated a variant of Deg1-Sec62 (Deg1-Sec62-N153D) that harbors an asparagine-to-aspartic acid mutation at this position from lysates of cells lacking the Hrd1 ubiquitin ligase. Purified Deg1-Sec62-N153D was separated by SDS-PAGE, and bands corresponding to Deg1-Sec62-N153D were excised (Fig. 1B) and proteolytically digested. Proteolytic processing of Deg1-Sec62-N153D with trypsin was complemented by further digesting one half of the trypsin-digested material with chymotrypsin to reduce large tryptic peptides into smaller sizes conducive to MS analysis. The trypsin and trypsin-chymotrypsin digestions were subjected to MS-based acetylpeptide mapping, resulting in a combined map that covered 73% of the protein sequence (Fig. 1C). Acetylation was identified with high confidence (with a false discovery rate of acetylated peptides of less than 1%) on the N-terminus of the protein, Lys3, and Lys7. Manual visualization of the results verified the high-confidence assignment of acetylation on the N-terminus and Lys3, while manual visualization indicated acetylation of Lys7 should be considered a lower-confidence result, in contradiction to the database search algorithm result. It is possible that additional lysine residues are acetylated but were not detected due to incomplete coverage of the Deg1-Sec62-N153D protein. Representative tandem mass spectra identifying sites of acetylation are shown in Figs. 1D–1I.

Acetylation of N-terminus and two internal residues does not influence Hrd1-dependent degradation of a model unstable translocon-associated protein

To determine if Hrd1-dependent degradation of Deg1-Sec62 is influenced by acetylation, we analyzed protein turnover in the context of mutations predicted to prevent acetylation. Certain conditions trigger a switch in E3 dependence for Deg1-Sec62 degradation from Hrd1 to Doa10. To evaluate Hrd1-dependent degradation exclusively, we used a version of Deg1-Sec62 specifically resistant to Doa10-dependent degradation. This variant, termed Deg1*-Sec62, harbors two point mutations (F18S and I22T) and behaves like Deg1-Sec62 with respect to Hrd1-dependent degradation (Johnson et al., 1998; Rubenstein et al., 2012). Deg1-Sec62 and Deg1*-Sec62 have been used interchangeably to evaluate degradation requirements for proteins that aberrantly engage the ER translocon (Rubenstein et al., 2012).

The N-terminal methionine of Deg1*-Sec62 is predicted to be acetylated by the NatB N-terminal acetyltransferase, which modifies proteins with the N-terminal Met-Asn dipeptide. Deleting the gene encoding the catalytic subunit of NatB (NAT3) is therefore predicted to abolish N-terminal acetylation of Deg1*-Sec62 (Hollebeke, Van Damme & Gevaert, 2012; Polevoda, Arnesen & Sherman, 2009). To test the hypothesis that N-terminal acetylation regulates substrate degradation, we analyzed Deg1*-Sec62 turnover by cycloheximide chase in wild-type yeast, yeast lacking HRD1, and yeast lacking NAT3 (Fig. 2A). Consistent with previous reports, deletion of HRD1 substantially increased steady state levels of Deg1*-Sec62 and delayed protein degradation (Buchanan et al., 2016; Rubenstein et al., 2012; Watts et al., 2015). By contrast, eliminating Nat3 from cells did not markedly impair Deg1*-Sec62 degradation. The control ERAD-L substrate CPY*-HA was stabilized in both hrd1Δ and nat3Δ cells (Fig. 2B), also consistent with previous reports (Hiller et al., 1996; Zattas et al., 2013).

We note marginally impaired degradation (without an increase in steady state abundance) of Deg1*-Sec62 in nat3Δ cells compared to wild-type yeast (Fig. 2A). This minor effect is likely indirect, as NAT3 deletion destabilizes the Hrd1 co-factor Der1, loss of which subtly stabilizes Deg1*-Sec62 (Rubenstein et al., 2012; Zattas et al., 2013). Degradation of a Deg1*-Sec62 variant with a mutation that abolishes the NatB consensus sequence (Deg1*-Sec62-3R in Fig. 2C, described below) supports the notion that N-terminal acetylation does not promote substrate degradation.

To determine if internal acetylation influences Deg1*-Sec62 degradation, we mutated both potentially acetylated lysine residues (at positions 3 and 7) to non-acetylatable arginine residues. The resultant protein is referred to as Deg1*-Sec62-2R (the version of the protein with both lysine residues intact is called Deg1*-Sec62-0R in Figs. 2C and 2D). Deg1*-Sec62-2R exhibited degradation kinetics similar to that of Deg1*-Sec62 in wild-type cells (Fig. 2C), and its rapid degradation was Hrd1-dependent (Fig. 2E). N-glycosylation of Deg1*-Sec62 and its derivatives only occurs following aberrant translocon engagement (Rubenstein et al., 2012). Sensitivity to Endo H, which removes all N-linked glycans from yeast proteins, confirmed that Deg1*-Sec62-2R engages the translocon in a manner similar to Deg1*-Sec62 (Fig. 2D). Therefore, acetylation of two lysine residues is dispensable for Hrd1-dependent degradation of a protein that aberrantly and persistently engages the translocon.

It was formally possible that acetylation at any one of three positions (Met1, Lys3, or Lys7) influences Hrd1-dependent degradation of Deg1*-Sec62. We simultaneously abolished all three acetylation events in two different ways. First, we generated a variant of Deg1*-Sec62 with point mutations predicted to prevent all three acetylation events. As for Deg1*-Sec62-2R, lysine residues at positions 3 and 7 were mutated to arginine. In addition, the second amino acid (asparagine) was mutated to arginine. Proteins with an N-terminal Met-Arg dipeptide are not predicted to be acetylated by any of the characterized N-terminal acetyltransferases (Polevoda, Arnesen & Sherman, 2009). The protein with three amino acids mutated to arginine is referred to as Deg1*-Sec62-3R. Deg1*-Sec62-3R is degraded at a rate similar to that of fully acetylatable Deg1*-Sec62 (Fig. 2C) in a Hrd1-dependent manner (Fig. 2F). Endo H sensitivity confirmed that Deg1*-Sec62-3R engages the translocon in a manner similar to Deg1*-Sec62 and Deg1*-Sec62-2R (Fig. 2D). Consistently, degradation of Deg1*-Sec62-2R occurred at a similar rate in the presence and absence of Nat3 (Fig. 2E).