mitchell et al jbc 072612 · 2012-08-17 · mechanism. in this report we show that erf4 functions...

Regulation of protein acyl transferases

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The Erf4 Subunit of the Yeast Ras Palmitoyl Acyl Transferase is Required for Stability of the Acyl-Erf2 Intermediate and Palmitoyl Transfer to a Ras2 Substrate

David A. Mitchell, Laura D. Hamel, Kayoko Ishizuka2, Gayatri Mitchell, Logan M. Schaefer and Robert J. Deschenes1

Department of Molecular Medicine, University of South Florida, Tampa, FL 33612

Running title: Regulation of protein acyl transferases

To whom correspondence should be addressed: Robert J. Deschenes, Department of Molecular Medicine,

MDC 7, Morsani College of Medicine, University of South Florida, Tampa, FL, USA, Tel.: (813) 974-6393, Fax: (813) 974-7357; Email: [email protected]

Keywords: Protein palmitoylation, Ras2, endoplasmic reticulum, posttranslational modification, membrane proteins

Background: The yeast Ras protein is pal-mitoylated by a DHHC protein acyl transferase comprised of Erf2 and Erf4. Results: Erf4 affects the stability, autopal-mitoylation, and palmitoyltransferase activity of Erf2. Conclusions: Erf4 regulates Erf2-dependent palmitoylation of Ras. Significance: This is the first example of regula-tion of a DHHC PAT. SUMMARY

Protein S-palmitoylation is a posttransla-tional modification in which a palmitoyl group is added to a protein via a thioester linkage on cysteine. Palmitoylation is a re-versible modification involved in protein membrane targeting, receptor trafficking and signaling, vesicular biogenesis and traffick-ing, protein aggregation, and protein degra-dation. An example of the dynamic nature of this modification is the palmitoylation-depalmitoylation cycle that regulates the sub-cellular trafficking of Ras family GTPases. The Ras Protein Acyl Transferase (PAT) con-sists of a complex of Erf2-Erf4 and DHHC9-GCP16 in yeast and mammalian cells, respec-tively. Both subunits are required for PAT activity, but the function of the Erf4 and Gcp16 subunits has not been established. This present study elucidates the function of Erf4 and shows that one role of Erf4 is to regulate Erf2 stability through an ubiquitin-

mediated pathway. In addition, Erf4 is re-quired for the stable formation of the pal-mitoyl-Erf2 intermediate, the first step of palmitoyl transfer to protein substrates. In the absence of Erf4, the rate of hydrolysis of the active site palmitoyl thioester intermedi-ate is increased resulting in reduced palmitoyl transfer to a Ras2 substrate. This is the first demonstration of regulation of a DHHC PAT enzyme by an associated protein.

Protein palmitoylation is involved in the

regulation of numerous cellular processes in-cluding cell growth and proliferation, protein trafficking, protein turnover, and vesicle fusion (1-4). Defects in protein palmitoylation have been linked to cardiovascular disease, infectious disease, and neurological disorders (5, 6) and more specifically, mutations in the protein acyl transferase (PAT) genes have been linked to colorectal and cervical cancers (7, 8), schizo-phrenia (9, 10), and X-linked mental retardation (11, 12).

In yeast, the Ras PAT comprises of a DHHC protein, Erf2, and a second subunit of unknown function, Erf4. Palmitoylation occurs by a two-step mechanism in which the Cys of the Erf2 DHHC motif undergoes autopal-mitoylation to create a thioester-linked palmitoyl intermediate, which can either undergo hydroly-sis (futile cycle) or transfer the palmitate from the enzyme cysteine to the cysteine of the Ras substrate (13). A similar mechanism has been

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.379297The latest version is at JBC Papers in Press. Published on August 16, 2012 as Manuscript M112.379297

Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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proposed for other members of the DHHC PAT protein family (14). To date, although Erf4 is required for Erf2-dependent palmitoylation, no specific role has been identified for the Erf4 subunit of the Ras PAT. The mammalian coun-terpart of Erf2, DHHC9, also co-purifies with a small auxiliary protein, GCP16 and is required for Ras PAT activity (15). Erf4 (also known as Shr5) was originally identified as a suppressor of the lethality resulting from the expression of a hyperactive Ras2 allele (corresponding to onco-genic mutations in mammalian Ras) in yeast (16), suggesting a potential regulatory role for Erf4. Based on sequence homology, putative Erf4 homologs are readily found in other fungal genomes, however, to date, only one metazoan homolog has been identified, GCP16. Although Erf4 and GCP16 associate with membranes, they do not contain the hydrophobic regions one would predict for an integral membrane domain. GCP16 associates with Golgi membranes via a dual palmitoylation site within its coding se-quence (17). Erf4, however, has no predictable trans-membrane or posttranslational modifica-tion consensus sequence motifs and does not require Erf2 for membrane localization (18). One explanation is that Erf4 associates with membranes through an interaction with an inte-gral membrane protein (18). Although it is clear that S-palmitoylation occurs primarily via the DHHC enzyme, the role of the auxiliary subunit remains unclear and the presence of these auxil-iary subunits has posed a long-standing question as to their function in the palmitoylation reaction mechanism. In this report we show that Erf4 functions in stabilizing Erf2 and the autopal-mitoylated Erf2 intermediate thioester. In the absence of Erf4, Erf2 undergoes degradation via ubiquitin-mediated mechanisms involving the ER quality control pathway (ERAD). However, stabilization of Erf2 in the absence of Erf4 does not restore palmitoyl transferase activity, demonstrating that Erf4 has multiple functions. This is the first example of an auxiliary subunit for DHHC PATs that can regulate pal-mitoylation. EXPERIMENTAL PROCEDURES Yeast strains, media, and microbiological tech-niques- The S. cerevisiae strains used in this study are described in (Table 1). Unless stated

otherwise, yeast cells were of the S288C genetic background. Yeast cultures were grown in rich medium (YEP) or synthetic minimal medium supplemented with amino acids to satisfy auxo-trophic requirements but maintain plasmids. Glucose was added to a final concentration of 2% (w/v) as carbon source and the cells were grown at 30°C with shaking. Yeast transfor-mation reactions were performed using the alkali cation method (19) using Frozen-EZ Yeast Transformation II kit (Zymo Research, Irvine, CA) according to the manufacturer’s instruc-tions. Genetic manipulations of yeast cells were as described (20). BY4742 is the WT strain for the deletion library obtained from Saccharomy-ces Genome Deletion Project (http://www-se-quence.stanford.edu/group/yeast_deletion_project/deletions3.html). RJY1620 has been de-scribed previously (18). RJY1888 was con-structed by mating yeast strains RJY1287 and RJY1620, sporulating the diploid parent and screening the meiotic progeny for G418 resistant cells that were MATa and able to grow on medi-um lacking tryptophan. Yeast strains lacking components of the quality control systems, e.g., ERAD, were constructed by knock out gene re-placement for each component in RJY1620 us-ing a PCR-mediated strategy. Wild type genes were replaced with KanMX-linked DNA frag-ments generated by PCR from genomic DNA isolated from the respective deletion mutant (Saccharomyces Genome Deletion Project). Primers to amplify the knock out region of inter-est were designed to include 100bps of genomic DNA sequence upstream and downstream of the start and stop codons, thus providing flanking sequences to permit homologous recombination. Knock out alleles were validated by PCR “Southern” analysis of the respective loci. Pri-mer deoxyribonucleotide sequences are availa-ble upon request. Plasmid construction- Plasmids used throughout this study are shown in Table 2. The sequences of the deoxyoligonucleotide primers used to construct ERF2 alleles are available upon re-quest. The Erf2-6R allele was synthesized de novo (Bio Basics, Inc, Amherst, NY). pUB221 was a gift from Daniel Finley, Harvard Universi-ty, Cambridge, MA (21). pESC-


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TrpPFA4:FLAG (B1165) was a gift from Mau-rine Linder, Cornell University, Ithaca, NY. B1821 was constructed by inserting a PCR fragment containing the terminal 174 bps of ERF2 (B1119 was used as the template) into B1164 using ligase-independent cloning. B1822 was constructed in a similar manner as B1821 except that B1835 (pUC57ERF2-6R) was used as the PCR template. B1275 was constructed using pEG(KG) as the vector backbone. The gene for Maltose Binding Protein (MBP) was amplified by PCR using pMAL (New England Biolabs, Inc, Ipswich, MA) as the template. This resulting product was used to replace the GST gene in pEG(KG) (ligase-independent cloning) while preserving the frame of Ras2CT35. The gene for mCherry was ampli-fied by PCR using pmCherry (Clontech,) as template. The resulting product was inserted between the genes for MBP and Ras2CT35 (lig-ase-independent cloning) to create the final fu-sion construct. Plasmids B1414 (18), B1302 (22), B924 (18), B1119 (23), B1258 (13), B1259 (13), B1250 (13), B529 (24), B374 (24) and B322 (25) have been described previously. B1825 (pESC-LeuFLAG:ERF2ΔC) was made by inserting the BglII-PacI fragment of B1259 into a similarly digested pESC-Leu. B1823 (pESC-LeuFLAG:ERF2-6R) was made by inserting the BglII-PacI fragment of B1835 into pESC-Leu. 6xHIS-ERF2 alleles were constructed using the Quikchange II Site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) per the manufacturer’s instructions. Isolates produced from the mutagenesis protocol were sequenced to confirm the ERF2 allele changes. Plasmids were rescued (26) and the DNA sequenced to confirm the presence of the mutations (Ge-neWiz, South Plainfield, NJ). Protein expression and purification- Overex-pression of Erf2 and Erf4 proteins was per-formed essentially as in Mitchell et al (13). Strain RJY1827 was co-transformed with pESC-Leu6xHIS:ERF2-(FLAG)ERF4 and B322 (pMA210) and grown in SC(-Leu-His) medium containing 2% (v/v) ethanol/ 2% (v/v) glycerol at 30˚C. The cultures were grown to 2 x 107

cells/ml and then induced by adding galactose to a final concentration of 4%. Cells were induced for 17 h (30˚C), then harvested by centrifugation

at 3000xg for 15 minutes, the pellet was re-suspended in breaking buffer (50 mM Tris-HCl pH 8, 500 ml NaCl, 1 mM EDTA, 1 mM DTT, 1x protease inhibitor cocktail (PIC), 8 µl/ml sat-urated PMSF) and the cells lysed by vortexing using glass beads (400-600 mesh, Sigma) for 40 minutes with 1.5 minute pulses. The resulting extract was spun at 3000xg for 15 minutes to remove cellular debris and unbroken cells, fol-lowed by a crude membrane fraction (P100) by centrifugation (100,000xg) for 1.5 h at 4˚C with a Beckman

50.2 Ti rotor. The supernatant was discarded and the pellet was re-suspended in Tris buffered saline, pH 7.4, with the aid of a Dounce homogenizer. The resulting extract was adjusted to a final concentration of 0.8% do-decylmaltoside (DDM), 500 mM NaCl, and 1 mM β-mercaptoethanol (β-ME). To solubilize the membranes, the extract was incubated at 4˚C (1.5 h). To aid in purification, urea and imidaz-ole were added to a final concentration of 2.4 M and 1 mM, respectively. Insoluble material was then removed by centrifugation (10,000xg) for 15 minutes at 4˚C. The supernatant was incu-bated with Ni-NTA resin at 4˚C for 1 h. The column was washed once with Solution W (50 mM Tris-HCl, pH 8.5, 0.08% DDM, 5 mM β-ME) containing 300 mM NaCl, and then twice with Solution W containing 150 mM NaCl. The protein was eluted with 50 mM Tris-HCl, pH8.5, 150 mM NaCl, 0.08% DDM, 5% glycerol and 250 mM imidazole. The eluted samples were desalted and the buffer changed to SPB (50 mM sodium phosphate buffer, pH6.8, 10% glycerol) using a column of G-25 resin. Fractions con-taining 6xHis-Erf2-Flag-Erf4 were pooled to obtain approximately 0.5 mg of purified Ras PAT per liter of culture.

For Flag-tagged Erf2 purifications, extracts were prepared using the glass bead break proce-dure detailed above in Tris buffered saline (TBS, 50 mM Tris-HCl pH 7.4 and 150 mM NaCl), containing 1x PIC and 8 µl/ml saturated PMSF. Unbroken cells and debris were removed by cen-trifugation at 3000xg for 15 minutes. A crude membrane fraction (P100) was obtained by cen-trifugation at 100,000xg for 1.5 h. The superna-tant was discarded and the resulting pellet re-suspended in Tris buffered saline, pH 7.4, con-taining 1x PIC and 8 µl/ml saturated PMSF. The extract was adjusted to a final concentration


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of 0.8% dodecylmaltoside (DDM) and incubated for 2 h at 4˚C with mild agitation. Insoluble ma-terial was removed by centrifugation at 10,000xg for 15 mins at 4˚C. 20 µl of pre-equilibrated Flag M2 antibody conjugated aga-rose (Sigma-Aldrich, St. Louis, MO) was added per 1 ml of clarified extract and the mixture in-cubated at 4˚C for 2 h with mild agitation. After incubation, the beads were washed 3x with Tris buffered saline, pH 7.4 containing 1x PIC, 8 µl/ml saturated PMSF and 0.8% DDM. The amount of Erf2 was determined empirically us-ing western blot analysis against a standard curve of known amounts of previously purified Erf2. MBP:mCherry:Ras2CT35 was expressed in RJY1842. The protein was purified by first isolating the membrane fraction in 1x PBS for 30 minutes at 13,000xg (P13). The P13 was then solubilized in 1x PBS containing 1% Tri-ton-X100 for 1 hour at 4˚C with gentle agitation. The extract was once again centrifuged at 13,000xg for 30 minutes to remove the insoluble fraction. The soluble fraction was incubated with pre-equilibrated amylose resin (New Eng-land Biolabs, Inc, Ipswich, MA) for 1 hour. The resin was washed three times with 1x PBS + 1% Triton-X100 and eluted from the resin using 1x PBS + 1% Triton-X100 + 10mM maltose. The resulting eluent was buffer exchanged to remove the maltose and concentrated. Erf2 stability assays- In the case of the GAL promoter driven Erf2 assays, yeast cells were seeded at an OD600 of 0.1 in 200 mls of synthetic medium supplemented with 2% glycerol and grown to an optical density of 1.0 at 30˚C with shaking. At this time, galactose was added to a final concentration of 2% and the cultures incu-bated at 30˚C with shaking for 3 h. After the incubation, glucose was added to a final concen-tration of 4% along with cycloheximide (25 µg/ml in DMSO) and 20 ml (20 OD600) volumes removed at various times and placed into chilled tubes containing 20 mM NaN3 (final concentra-tion) to stop growth and metabolism. In the case of strains expressing ERF2:13xMYC, cultures were seeded at an OD600 of 0.1 in synthetic me-dium supplemented with 2% glucose and grown at 30˚C with shaking to an OD600 of 1.0. Cyclo-heximide (25 µg/ml in DMSO) was added and 20 ml volumes removed at various times and

placed into chilled tubes containing NaN3 (20 mM final). For both procedures, after all the time points were collected, cells were harvested by centrifugation (3500xg) and the cell pellets washed with Tris buffered saline, pH 7.4 con-taining 20 mM NaN3. The cells were broken in Thorner buffer (40 mM Tris-HCl, pH 6.8, 5% SDS, 8 M Urea, 100 µM EDTA, 5% β-mercaptoethanol and 0.0004% bromophenol blue) plus glass beads. Samples were analyzed by Western blot. Flag-tagged proteins were transferred to a nitrocellulose membrane and visualized by ECL (Pierce, Milwaukee, WI) us-ing mouse anti-FLAG M2 antibody (Sigma-Aldrich, St. Louis, MO) as primary antibody and anti-mouse IgG horseradish peroxidase conju-gate as secondary antibody. Coupled PAT Assay- The Coupled Fluorescence Assay was performed as in Mitchell et al (13). Briefly, the production of NADH was monitored with a Biotek Mx fluorimeter (Biotek, Winoo-ski, VT) using 340 nm excitation / 465 nm emis-sion. The 200 µl reaction contained 2 mM 2-Oxoglutarate (α-ketoglutamic acid), 0.25 mM NAD+, 0.2 mM thiamine pyrophosphate, 0.4-1 µg of purified 6XHis:Erf2-Erf4 complex, 1 mM EDTA, 1 mM dithiothreitol, 32 mU 2-oxogluarate dehydrogenase (α-ketoglutarate de-hydrogenase), 50 mM sodium phosphate, pH6.8. The reaction was initiated by the addition of dif-ferent concentrations of palmitoyl-CoA and monitored for 30 mins at 30˚C. The first 10 mins of the reaction was analyzed to determine the initial rates of CoASH release. Autopal-mitoylation activity was determined from a standard curve of NADH production as a func-tion of CoASH concentration. In these reac-tions, CoASH was added to the standard PAT reaction mixture (without Erf2-Erf4 complex or palmitoyl-CoA) and the reaction was allowed to proceed to equilibrium (10 s) before fluores-cence was measured (ex. 340 nm / em. 465 nm). Assays using the catalytically dead C203S de-rivatives of the Erf2 complexes were performed and the values subtracted from the rates obtained for the respective Erf2 complexes. The values obtained for the varying concentrations of pal-mitoyl-CoA were fitted using Graphpad Prism software v4.0.


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Bodipy C12:0 Transfer Assay- Bodipy C12:0-CoA (40 µM final) was added to a 25µl reaction containing approximately 2.0 µg enzyme (FLAG-Erf2-Erf4) bound to anti-FLAG tagged agarose and 100 pmol purified MBP:mCherry:Ras2CT35 (wt) in 0.05 M sodi-um phosphate buffer, pH 7.4, and incubated at 30˚C. After 30 mins 5x non-reducing loading buffer was added to each sample. Each reaction was heated at 65˚C for three mins and then sub-jected to SDS-PAGE (12%). The gel was washed three times in ddH2O and visualized on the Typhoon 9410 Variable Mode Imager (GE Healthcare, Piscataway, NJ) for Bodipy fluores-cence (ex. 485 nm / em. 528 nm) to visualize transfer of the Bodipy signal to MBP:mCherry:Ras2CT35 and for mCherry fluo-rescence (ex. 520 nm/ em. 610 nm) to determine the amount of MBP:mCherry:Ras2CT35 loaded per lane. The amount of FLAG-Erf2 was deter-mined empirically using SDS-PAGE under re-ducing conditions. Complementation Assay- The in vivo function of the mutants along with the wild type protein was investigated using our previously described complementation assay (27). Briefly, in this assay, cells contain a defective allele of RAS2 that is balanced by an episomal copy of RAS2 linked to URA3. Under these conditions, the loss of ERF2 gene is permissible as long as the cell maintains the RAS2-URA3-based episome. This is detected by the ability or inability of the strain to grow on medium supplemented with 5’-Fluoroorotic acid (FOA) (28). Cells carrying ERF2 alleles were transformed into RJY1888 and plated on synthetic medium containing glu-cose and lacking leucine. Colonies were inocu-lated into liquid synthetic medium containing glucose or raffinose (both lacking tryptophan) and grown to an OD600 of between 0.8 and 1.2. The cell density was determined by monitoring absorbance using a spectrophotometer at 600 nm. 1:10 serial dilutions were made starting with 10,000 cells. The cells were spotted onto syn-thetic medium containing or lacking 5’-FOA as presented in the figures. Synthesis of Bodipy C12:0 CoA- The synthesis of Bodipy C12:0-CoA from Bodipy C12:0 (Invi-trogen/Life Technologies, Grand island, NY)

and CoASH (Sigma-Aldrich, St. Louis, MO) was based on the procedure of Berthiaume et al (29). 2 µmol of Bodipy C12:0 were dissolved in 500 µL of a solution of methanol and 1% triton X-100, transferred to a glass vial and dried under a stream of nitrogen. The residue was re-suspended in 720 µL of a solution of 300 mM MOPS-NaOH, pH 7.5, 30 mM MgCl2 and soni-cated for 10 mins in a bath sonicator. After son-ication, 200 µL of 100 mM ATP, 8 mg CoASH (trilithium salt, Sigma-Aldrich, St. Louis, MO), 45 µL of 10 mM adenophosphate, 20 µL of 1 M DTT, 1 U (500 µL) Acyl CoA Synthetase (Sig-ma-Aldrich, St. Louis, MO) and 5 U ATP Sulfu-rylase (New England Biolabs, Inc, Ipswich, MA) in a total volume of 1.6 mls. The reaction was incubated at 35˚C for 14 h. Bodipy C12:0-CoA was precipitated from the reaction by adding perchloric acid to 1.3%. The precipitant was washed once with acetone and twice with ethyl ether to remove unreacted Bodipy C12:0. The precipitant was dried under a stream of nitrogen and re-suspended in 10 mM sodium phosphate, pH 6.0. The amount of compound was deter-mined at 260 nm using an extinction coefficient of 15,400 (L/mol-cm). RESULTS Erf4 and the C-terminal 58 amino acids of Erf2 are required for Erf2 stability- The DHHC pro-tein, Erf2, and its associated protein, Erf4, are required for Ras PAT activity (23, 27). Exten-sive mutagenesis has defined the regions of Erf2 that are important for palmitoyl transferase ac-tivity (13). In contrast, the function of Erf4 is less well defined. To begin to address the role of Erf4, we attempted to isolate Erf2 for enzy-matic analysis in the presence and absence of Erf4. However, we observed that the steady-state amount of Erf2 was drastically decreased (approximately 40-fold) in the absence of Erf4 (data not shown). To investigate this further, we performed pulse-chase experiments using a Myc-epitope tagged Erf2 expressed in ERF4 and erf4Δ yeast strains. Cells were grown to mid-log phase, cycloheximide was added (t0), and Erf2 levels analyzed using anti- Myc epitope tag anti-bodies (Fig. 1A). The amount of phospho-glycerate kinase (PGK) was used for normaliza-tion of the samples. In the ERF4 wild type strain, the half-life of Erf2 was153 minutes,


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whereas in the strain lacking ERF4 (erf4Δ), the half-life of Erf2 was reduced to 53 minutes (Fig. 1B).

One possible explanation for the instability of Erf2 in the absence of Erf4 is that the loss of the Erf4 interaction may uncover a domain that is linked to Erf2’s instability. To map the region of Erf2 that may contribute to its degradation, we constructed Erf2 mutants lacking the first 119 amino acids (N-terminal deletion, Erf2ΔN) or the final 58 amino acids (C-terminal deletion, Erf2ΔC) that were under the control of the GAL1,10 promoter (13). Deletion of the N-terminus had no effect on increasing the half-life of Erf2 in the absence of ERF4 (22 mins) when compared to wild type Erf2 (25 mins) under the same conditions. However, when the C-terminal 58 amino acids were truncated, the half-life of Erf2 increased to 361 mins, approximately 2-fold that of wild-type Erf2 in the presence of ERF4 (169 mins). A possible explanation for the increased stability of Erf2ΔC is that it may not be properly localized and that the stability may reflect non-native sub-cellular localization where there is a different molecular degradation mechanism. However, like the WT GFP:Erf2-Erf4 complex, the bulk of GFP:Erf2ΔC, in the absence of Erf4, localized to the peri-nuclear ER (Ishizuka and Deschenes, unpublished results). Ubiquitin-dependent clearance of Erf2 by the ERAD system- The Erf2-Erf4 complex localizes to the ER where it palmitoylates Ras (18, 27, 30). We speculated that ER quality control mechanisms would be involved in Erf2 turnover and therefore examined Erf2 for the presence of covalently attached ubiquitin (31). FLAG:ERF2 was co-expressed with 6xHIS:Ubiquitin (pUb221) in a strain lacking ERF4 (21). The cell extract was enriched for ubiquinylated pro-teins using Ni-NTA agarose and immunoblotted for Erf2 using anti-Flag M2 antibodies (Fig. 2A). The signal was specific for both Flag:Erf2 and 6xHIS:Ub and appeared as a smear above the band representing Flag:Erf2 alone, presuma-bly due to heterogeneous poly-ubiquitinylated Flag:Erf2 (Fig. 2A). These data are consistent with the role of the quality control system in degrading Erf2 molecules that are not in com-plex with Erf4.

To investigate whether the ERAD pathway is involved in Erf2 turnover, the steady-state half-life of Erf2 was measured in wild type yeast strains or strains harboring a deletion in key ERAD genes (Fig. 2B). The ERAD pathway has been defined by mutation in genes encoding proteins that monitor cytosolic-facing, ER lu-men-facing, or general features of ER membrane proteins (32-34). Erf2 is predicted to be an inte-gral membrane protein with 4 trans-membrane spanning segments, short ER lumen loops be-tween TM1 and 2, and TM3 and 4 and a largely cytosolic facing catalytic domain (35). The first indication that the ERAD system was involved in monitoring Erf4-dependent structural integrity of Erf2 came from deleting the ubiquitin-conjugating enzyme, UBC7, which is considered a general ERAD component (36). Deletion of UBC7 resulted in increased stability of Erf2 in a strain lacking ERF4 (Fig. 2B). To investigate further what part of Erf2 was engaging the ERAD system, we deleted YOS9, an ERAD component that monitors domains of proteins within the ER lumen (37). Loss of YOS9 in an erf4Δ strain background had no effect on the half-life of Erf2 when compared to the erf4Δ alone. In contrast, deletion of DOA10 (33), the cytosolic specific ERAD ubiquitin-conjugating enzyme component had the greatest stabilizing effect on Erf2 in a strain lacking ERF4. We ob-served similar results of Erf2 stabilization by deleting the gene for the membrane specific ERAD ubiquitin-conjugating enzyme, HRD1 (38), or the gene for RPN10 (39), a component of the 26S proteosome, in an erf4Δ strain back-ground. Together, these data imply that in the absence of Erf4 more than one domain of Erf2 is not properly folded, thereby activating more than one ERAD-specific pathway. The C-terminal 58 amino acid residues of Erf2 are necessary and sufficient for ubiquitin-dependent, ERAD mediated clearance of DHHC PATs- The C-terminus of Erf2 has 6 lysine resi-dues that could serve as possible ubiquitinyla-tion sites (Fig. 3A) (40). Changing all of the lysines to arginines (Erf2-6R) resulted in stabili-zation of Erf2 in the strain lacking ERF4, con-sistent with C-terminus of Erf2 being necessary of ubiquitin-dependent degradation (Fig. 3B and 3C). To determine if the C-terminal 58 amino


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acids containing the 6 lysines is sufficient to confer degradation, we constructed a fusion pro-tein that placed the 58 amino acids of Erf2 onto the C-terminus of Pfa4, another DHHC enzyme that resides in the ER membrane (Fig. 3A). The half-life of Pfa4 with the C-terminal FLAG (Pfa4:Flag) epitope was 46 mins (Fig. 3B and 3C). When the Erf2 58 amino acid C-terminal sequence was added to the Pfa4:Flag construct (PFA4:FLAG:Erf2C58), the half-life of the pro-tein decreased to 8 mins (Fig. 3B and 3C). This decrease was independent of the presence of Erf4 (data not shown). As with Erf2, creating the six Lys to Arg mutations in the Pfa4-Erf2C58 fusion increased the half-life of the Pfa4:Flag:Erf2C58-6R fusion protein by 4-fold (23 mins) (Fig. 3B and 3C). Three combinations of 5 arginines (R304R311R316R335R355K358, R304R311R316R335K355R358 and

K304R311R316R335R355R358) and one lysine were constructed and produced similar results as the 6 arginine change (R304R311R316R335R355R358 ) (data not shown). Although a specific context of a ubiquitinylation-directed sequence was not de-termined, the substitution of arginines for ly-sines within the wild type Erf2 and Pfa4 fusion constructs confirmed the ubiquitin-mediated degradation of Erf2 and demonstrated that the Erf2 C-terminal 58 amino acid residues are nec-essary and sufficient to direct Erf2 degradation. Strains harboring stabilized Erf2 still require Erf4, suggesting an additional function for Erf4- The loss of a subset of the ERAD components increased the stability of Erf2 in the absence of ERF4. We therefore asked whether, under these conditions, there was a restoration of Erf2 func-tion. In other words, was Erf4 required for Ras palmitoylation beyond its role in Erf2 stabiliza-tion. We utilized the plasmid shuffle assay that was originally used to identify ERF2 and ERF4 (27) using RJY1620 (18) deleted for the ERAD components (above). This strain has the pal-mitoylation-dependent Ras2 (RAS2 CS-Ext) al-lele at the RAS2 locus and harbors a plasmid expressing wild type RAS2 that can be lost by asymmetric segregation if Ras2 CS-Ext is pal-mitoylated. We observed that only the presence of wild type ERF4 would allow the loss of the RAS2-based plasmid on 5’-Fluoroorotic acid (5’-FOA) (Fig. 4A). Although some of the ERAD

mutants could increase the stability of Erf2 in the absence of Erf4 (Fig. 2B), these mutants were unable to suppress the loss of ERF4 under these conditions (Fig. 4A). We also examined the ability of stabilized Erf2-6R to suppress the loss of ERF4. As with the loss of the ERAD components, expression of FLAG:ERF2, FLAG:ERF2ΔC or FLAG:ERF2-6R,was unable to suppress the growth defect of strains lacking Erf4 (Fig. 4B). Together, these in vivo data demonstrate that the role Erf4 plays in palmitoyl transferase activity extends beyond solely stabi-lizing Erf2. We were surprised to observe that FLAG:ERF2ΔC-ERF4 could also suppress the erf2Δerf4Δ phenotype of strain RJY1888, albeit at a low level (approximately 1/1000 that of FLAG:ERF2-ERF4). This suppression was de-pendent on the FLAG:ERF2ΔC-ERF4 plasmid (data not shown). Erf4 is required for stable formation of the Erf2-palmitoyl intermediate- The Erf2-Erf4 enzyme reaction proceeds via a two-step mechanism (13). In the first step, Erf2 is autopalmitoylated, using palmitoyl-CoA as substrate, releasing re-duced CoASH (1, 4). The second step of the reaction transfers the palmitate from Erf2 to the protein substrate. The autopalmitoylation reac-tion can be assessed by two assays. In the first, the steady state amount of palmitoylated Erf2 is determined by performing the autopal-mitoylation reaction using a tagged palmitoyl-CoA probe, and the reaction products separated by non-reducing SDS-PAGE (23). The limita-tion of this assay is that one cannot differentiate between an enzyme that can undergo autopal-mitoylation with rapid hydrolysis of the thioester linkage and an enzyme that does not get auto-palmitoylated. In the second assay, the rate of palmitoyl-CoA reacted is monitored by measur-ing the production rate of CoASH, a product of the reaction (13). This assay takes into account the formation and subsequent hydrolysis of the palmitoyl-Erf2 thioester intermediate. Together, these assays provide an accurate measure of au-topalmitoylation and thioester hydrolysis. FLAG:ERF2, FLAG:ERF2-6R and FLAG:ERF2ΔC were expressed in yeast cells (RJY1828) with and without ERF4 (GST:ERF4). The PAT complexes were partial-


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ly purified and the amount of enzyme deter-mined by SDS-PAGE using a known standard. To determine the steady state amount of acyl-Erf2 intermediate, we reacted the isolated Flag:Erf2 molecules with Bodipy C12:0-CoA, an analog of palmitoyl-CoA, and the products of the reaction separated by non-reducing SDS-PAGE (Fig. 5A). The steady state amount of Bodipy C12-Erf2 was greater in the full-length stabilized mutant (Erf2-6R-Erf4) when com-pared to wild type Erf2-Erf4. When Erf4 is ab-sent, the steady state amount of Bodipy C12-Erf2-6R intermediate drops more than 2 orders of magnitude. A similar phenomenon is ob-served for wild type Erf2 in the absence of Erf4. Interestingly, the steady state amount of Erf2ΔC is independent of Erf4, albeit at approximately 30% the level of wild type Erf2-Erf4, suggesting that loss of the C-terminal 58 amino acids of Erf2 may influence its association with Erf4.

On the surface, these data seem to argue for a diminution of autopalmitoylation activity for Erf2 in the absence of Erf4. However, when the rate of CoASH production is measured after the steady-state of the reaction is reached, we ob-serve an increase in autopalmitoylation and the palmitoyl-Erf2 intermediate thioester hydrolysis rate for Erf2 (closed circles) (3.3-fold) and Erf2-6R (closed squares) (2.4-fold) in the absence of Erf4 (Table 3) as determined by an increase in the VMAX of the respective reactions. We also observed the greatest CoASH production rates using the Erf2ΔC (open triangles) and Erf2ΔC-Erf4 (closed triangles) proteins implying that autopalmitoylation and thioester hydrolysis are greater for these mutants. To ensure that the activity signal we were detecting is due to the formation and hydrolysis of the palmitoyl-Erf2 thioester intermediate (located at residue C203 of Erf2), we mutated the codon for C203 to a serine codon (C203S) for all the ERF2 alleles (with and without ERF4) and repeated the exper-iment, subtracting the rates obtained for the C203S proteins from the corresponding Erf2-dependent activities. These data demonstrate that a) the Erf2 protein is capable of forming and hydrolyzing the palmitoyl-Erf2 thioester inter-mediate in the absence of Erf4 and b) in the cas-es of Erf2 and Erf2-6R, the presence of Erf4 de-creases the VMAX of the reaction suggesting that Erf4 has the potential for regulating autopal-

mitoylation/hydrolysis, possibly by controlling access to the active site. Erf4 is required for palmitoyl transfer to Ras- Finally, we determined the ability of Erf2 to transfer the fluorescent palmitoyl-CoA analog, Bodipy C12:0-CoA, to a Ras2 substrate (Fig. 6). In these experiments, Erf2, with or without Erf4, were affixed to beads through antibodies to the Flag epitope. MBP:mCherry:Ras2CT35 (100 pmol) was added and the reaction was initiated by the addition of Bodipy C12:0-CoA (1.2 nmol). Normalizations for the amount of Erf2 protein added to the reactions are shown in the lower panel of Figure 5A. The number of moles of Bodipy C12:0-CoA transferred was deter-mined empirically using a standard curve (data not shown). As expected, more Bodipy C12 was transferred when Erf4 was present with Erf2 and Erf2-6R. Previously, we demonstrated that alt-hough Erf2-Erf4 autopalmitoylation proceeds with burst kinetics, the transfer of palmitate to a Ras2 substrate proceeds by what appears to be first order kinetics (13), in that the reaction sig-nal is linear with respect to time, does not demonstrate a burst of activity and appears de-pendent on the concentration of Ras2. We de-termined the amount of Bodipy C12 transferred to Ras2 to be approximately 4.8 pmol (1739 pmol/min/µmol MBP:mCherry:Ras2CT35) for Erf2-Erf4 and 4.0 pmol (1515 pmol/min/µmol MBP:mCherry:Ras2CT35) for Erf2-6R-Erf4. In comparison, a value of 940 pmol/min/µmol GST:Ras2 was obtained using 3H-palmitoyl-CoA and GST:Ras2 as substrates (23). We were intrigued to observe a small, yet detectable amount of Bodipy C12 transferred to Ras2 using Erf2ΔC-Erf4 of 0.6 pmol (182 pmol/min/µmol MBP:mCherry:Ras2CT35). One could imagine that the relatively small amount transferred to the Ras2 substrate may explain the detectable, yet reduced viability we observed for our growth assay (Fig. 4B) for Erf2ΔC-Erf4. We also de-tected amounts of transfer for Erf2ΔC and Erf2-6R of 0.4 pmol (150 and 152 pmol/min/µmol MBP:mCherry:Ras2CT35, respectively) in the absence of Erf4 that were greater than with Erf2 (0.2 pmol; 88 pmol/min/µmol MBP:mCherry:Ras2CT35) alone. The amount of background fluorescence, presumably non-catalytic palmitoylation, observed for the Ras2


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substrate alone was well below the detection limit for the assay and was estimated, based on background subtraction, to be less than 5 percent of the Erf2-Erf4-dependent transfer signal. Alt-hough the MBP:mCherry:Ras2CT35 protein migrates as a doublet, we detected palmitate transfer in only the top band of the doublet sug-gesting that the doublet is formed from proteoly-sis of the CaaX box tail of the full length pro-tein. In addition to the palmitoylated Ras2 gel band, we also observe non-specific bands that appear to be contaminants from the Bodipy C12:0-CoA synthesis. DISCUSSION

The Ras protein acyl transferase (PAT), Erf2-Erf4, is a member of a large family of en-zymes that has at least seven members in fungi and over twenty members in higher eukaryotes (1). Like the vast majority of members of the PAT family, the Erf2 component of this com-plex contains the canonical DHHC motif and is the subunit involved in the formation of the palmitoyl-enzyme intermediate (13, 23, 41, 42). However, Erf2 requires an accessory subunit, Erf4, for palmitoyl transfer activity (23, 27). ERF4/SHR5 has been identified twice in genetic screens aimed at identifying regulators of the Ras pathway in S. cerevisiae (16). This hetero-dimer stoichiometry is also observed for the mammalian homologue of Erf2, DHHC9, and its accessory subunit, GCP16 (15). The identifica-tion of GCP16, the accessory subunit of mam-malian Ras PAT, DHHC9, was based on the primary amino acid sequences of the fungal Erf4 family. Currently, only the Ras PAT enzymes have been shown to require an accessory subunit in addition to the DHHC subunit. In previous reports, we demonstrated that a) Erf2 and Erf4 interact (18, 27), b) the Erf2-Erf4 (and DHHC9-GCP16) interaction is required for the enzymatic activity of the Ras protein acyl transferase (15, 23) and c) the Erf2-Erf4 protein acyl transferase complex transfers palmitate from a donor (pal-mitoyl-CoA) to a protein substrate (Ras2) using a two-step reaction mechanism (13). Although the reaction mechanism has been determined, the contribution of the individual subunits to palmitate transfer has not been addressed prior to this study.

Here we show that Erf4 regulates the auto-palmitoylation state of the enzyme by stabilizing the palmitoyl-Erf2 intermediate and also is re-quired for the second transfer step of the reac-tion. Erf4, therefore, potentially plays a role in transfer catalysis, substrate recognition or both. In the past, the role of Erf4 in palmitoylation has been clouded by the inability to accurately measure the palmitoylation activity of Erf2 in the absence of Erf4 (22, 23). Steady state amounts of Erf2 are decreased approximately 40-fold in the absence of Erf4 in vivo, an obser-vation that implies Erf4 stabilizes or impedes the degradation of Erf2 in the cell. We show this to be the case. The half-life of Erf2 is reduced from 153 min to 50 minutes in the absence of Erf4. In order to determine if specific amino acid sequences or domains are involved in Erf2 degradation, we parsed Erf2 into three domains; the N-terminal (amino acids 1-119), DHHC (amino acids 120-300) and C-terminal (amino acids 301-359). The DHHC domain serves as the catalytic core of the PAT, being able to per-form the first step of palmitoylation, autopal-mitoylation, but it is not sufficient to have PAT transfer activity by itself (13). Unlike the DHHC domain, N- and C-terminal domains of Erf2 do not share significant homology with other PATs or any known proteins (1). Deletion of the N-terminal domain had no effect on stabi-lizing Erf2 in the absence of Erf4. However, deletion of the C-terminal domain in the absence of Erf4 increased the half-life of Erf2 to 2-fold greater than that of the wild type enzyme in the presence of Erf4. The degradation of Erf2 is an ERAD mediated event resulting in poly-ubiquitinylation of and is facilitated by the C-terminal 58 amino acids of Erf2. This was con-firmed by creating an Erf2 C-terminal fusion of this sequence with another endoplasmic reticu-lum localized PAT, Pfa4 (Pfa4:FLAG:Erf2 C58). The addition of the C-terminal domain decreased the half-life of Pfa4 by 75%. Stabiliz-ing Erf2 either by removing the C-terminal 58 amino acids or changing the 6 lysines within that domain to arginines, however, does not suppress the loss of Erf4 in vivo. Additionally, deletion of the 58 C-terminal amino acids has considera-ble influence on the palmitoyl transfer activity of the enzyme in vitro. Finally, we observed that Erf4 has a negative regulatory effect on the post


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steady-state autopalmitoylation/hydrolysis activ-ities of Erf2. One possible molecular explana-tion is that Erf4 binds Erf2 through the C-terminal domain. To date, we have been unable to detect an interaction between Erf4 and the Erf2 C-terminal domain (data not shown). Al-ternatively, the association of Erf4 with Erf2 could cause a conformational change that buries or masks the C-terminal domain in some way that does not involve association with Erf4, making it inaccessible to the degradation ma-chinery. It is difficult to say at this point which model reflects reality. However, it is clear that the association of Erf4 with Erf2 regulates the amount of Erf2 and therefore controls the amount of palmitate transferred to Ras sub-strates. Taken together, these data support the notion that Erf4, while important for stabilizing Erf2, is required for palmitate transfer to protein substrates and is consistent with a role in cataly-sis and/or substrate recognition.

We were intrigued to observe that Erf4 had an inhibitory effect on the rate of autopal-mitoylation/ hydrolysis cycling. Previously, we had formulated the hypothesis that Erf2, in the absence of Erf4, was unable to perform either step of the palmitoylation reaction based on the inability to detect 3H-palmitoyl-labelled Erf2 by autoradiography (23). However, we developed an assay (13) that monitors the production of CoASH, one of the products of autopal-mitoylation, by coupling its formation to the re-duction of NAD+ using α-ketoglutarate dehy-drogenase as the catalyst. Although this assay does not measure the pre-steady state burst ki-netics, it does monitor the post-steady state ki-netics of the autopalmitoylation and hydrolysis reactions, which can give insights into the en-zyme’s molecular mechanism. Based on the result of this assay and the palmitoyl transfer assay, three conclusions can be drawn. First, the absence of Erf4 increases the VMAX of the auto-palmitoylation reaction for wild type Erf2 and Erf2-6R by 3.3- and 2.4-fold, respectively, while having little effect on the KM. One possible ex-planation is that Erf4 protects the active site in-termediate thioester from hydrolysis, potentially by limiting access to water. Secondly, deletion of the C-terminal 58 amino acids has a greater effect on increasing the autopalmitoylation reac-tion VMAX and appears to be independent of the

presence of Erf4, implying that the C-terminus of Erf2 also participates in protecting the active site. This may occur directly by forming a do-main capable of shielding the active site or indi-rectly by affecting the overall folding of the en-zyme. Finally, Erf4 is required for the transfer of the palmitoyl group from Erf2 to the protein substrate. However, it remains to be determined whether Erf4 participates in substrate recogni-tion, transfer catalysis or both.

We have demonstrated that Erf4 plays a role not only in stabilizing Erf2 but also in pro-moting the transfer of palmitate from the pal-mitoyl-enzyme intermediate to the protein sub-strate. Can this observation be extended to the other PATs? DHHC-mediated protein pal-mitoylation occurs in two steps; autopal-mitoylation of the DHHC molecule to form a palmitoyl-enzyme intermediate, which appears to be a universal activity (4, 42, 43), and transfer of the modifying palmitate to a protein substrate. Our data highlights a subset of functions for the Erf4 subunit. First, Erf4 protects Erf2 from ubiquitin-mediated degradation. Although the mechanism is not immediately clear, one possi-ble explanation is that Erf4 is involved in providing the proper conformation of Erf2 with-in the endoplasmic reticulum. Aside from its effect on Erf2 stability, the surprising aspect of Erf4 function is its effect on autopalmitoylation. The loss of Erf4 does not abolish autopal-mitoylation, as would be the case if residues of Erf4 participated in autopalmitoylation catalysis. Recently, we demonstrated that autopal-mitoylation of the Erf2 active site, which occurs with burst kinetics, could be followed by hy-drolysis of the palmitoyl-Erf2 intermediate thi-oester linkage in the absence of a protein sub-strate (13). It appears that loss of Erf4 may in-crease the hydrolysis rate of the thioester of the intermediate causing the enzyme to undergo rap-id cycles of autopalmitoylation and hydrolysis. Erf4, therefore, acts to limit the access of water to the active site. This observation is also true for the effect of GCP16 on DHHC9 (Mitchell and Deschenes, unpublished results). An in-crease in the hydrolysis rate would come at the expense of the steady state amount of palmitoyl-enzyme intermediate and ultimately, decrease the amount of palmitate that gets transferred to the protein substrate. The juxtaposition of the


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DHHC domain with the hydrophobic milieu of the membrane, hypothesized for all DHHC en-zymes (35), may be a mechanism for limiting water molecules from invading the active site. In addition, Erf4 is required for the transfer of palmitate to the protein substrate. One possibil-ity is that Erf4 is involved in substrate recogni-tion. Another, non-mutually exclusive hypothe-sis is that residues of Erf4 are required for catal-ysis of the palmitoyl transfer step. It is evident, however, that the mechanism of autopal-mitoylation of DHHC enzymes requires shield-ing the active site from water, a job which may

be performed by residues/domains of the DHHC molecule or by another accessory protein subu-nit. It is therefore conceivable that accessory subunits exit for many, if not all, DHHC en-zymes and that their identification has gone un-detected. Taken together, these data provide an initial elucidation of the molecular mechanism underlying the role of accessory proteins in pro-tein palmitoyl transfer and we are now begin-ning to address some of the questions directed at the activity and substrate recognition of pal-mitoyl transferases as a whole.

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of protein palmitoylation in yeast. Cell. 125, 1003-1013.

Acknowledgements- We would like to thank Vladimir Valdez and Krishna Reddy for critical reading of

the manuscript and many helpful discussions. This work is dedicated to the memory of our friend and col-

league, Kayoko Ishizuka, PhD.


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FOOTNOTES

* This work was supported by NIH grants CA50211 and GM73976 to RJD.

1. To whom correspondence should be addressed: Robert J. Deschenes, Chairman, Department of Molec-

ular Medicine, MDC 7, Morsani College of Medicine, University of South Florida, Tampa, FL, USA,

Tel.: (813) 974-6393, Fax: (813) 974-7357; Email: [email protected]

2. Deceased

3. The abbreviations used are: PAT, Protein Acyl Transferase; PCR, Polymerase Chain Reaction; 5’-

FOA, 5’-Fluoroorotic Acid; DDM, n-Dodecyl Beta-D-Maltoside; β-ME; Beta-Mercaptoethanol; PMSF,

Phenylmethylsulfonylfloride; PIC, Protease Inhibitor Cocktail; DMSO, Dimethyl Sulfoxide.

FIGURE LEGENDS

FIGURE 1. Erf2 is destabilized in the absence of Erf4. Strain RJY1620 expressing 13xMyc:ERF2 and

B1414 (ERF4) or 13xMyc:ERF2 alone was grown to an OD600 of 1.0, treated with cycloheximide (25

µg/ml) to inhibit protein translation and samples removed at the indicated times after cycloheximide addi-

tion. A) Representative western analysis of ERF2 ERF4 expressing strains (i) or ERF2 erf4∆ strains (ii)

probed with antibodies to the c-Myc epitope that tags Erf2. The blots were also probed with antibodies to

phosphoglycerate kinase (PGK) as a lane loading control. B) Semi-log graphical representation of west-

ern blot shown in (A) after densitometry (ImageJ, NIH) to empirically determine the half-life of Erf2 in

the presence of Erf4 (153 min) and in the absence of Erf4 (25 min). C) Comparison of half-lives of Erf2

in the presence of Erf4 and Erf2, Erf2∆N and Erf2∆C in the absence of Erf4. The bar graph shows the

data from two independent experiments (black and gray bars) determining the half-life of Erf2.

FIGURE 2. Degradation of Erf2 involves polyubiquitinylation and the ERAD system. A) (Upper panel)

To determine if Erf2 undergoes ubiquitinylation, extracts from diploid erf4D strains expressing

FLAG:ERF2 with and without pUb221 (6XHIS:Ubiquitin) or FLAG:ERF2-6R with and without pUb221


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were treated with 6 M guanidine-hydrochloride (to denature and dissociate all non-covalently associated

proteins) and ubiquitinylated proteins were isolated using Ni-NTA agarose, separated by SDS-PAGE and

immunoblotted with ant-Flag antibodies (to detect Erf2). Strains expressing both plasmids produced

polyubiquitin-conjugated Erf2 molecules as seen by a smear larger than the apparent molecular weight of

Flag:Erf2. (Lower panel) Whole cell extracts from the strains used in the upper panel were separated by

SDS-PAGE and immunoblotted with anti-Flag antibody to demonstrate the presence of Flag:Erf2. B)

The half-life of 13xMyc:Erf2, in the absence of Erf4, is increased by deleting the ER quality control com-

ponents. The bar graph shows the data from two independent experiments (black and gray bars) for the

half-life of Erf2 in isogenic strains erf4, yos9 erf4, rpn10 erf4, hrd1 erf4, ubc7 erf4 and doa10 erf4 com-

pared to the wild type (ERF4) strain.

FIGURE 3. The C-terminal 58 amino acids of Erf2 are sufficient to promote degradation. A) Schematic

representation of ER localized acyl transferase, Pfa4, with C-terminal Flag epitope and Erf2 58 amino

acid additions. The amino acid sequence below the schematic compares the wild type Erf2 C-terminal 58

amino acids with that of Erf2-6R in which the six lysines are mutated to arginines (asterics). B)

Representative western analysis comparing the amounts of Pfa4:Flag, Pfa4:Flag:Erf2C58,

Pfa4:Flag:Erf2C58-6R, Flag:Erf2 and Flag:Erf2-6R at the indicated times after cycloheximide (25 µg/ml)

addition, probed with antibodies to the Flag epitope (Sigma-Aldrich, St. Louis, MO). C) Comparison of

the half-lives of Pfa4:Flag, Pfa4:Flag:Erf2(C58), Pfa4:Flag:Erf2(C58-6R), Flag:Erf2 and Flag:Erf2-6R.

The bar graph shows the data from two independent experiments (black and gray bars) determining the

half-life of the indicated fusion proteins.

FIGURE 4. Stabilized Erf2 cannot suppress the loss of ERF4. Functional plasmid shuffle assay to

determine the ability different Erf2 stabilizing conditions to suppress the loss of ERF4. A) The genes for

ERAD components were deleted from RJY1620 and plated on synthetic medium lacking uracil to

demonstrate the presence of the sectoring plasmid. These strains, harboring pRS314 (TRP1) or B1414


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(pRS314ERF4), were replicated to synthetic medium lacking tryptophan and supplemented with 5’-

fluoroorotic acid (5’-FOA) to select for those strains capable of losing the URA3 linked RAS2 episome.

B) Series dilution of strain RJY1888 cultures harboring plasmids expressing the ERF2 alleles with and

without ERF4. Cells were spotted in 10-fold dilutions (104 initial CFUs) on medium lacking leucine (left

panel) and medium lacking leucine supplemented with 5’-FOA (right panel) and incubated for 4 days at

30˚C.

FIGURE 5. Erf4 dependence of Erf2 autopalmitoylation. A) In vitro autopalmitoylation reactions using

Bodipy C12:0-CoA as the acyl donor. (Top panel) In vitro autopalmitoylation reactions were separated

using SDS-PAGE under non-reducing conditons and the fluorescence visualized using ex. 488nm / em.

520nm filters (Typhoon, GE). The middle panel of (A) shows a representative western blot analysis used

to quantify the amount of Erf2 and Erf2 mutants. The bar graph shows the amount of autopalmitoylation

normalized to the amount of Erf2 protein present in each sample (bottom panel). The asterix denotes

cross reactivity of anti-Flag with reduced small chain IgG from the antibody coated agarose beads. B)

Post-steady state autopalmitoyation and hydrolysis fluorescence assay that couples the production of

CoASH, a product of the autopalmitoylation reaction, with the reduction of NAD+ (NADH) using α-

ketoglutarate dehydrogenase (KDH). Assays were performed varying the amount of palmitoyl-CoA. The

background values were determined by performing the assays using the analogous catalytically impaired

Erf2 enzymes (Erf2 C203S) and those values subtracted from the values obtained using the active Erf2

enzymes. The data represent Erf2ΔC (open triangle), Erf2ΔC-Erf4 (closed triangle), Erf2-Erf4 (open

circle), Erf2 (closed circle), Erf2-6R-Erf4 (open square) and Erf2-6R (closed square). The data were fit

using Prizm software, n=4. KM, VMAX and kCAT/KM values are shown in Table 3.

FIGURE 6. Erf2 requires Erf4 to transfer palmitate to Ras2. In vitro transfer reactions using Bodipy

C12:0 CoA as the acyl donor. (First panel) The products of the in vitro acyl transfer reactions were

separated using SDS-PAGE under non-reducing conditions. MBP:mCherry:Ras2CT35 (100 pmol) was


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used as the protein substrate. The amount of Bodipy C12:0 transfer was determined by scanning the gel

using ex. 488nm/ em. 520nm filters (Typhoon, GE). (Second panel) The amount of

MBP:mCherry:Ras2CT35 is identical in all cases as determined by measuring the fluorescence of the

mCherry chromophore using ex. 520nm/ em. 610nm filters (Typhoon). (Third panel) The same enzyme

preparations used to determine the autopalmitoylation activity (Fig. 5A) were also used for determining

the acyl transfer activity and were quantified by western blot analysis using antibodies to the Flag epitope.

The asterix denotes cross reactivity of anti-Flag with reduced small chain IgG from the antibody coated

agarose beads. (Fourth panel) The bar graph below shows the amount of Bodipy C12:0 transfered

relative to the amount of MBP:mCherry:Ras2CT35:Ras2 substrate used in the reaction (pmol/min/µmol

MBP:mCherry:Ras2CT35) normalized to the amount of Erf2 protein (Fig. 5A, bottom panel) present in

each sample. The background fluorescence of the MBP:mCherry:Ras2CT35 alone lane (no enzyme) was

subtracted from the sample lanes.


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Table 1: Saccharomyces cerevisiae strains used in this study Source

RJY1620 MATα leu2-3,112 ura3-52 his3Δ200 trp1Δ63 lys2Δ801 ade2-101 ade8Δ ras1::HIS3 Ras2CS-ext erf4::KanMX [YCp52-RAS2] (18)

RJY1788 (W303) MATa/α leu2/leu2 ura3/ura3 his3/his3 trp1/trp1 GAL+/GAL+ (13)

RJY1842 (W303) MATa/α leu2/leu2 ura3/ura3 his3/his3 trp1/trp1 GAL+/GAL+ erf4::NAT/erf4::NAT This study

RJY1888 MATα leu2-3,112 ura3-52 his3Δ200 trp1Δ63 lys2Δ801 ade2-101 ade8Δ ras1::HIS3 Ras2CS-ext erf2::TRP1 erf4::KanMX [YCp52-RAS2] This study

RJY1883 MATα leu2-3,112 ura3-52 his3Δ200 trp1Δ63 lys2Δ801 ade2-101 ade8Δ ras1::HIS3 Ras2CS-ext erf4::KanMX yos9::NAT [YCp52-RAS2] This study

RJY1884 MATα leu2-3,112 ura3-52 his3Δ200 trp1Δ63 lys2Δ801 ade2-101 ade8Δ ras1::HIS3 Ras2CS-ext ef4::KanMX rpn10::NAT [YCp52-RAS2] This study

RJY1885 MATα leu2-3,112 ura3-52 his3Δ200 trp1Δ63 lys2Δ801 ade2-101 ade8Δ ras1::HIS3 Ras2CS-ext erf4::KanMX hrd1::NAT [YCp52-RAS2] This study

RJY1886 MATα leu2-3,112 ura3-52 his3Δ200 trp1Δ63 lys2Δ801 ade2-101 ade8Δ ras1::HIS3 Ras2CS-ext erf4::KanMX ubc7::NAT [YCp52-RAS2] This study

RJY1887 MATα leu2-3,112 ura3-52 his3Δ200 trp1Δ63 lys2Δ801 ade2-101 ade8Δ ras1::HIS3 Ras2CS-ext erf4::KanMX doa10::NAT [YCp52-RAS2] This study

RJY1287 MATa leu2-3,112 ura3-52 his3Δ200 trp1Δ63 lys2Δ801 ade2-101 ade8Δ ras1::HIS3 Ras2CS-ext erf2::TRP1 [YCp52-RAS2] (27) by guest on March 11, 2020

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Table 2: Plasmids Source B1820 YEp351ERF2:13xMYC This study B1821 pESC-Trp PFA4:FLAG:Erf2CT58 This study B1822 pESC-Trp PFA4:FLAG:Erf2CT58-6R This study B1823 pESC-Leu FLAG:ERF2-6R This study B1825 pESC-Leu 6XHIS:ERF2 This study B1826 pESC-Leu 6XHIS:ERF2 C203S This study B1827 pESC-Leu 6XHIS:ERF2ΔC This study B1828 pESC-Leu 6XHIS:ERF2ΔC C203S This study B1829 pESC-Leu 6XHIS:ERF2ΔC/FLAG:ERF4 This study B1830 pESC-Leu 6XHIS:ERF2ΔC C203S/FLAG:ERF4 This study B1831 pESC-Leu 6XHIS:ERF2-6R This study B1832 pESC-Leu 6XHIS:ERF2-6R C203S This study B1833 pESC-Leu 6XHIS:ERF2-6R/FLAG:ERF4 This study B1834 pESC-Leu 6XHIS:ERF2-6R C203S/FLAG:ERF4 This study B1835 pUC57 ERF2-6R This study B1836 pEG-MBP:mCherry:Ras2CT35 This study


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Table 3: Erf2 Complex Autopalmitoylation/ Thioester Hydrolysis Activities Erf2 Complex KM (µM) VMAX (pmol/min/µg) kCAT/KM (min-1M-1)

Erf2-Erf4 43 +/- 8 43 +/- 3 66,667

Erf2 20 +/- 1 143 +/- 11 476,667

Erf2ΔC-Erf4 41 +/- 4 460 +/- 21 659,971

Erf2ΔC 29 +/- 8 385 +/- 54 780,933

Erf2-6R-Erf4 20 +/- 3 52 +/- 6 173,333

Erf2-6R 16 +/- 2 125 +/- 4 520,833


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and Robert J. DeschenesDavid A. Mitchell, Laura D. Hamel, Kayoko Ishizuka, Gayatri Mitchell, Logan M. Schaefer

Stability of the Acyl-Erf2 Intermediate and Palmitoyl Transfer to a Ras2 SubstrateThe Erf4 Subunit of the Yeast Ras Palmitoyl Acyl Transferase is Required for

published online August 16, 2012J. Biol. Chem.

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mitchell et al jbc 072612 · 2012-08-17 · mechanism. in this report we show that erf4 functions...

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