Insulin-dependent Interactions of Proteins with GLUT4 Revealed through Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)* Leonard J. Foster,†,‡,# Assaf Rudich,§,#,⊥ Ilana Talior,§,# Nish Patel,§ Xudong Huang,§ L. Michelle Furtado,§ Philip J. Bilan,§ Matthias Mann,†,| and Amira Klip*,§ Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark, UBC Centre for Proteomics and Department of Biochemistry and Molecular Biology, 301-2185 East Mall, University of British Columbia, Vancouver, BC, V6T 1Z4, Program in Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8, and Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried, Germany D-82152 Received August 10, 2005
The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein-protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulindependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas components of the ubiquitin-proteasome degradation system were generally reduced. Proteins participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related proteins, R-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 ( 0.5-fold by SILAC and 2.9 ( 0.8-fold by immunoblotting. Insulin caused GLUT4 and R-actinin-4 co-localization as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably R-actinin-4, associate with the transporter, potentially to facilitate its routing to the plasma membrane. Keywords: alpha-actinin 4 • L6 muscle cells • protein-protein interactions • mass spectrometry • quantitative proteomics • insulin • GLUT4 binding proteins
Introduction The insulin-regulated glucose transporter GLUT4 is a member of the SLC2 family comprising GLUTs 1-12,1 and is responsible for the majority of glucose uptake into skeletal muscle and fat cells.2 Upon insulin stimulation, GLUT4 is * To whom correspondence should be addressed. Tel.: (416) 813-6392. Fax: (416) 813-5028. E-mail:
[email protected]. † University of Southern Denmark. ‡ University of British Columbia. § Hospital for Sick Children. | Max-Planck Institute for Biochemistry. # These authors contributed equally to this work. ⊥ AR current address: S. Daniel Center for Health and Nutrition, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84103, Israel.
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recruited to the plasma membrane from intracellular vesicular pools, a process requiring dynamic changes in the actin cytoskeleton.3-5 Insulin-induced stimulation of glucose uptake is critical for the maintenance of glucose homeostasis. Indeed, this process is impaired in insulin resistance, a hallmark of type 2 diabetes. In this disease, GLUT4 expression is attenuated in adipose tissue and type 1 oxidative muscle fibers,6 sparking interest in processes regulating the half-life of the transporter. Since a protein’s intracellular localization, traffic, activity, stability, and degradation may all be controlled through interactions with binding protein partners, identifying GLUT4interacting proteins is of biological and clinical interest. Several potential GLUT4-binding partners have been proposed, mostly by pull-down experiments using GLUT4 C-terminal sequences 10.1021/pr0502626 CCC: $33.50
2006 American Chemical Society
Insulin-Dependent Protein Interactions with GLUT4
fused to glutathione-s-transferase.7-13 While providing important insights, these studies may be limited in their capacity to detect interactions with the complete protein sequence, which includes a large cytosolic loop as well as C- and N-terminal intracellular domains. Unfortunately, anti-GLUT4 antibodies are directed only against intracellular epitopes, which may be occupied by ligand proteins. Such a situation limits the use of anti-GLUT4 antibodies as a tool for a comprehensive analysis of the array of GLUT4-interacting proteins that are dynamically regulated by acute insulin stimulation. Mass spectrometry-based proteomics has emerged as a powerful method for determining the composition of immunoprecipitated protein complexes.14,15 Commercially available mass spectrometers and database search tools have advanced to the point where experienced laboratories can identify hundreds or thousands of proteins in exceedingly complex mixtures using nano-flow high performance liquid chromatography (HPLC)/ tandem mass spectrometry (LC-MS/MS).16,17 The reliability of these protein identities is markedly augmented through the use of stringent acceptance criteria, such as enzyme specificity and high mass accuracy.18 The classical approach of independent immunoprecipitations from different conditions can be confounded by the presence of nonspecifically interacting proteins and the nonquantitative nature of mass spectrometry restricts the ability to detect dynamic stimulus-induced alterations in the composition of a protein complex. Recently, we have applied stable isotope labeling by amino acids in cell culture (SILAC,19) to proteomic studies of protein complexes as a method for distinguishing functional (dynamic) protein-protein interactions from nonfunctional or nonspecific interactions with a solid-phase matrix or affinity bait.20-22 Proteins that bind nonspecifically from labeled and unlabeled samples will bind equally well to the bait, presenting a ratio near 1.0, whereas proteins whose binding depends on the functional condition of the experiment will yield a ratio of greater than or less than 1.0. Independently, Ranish et al. developed a similar approach23 using isotope coded affinity tags.24 The incorporation of the quantitative dimension provided by SILAC or isotope tags is critical as, in our experience, the fraction of nonspecific or nonfunctional interactions present in an isolated complex can be as high as 99% and can easily mask biologically relevant interactions of low affinity.20-22 Here, we apply SILAC to identify proteins interacting with GLUT4 in an insulin-dependent manner using a skeletal muscle cell line that stably expresses GLUT4 tagged with a myc epitope in its first exofacial loop (GLUT4myc). Immunoprecipitation via antibodies that recognize the noncytosolic tag on GLUT4myc allows for the identification of proteins that could associate with the transporter throughout its cytosol-facing sequences.
Experimental Procedures Materials. Sequencing grade modified porcine trypsin was purchased from Promega (Mannheim, Germany). Endopeptidase LysC was obtained from Wako (Osaka, Japan). Chromatography solvents were purchased from Sigma-Aldrich Denmark A/S (Copenhagen, Denmark). L-leucine-5,5,5-D3 was purchased from Cambridge Isotope Laboratories, Andover, MA). Normal isotopic abundance leucine and Protease Inhibitor Cocktail were from Sigma (St. Louis, MO). Fetal bovine serum, R-MEM standard culture medium and other tissue culture reagents were purchased from Wisent Inc. (Toronto, ON). Leucine-deficient R-MEM media was purchased from JRH Biosciences Inc., (Lenexa, Kansas, USA). N-hydroxysuccinim-
research articles ide-activated Sepharose 4 FastFlow beads and Sepharose CL6B beads were from Amersham Biosciences/ Pharmacia (Piscataway, NJ). Rabbit polyclonal antibodies for R-actinin-4, was from Alexis Biochemicals (San Diego, CA). Antibody to GAPDH (sc-25778) and Monoclonal (9E10) anti-myc were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Stable Isotope Labeling of Cultured L6 Myoblasts. L6GLUT4myc myoblasts were grown and differentiated into myotubes as described25 but with the following modifications: Cells were seeded on 10 cm dishes and grown in leucinedeficient R-MEM (JRH Biosciences Inc., Lenexa, Kansas, USA) supplemented with 2% (v/v) fetal bovine serum (FBS) that was dialyzed against PBS for 4 h at 4 °C to deplete it of amino acids (10 000 molecular weight cutoff), 1% (v/v) ampicillin-streptomycin antibiotic solution, and 52.4 mg/L of either normal isotopic abundance leucine (LeuD0, Sigma) or leucine with three deuterium atoms (L-Leucine-5,5,5-D3, LeuD3). Seven days after seeding, myotubes were serum-starved for 3 h in the same medium devoid of FBS, and then stimulated for 10 min without or with 100 nM insulin, respectively. To eliminate the possibility of isotopic effects, we switched the LeuD0 and LeuD3 labels between the basal and insulin-stimulated conditions several times and this did not impact the insulin effect observed on GLUT4-interacting proteins. In addition, the 7 days incubation of L6-GLUT4myc cells with LeuD3 had no effect on the cultures compared to cells cultured with LeuD0. In preliminary experiments we found that immunoprecipitating GLUT4myc directly from whole cell lysates resulted in excessive levels of myosin in the immune complexes that masked many less abundant proteins. We found that by initially isolating total membranes (TM) from LeuD0- and LeuD3-labeled myotubes cultures the levels of highly abundant muscle cytoskeletal proteins were markedly reduced. To prepare TM, myotubes (10 or 30 × 10 cm dishes per condition for regular or large-scale experiments, respectively) were washed with ice-cold PBS and then with homogenization buffer (250 mM sucrose, 20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM sodium vanadate, 10 nM okadaic acid and 1:1000 dilution of Protease Inhibitor Cocktail). Cells were then scraped in the same buffer, and homogenized by 12 strokes in a Teflon-glass Wheaton homogenizer, followed by centrifugation (700 × g for 10 min at 4 °C). The supernatants were collected and pellets were re-suspended and homogenized twice more as described above. To obtain TM, supernatants were centrifuged (195 000 × g for 75 min at 4 °C) and pellets were collected in 1.5 mL PBS containing 0.2% (v/v) C12E8, phosphatase and protease inhibitors as above, homogenized, and rotated for 15-30 min at 4 °C to allow solubilization by the detergent (∼3 mg protein/mL). Thereafter, TM were centrifuged in a tabletop microfuge (14 000 × g for 15 min at 4 °C), the supernatant was collected and protein content determined (bicinchoninic acid method). This final centrifugation removed large cytoskeletal structures prior to immunoprecipitation of GLUT4myc. Immunoprecipitation of GLUT4myc. Mouse monoclonal 9E10 IgG anti-myc antibody (200 µg) were first passed through protein G columns (Pierce Biotechnology Inc., Rockford, IL), eluted, and dialyzed against PBS prior to being covalently linked to N-hydroxysuccinimide-activated Sepharose 4 FastFlow beads (1 mL wet volume) according to the manufacturer’s instructions. The Sepharose beads conjugated to anti-myc were Journal of Proteome Research • Vol. 5, No. 1, 2006 65
research articles suspended in PBS pH 7.4 (50% v/v; 100 µg antibody/ml bead suspension). Equal amounts of TM protein from LeuD3-labeled, insulinstimulated cells, and unlabeled nonstimulated (basal) cells were mixed for the immunoprecipitation of GLUT4 (0.55 and 5.0 mg protein of each for regular and large-scale experiments, respectively). The mixed TM samples were first pre-cleared with Sepharose CL-6B beads (0.05 or 0.5 mL of 50% v/v suspension, as required) with rotation for 30 min at 4 °C, then immunoprecipitation was carried out overnight at 4 °C with Sepharose beads containing anti-myc antibody (0.04 or 0.4 mL of 50% v/v suspension, as required). Thereafter, beads were precipitated by slow (30 s, 950 × g) spins, washed twice with PBS containing inhibitors and 0.2% C12E8, and once with PBS before addition of 150 µL of modified SDS-PAGE sample buffer (5% (w/v) SDS, 25% (v/v) glycerol, and 156 mM Tris-HCl pH 6.8, without β-mercaptoethanol and bromophenol blue) and incubation for 15 min at 65 °C. LC-MS/MS, Database Searching and Quantification. In initial experiments, the immune complex was extracted in 200 µL 6 M urea/2 M thiourea (in 20 mM Tris, pH 8.0) and prepared for LC-MS as described.26 In later experiments, the immune complexes were solubilized in modified SDS-PAGE sample buffer and subjected to one-dimensional gel electrophoresis. Proteins were then visualized with colloidal Coomassie blue, each lane cut into 10 slices and then subjected to in-gel digestion.27 Peptide digests were desalted using STAGE tips28 and loaded onto reversed phase analytical columns for liquid chromatography.29 Peptides were eluted from the analytical columns by 100 min gradients comprised of three linear steps running from 5.6% to 64% acetonitrile and sprayed directly into the orifice of a linear ion-trap-Fourier transform mass spectrometer (Finnigan LTQ-FT, Thermo Electron). The LTQ-FT was set to acquire tandem mass spectra of the five most abundant multiply charged peptides per ∼2 s cycle. Survey scans were acquired at 100 000 resolution using the FT detector while fragment spectra were simultaneously acquired on the linear ion-trap detector. All fragment spectra were then searched, using Mascot Server (Matrix Science), against the most recent rat IPI protein database (v1.19, 33 572 entries) that had been supplemented with sequences for human keratins, mouse immunoglobulins, bovine serum albumin, porcine trypsin and endopeptidase LysC. The following search criteria were used in all cases: tryptic cleavage with a maximum of one missed cleavage, ion-trap fragmentation characteristics, 10 parts per million (ppm) error tolerance for precursor ion masses (measured in FT), 0.5 Da error tolerance for product ion masses (measured in LTQ), methionine oxidation and leucine with three deuterium atoms as variable modifications and cysteine carbamidomethylation as a fixed modification. Fragment spectra were extracted from Xcalibur RAW files and charge states assign using the Extract_MSN script (Thermo Electron), requiring a minimum ion intensity of 10 for each peak and a minimum of 5 peaks per spectrum. Charge states and monoisotopic peak assignments were then double-checked using DTASuperCharge, an in-house script, before DTA files were combined into a single Mascot Generic Format file. Acceptance criteria for peptide and protein identifications were determined by searching 5400 random fragment spectra against forward and reversed rat IPI databases (see ref 17 for an introduction). After removing redundant peptide sequences, 929 unique peptide sequences with scores greater than 24, more than 7 amino acids in length and with mass accuracies 66
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better than 10 ppm were identified in the normal database, while only 8 similar sequences were identified in the reversed database. These results indicate a false positive identification rate of 8/929, or 0.9%, at the peptide level. Therefore, proteins were only considered identified if at least two unique peptides of this quality were sequenced among all experiments, giving a statistical confidence of more than 99.99% at the protein level, or less than one false positive protein identification in ten thousand. Application of these criteria to the entire data set led to the identification of 970 unique protein sequences. Multiple isoforms of a protein were only kept in the final list if they were differentiated by at least one unique peptide meeting the above criteria; however, each isoform was still required to have at least two peptides identifying it. Insulin:control ratios were calculated based on the integration of extracted ion chromatograms generated from survey scans using MSQuant22 (http://msquant.sourceforge.net), after accounting for the reversed phase shift due to deuterated leucine. The average measured ratio of GLUT4myc was initially 0.91 so this was adjusted to 1.0 and all other ratios were corrected accordingly. Since quantification was based on leucine, peptides that did not contain any leucine residues were not quantifiable. Additionally, where the peptide ion signal was too close to the noise level no reliable quantification was possible. Nonetheless, of the 970 identified proteins we were able to quantify 603. All peptide and protein information was stored in an in-house relational database for subsequent querying. Gene Ontology Classification. UniProt accession numbers of all 603 quantified proteins and the subset of 36 proteins displaying a ratio greater than 1.5 or less than 0.66 based on at least three peptides were classified using the GOMiner tool (http://discover.nci.nih.gov/gominer/) as described.30 Distributions of these two sets of proteins within the subcategories of the Cellular Physiological Process category (GO:0050875) were then graphed. Detection of r-Actinin-4 and GAPDH in GLUT4myc Immunoprecipitates by Immunoblotting. L6 myoblasts seeded in four 10-cm dishes and grown to confluence or differentiated into myotubes were deprived of serum for 4.5 h at 37 °C, and then treated without or with 100 nM insulin for 20 min at 37 °C. The cells were washed with cold PBS, scraped with lysis buffer (150 mM NaCl, 2 mM EDTA, 1% (vol/vol) Triton X-100, 20 mM iodoacetamide, 50 mM N-octylglucoside and Protease Inhibitor Cocktail, 20 mM Tris-HCl, pH 8.0), passed through 27.5 gauge syringe and incubated on ice for 2 h. The lysates were centrifuged (13 000 × g) for 15 min at 4 °C and supernatants were quantified for protein concentration using the bicinchoninic acid method. Equal amounts of protein (1.5 mg of protein/reaction) from each sample were immunoprecipitated with 40 µL of myc antibody conjugated to Sepharose beads with rotation overnight at 4 °C. Beads were collected by centrifugation for 30 s at 950 × g at 4 °C and washed 5× with wash buffer (150 mM NaCl, 2 mM EDTA, 1% (vol/vol) Triton X-100, 20 mM iodoacetamide, Protease Inhibitor Cocktail, 20 mM Tris-HCl, pH 8.0) by gentle rotation for 10 min at 4 °C. Beads were then resuspended in 20 µL of lysis buffer plus 20 µL SDS-PAGE sample buffer (2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, 0.005% (w/v) bromophenol blue and 62.5 mM Tris-HCl pH 6.8) and heated at 100 °C for 3 min. Proteins were separated by 5 or 10% polyacrylamide (vol/ vol) SDS-PAGE, transferred onto PVDF membranes and blotted with anti-R-actinin-4 (1:2000), anti-GAPDH (1:1000) or antimyc (1:500) antibodies. Immunoreactive bands were visualized
Insulin-Dependent Protein Interactions with GLUT4
with Horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Fluorescence Microscopy. L6-GLUT4myc myotubes grown on 25 mm diameter glass coverslips were deprived of serum for 4 h, and then treated with 100 nM insulin for 5 min at 37 °C. Labeling of actin filaments with rhodamine-coupled phalloidin and immunostaining of specific antigens was carried out as described previously.31 It was essential to fix the cells at 4 °C immediately after insulin stimulation and to permeabilize in 0.1% (vol/vol) Triton X-100 for 3 min in order to preserve actin morphology. The primary antibody dilution factors were as follows: myc monoclonal, 1:150 or R-actinin-4 polyclonal, 1:250 in 0.1% (wt/vol) BSA/PBS. To label actin filaments simultaneously with any of the above listed molecules, fixed and permeabilized cells were incubated for 1 h at room temperature with the fluorophore-conjugated phalloidin indicated in the figure legends (0.01 U/ coverslip) during the incubation with the secondary antibody. To eliminate artifactual “co-localization” in these co-staining experiments, fluorophores with maximal spectral separation were chosen. The staining pattern observed in co-staining experiments was compared to that with single-staining experiments using the same antibody. Cells were examined with a Zeiss LSM 510 laser scanning confocal microscope using multichannel scanning to reduce the possibility of cross-talk between fluorophores. Acquisition parameters were adjusted to exclude saturation of the signal.
Results SILAC Approach to Immunoprecipitations. Affinity-based methods are often used to isolate proteins interacting with a target of interest and powerful, mass spectrometry-based proteomics tools32 employed to analyze the resulting complexes. However, biologically interesting protein-protein interactions may be weak or transient, so excessive washing of isolated complexes can interfere with their detection. Alternatively, light washing can preserve such interactions but highly abundant or generally ‘sticky’ proteins that bind nonspecifically to the affinity matrix present a high background that can easily mask specific interactions. We used SILAC to overcome some of these issues, and the principle of this approach is depicted in Figure 1. GLUT4myc was immunoprecipitated from insulinstimulated cells whose proteome had been SILAC-labeled with LeuD3 (L-Leucine-5,5,5-D3), and from nonstimulated cells whose proteome had been labeled with LeuD0 (normal isotopic abundance leucine). Eluates were mixed and two versions of each Leu-containing peptide were detected. By calculating the ratio between the MS peak derived from a peptide with LeuD3 (in this case, from insulin-stimulated cells) and the peak from the respective LeuD0 (basal), a ratio value of 1.0 would indicate no change in the abundance of this protein in the basal compared to the insulin-stimulated state within the GLUT4myc immunoprecipitate, whereas a value higher or lower than one would indicate that insulin increased or decreased, respectively, the abundance of the specific peptide. Protein Identification and Quantification. Isolated GLUT4myc immune complexes were analyzed by one-dimensional gel electrophoresis followed by liquid chromatography/ mass spectrometry, as described in Experimental Procedures. Figure 2a shows the total ion chromatogram from a typical analysis of one gel slice. Survey scans (Figure 2b) at 100 000 resolution were measured approximately every 2 s throughout the LC gradient and peptide ions detected (such as TFDQI-
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Figure 1. Application of SILAC to identify specific, insulindependent binding partners from background binders. Two populations of GLUT4myc -expressing L6 myotubes are cultured in media containing either leucine with three hydrogen atoms replaced by deuterium (L-leucine-5,5,5-D3; LeuD3) or normal isotopic abundance leucine (LeuD0). One population (the LeuD3 population in this study) is stimulated for 10 min with 100 nM insulin before both populations are processed for total membranes, solubilized, combined and subjected to immunoprecipitation with 9E10 anti-myc antibody as detailed in Experimental Procedures. Isolated immune complexes are eluted, resolved by one-dimensional SDS-PAGE and digested to peptides for analysis by LC/MS. Peptides from proteins whose interactions with GLUT4myc are insulin-dependent will show higher or lower abundance in the LeuD3 form (red) if insulin enhances or reduces, respectively, their interactions with GLUT4myc. Peptides from proteins whose binding are not affected by insulin or are bound nonspecifically (blue) will be present at equal proportions of LeuD0 and LeuD3 labeling in the GLUT4myc immunoprecipitates.
SATFR from GLUT4 depicted in Figure 2c), were fragmented in the LTQ. After discounting modifications and charge states, 9754 unique peptides were sequenced and their masses measured to an average accuracy of 2.39 ppm. Nine-hundred seventy proteins, their sequences covered to an average of 24%, were identified from these peptides (Supporting Information Table 1) with an estimated false:positive ratio < 1:10 000 (see Experimental Procedures). Sequenced peptides were quantified on the basis of their extracted ion chromatograms (Figure 2d) and insulin:control ratios for each protein were calculated as the average ratios observed in all experiments for 603 proteins (Supporting Table 1). While the ratios ranged from 0.3 to 4.0 (Figure 2e), the presence of most proteins in the immune complex was not affected by insulin, consistent with previous estimations of the prevalence of ‘bona fide’ interactions in a protein complex.20-22 The average relative standard error of the mean for the in insulin:control ratios was 26%, similar to previous reports using SILAC (LeuD3: LeuD0 ratios) with other types of stimuli.26,33 Thirty-six proteins (Tables 1A and 1B) with insulin:control ratios greater than 1.5 or lower than 0.66 (1/1.5) for at least three peptides were identified. A cutoff value of 1.5 was selected as this was approximately twice the standard error of the populaJournal of Proteome Research • Vol. 5, No. 1, 2006 67
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Figure 2. Application of SILAC to determine specific binding partners. (a) Peptide mixtures from in-gel digested proteins were resolved by reversed phase HPLC (see Experimental Procedures) and sprayed directly into the orifice of an LTQ-FT. (b) Survey scans of peptides eluted from reversed phase columns were acquired and used to select peptide ions for fragmentation. Fragment spectra (c) of the 593.3 ion from (b) was found to match the TFDQISATFR peptide from GLUT4. (d) Representative extracted ion chromatogram for the LISEEDLLK peptide from GLUT4myc demonstrating an insulin:control (LeuD3/LeuD0) ratio of 0.91. (e), Plot of insulin:control ratios for all proteins quantified in this study, ranked from largest to smallest.
tion and was deemed to represent the minimum biologically significant change.20,21,33 Moreover, the insulin-dependent changes in surface GLUT4 levels are in the range 1.5 to 2.2fold in muscle cells. As an additional measure of confidence, we only considered those proteins where a standard deviation could be calculated (i.e., based on at least three peptides). Proteins Exhibiting Dynamic, Insulin-Regulated Association with GLUT4 as Identified by SILAC. Some general trends were observed for the entire cohort of 603 proteins. For example, several ubiquitin-proteasome components or other proteases showed reduced interaction with GLUT4 in the insulin-treated samples (ratio < 1), whereas those involved in protein synthesis and protein folding tended to display enhanced association with GLUT4 (ratio > 1) (Supporting Information Table 1). Proteins with insulin:control ratio > 1.3 and 1.5, suggesting that insulin augmented their association with GLUT4. Similarly, proteins involved in intracellular traffic, had either insulin:control ratios >1.5 and increased association with GLUT4 in response to insulin (Arfs 3 and 5) or insulin:control ratios 1.3 and
