Reagents and antibodies
General laboratory chemicals were obtained from Sigma-Aldrich and Thermo Fisher Scientific. Antibody to EGFP was raised in sheep against the entire coding region of EGFP and affinity purified. Rabbit anti–golgin-160 antibodies were raised and affinity purified against the entire coding region of rat golgin-160 expressed as a His6-tagged protein in bacteria. Mouse anti-clathrin clone X22 was a gift from S. Royle (University of Liverpool, Liverpool, England, UK). Commercially available antibodies were used to α-tubulin (mouse DM1A; Sigma-Aldrich), actin (mouse 2Q1055; Abcam), α-adaptin (clone 8; BD), γ-adaptin (mouse clone 88; BD), δ-adaptin (mouse clone 18; BD), EEA1 (rabbit 2411; Cell Signaling Technology), FLAG antibodies (mouse M2; Sigma-Aldrich), GM130 (mouse clone 35; BD), human LAMP1 (mouse clone 25; BD), TGN46 (sheep AHP500; Serotec), TfR (rabbit CBL47; Millipore), and CI-MPR (mouse 2G11; Abcam). Secondary antibodies raised in donkey to mouse, rabbit, sheep/goat, and human conjugated to HRP, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 568, and Alexa Fluor 647 were obtained from Invitrogen and Jackson ImmunoResearch Laboratories.
Molecular biology and protein purification from bacteria and insect cells
Human DENNs were amplified from image clones (Source Bioscience Geneservice) or human fetal cDNA (Marathon ready cDNA; Takara Bio Inc.) using KOD polymerase (EMD). Mutagenesis was performed using the QuikChange method according to the protocol (Agilent Technologies). Duplexes for siRNA were obtained from QIAGEN or Thermo Fisher Scientific. Mammalian expression constructs were made using pcDNA4/TO and pcDNA5/FRT/TO vectors (Invitrogen). Bacterial expression constructs were made using pQE32 (QIAGEN), pMal (New England Biolabs, Inc.), and pFAT2 encoding the His6
–maltose-binding protein, and His6
-transferase, respectively. His6
-transferase–tagged Rab proteins in pFAT2 were expressed in BL21 (DE3) pRIL or BL21 (DE3) pG-KGE8 (Takara Bio Inc.) at 18°C for 12–14 h, then purified using Ni-NTA agarose as described previously (Fuchs et al., 2005
). In brief, cell pellets were lysed for 20 min in 10 ml IMAC5 (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM imidazole, 0.2% Triton X-100, and protease inhibitor cocktail; Roche) containing 0.5 mg/ml lysozyme, and then sonicated at 70% power four times for 30 s with a 30-s rest period. Lysates were clarified by centrifugation at 14,000 rpm in a JA-17 rotor for 30 min. To purify the tagged protein, 0.5 ml of nickel-charged NTA-agarose (QIAGEN) was added to the clarified lysate and rotated for 2 h. The agarose was washed three times with IMAC20 (IMAC5 with 20 mM imidazole), then the bound proteins were eluted in IMAC200 (IMAC5 with 200 mM imidazole), collecting 0.5 ml fractions. All manipulations were performed on ice or in an 8°C cold room. His6
-tagged Rabex5, Rabin3/8, Rabin3-like/GRAB, DENND1B-S, DENND1B-L, and DENND2A-D in pQE32 were expressed in JM109 at 18°C for 12–14 h, then purified using nickel-charged NTA agarose using the same procedure as the Rabs. Rab12 and Rab39 were expressed using the pAcHis-GST vector encoding as His6
-transferase tag, which is equivalent to that in pFAT2 in the baculovirus/Sf9 cell expression system (BD). For virus infection, 10 × 15-cm dishes of containing 1.5 × 108
at 5 × 105
cells/ml of Sf9 cells were infected with a virus moiety of infection of 1.0 for 48 h (Neef et al., 2005
). The infected cells were harvested, washed once in PBS, and then lysed in IMAC5 for 20 min on ice. Lysates were clarified by centrifugation at 14,000 rpm in a JA-17 rotor (Beckman Coulter) for 30 min. Proteins were purified with the same protocol used for bacterial expression with minor modifications: 100 µl of nickel-charged NTA agarose was used for the purification, and 100-µl fractions of the elution were collected. Purified proteins were dialyzed against TBS (50mM Tris-HCl, pH 7.4, and 150 mM NaCl) and then snap frozen in liquid nitrogen for storage at −80°C. Protein concentration was measured using the Bradford assay.
Recombinant STxB was expressed untagged from pTrc99A in Escherichia coli BL21 (DE3) grown in lysogeny broth for 14 h at 37°C. All subsequent steps were performed at 4°C. The bacterial cell pellet from a 1 liter culture was resuspended in 20 ml of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1 mg/ml polymyxin B. After mixing for 30 min on a roller, the sample was sonicated twice at 70% power for 10 s. A periplasmic lysate was prepared by removing the cell debris by centrifugation, first at 13,000 g for 15 min then again at 90,000 g for 35 min. This lysate was diluted by the addition of two volumes of 20 mM Tris-HCl, pH 7.5, then loaded on to a 5 ml HiTrapQ column (GE Healthcare). The column was washed with 20 ml of 20 mM Tris-HCl, pH 7.5, then eluted with a 50-ml linear gradient from 0–600 mM NaCl in the same buffer. The flow rate was 2 ml/min throughout. Fractions of 2.5 ml were collected and analyzed by SDS-PAGE, and the peak of STxB pooled to give a 15-ml solution at 1 mg/ml. This was then concentrated to 5 mg/ml using Centricon 10K units (Millipore) centrifuged at 3,000 g. A 500 µl aliquot was then applied at 0.15 ml/min to a Superose 12 gel filtration column equilibrated in PBS, and 1-ml fractions were collected. The peak fractions of pentameric STxB were pooled to give a 1.1 mg/ml solution. A 1-ml aliquot of this solution was labeled by the addition of one vial of monoreactive N-hydroxysuccinimidyl Cy3 (GE Healthcare). After 5 min at room temperature, 100 µl of 1 M Tris-HCl, pH 8.0, was added to stop the reaction. Excess dye was removed by desalting over PD10 columns (GE Healthcare). Fractions of 500 µl were collected, and absorption was measured at 280 and 552 nm. Using molar extinction coefficients of 150,000 M−1cm−1 for Cy3 and 170,000 M−1cm−1 for pentameric STxB, dye labeling was calculated as 1 Cy3 per toxin subunit with a concentration of 0.7 mg/ml.
Cell culture and protein purification from mammalian cells
HeLa and HEK293 cells were cultured in DME containing 10% bovine calf serum (Invitrogen) at 37°C and 5% CO2. For plasmid transfection and siRNA transfection, Mirus LT1 (Mirus Bio LLC), and Oligofectamine (Invitrogen), respectively, were used according to the manufacturers’ instructions. FLAG-tagged forms of DENND1A, DENND1C, DENND3, DENND4A, DENND4B, DENND4C, DENND5A, DENND5B, MTMR5, MTMR13, and MADD in pcDNA5/FRT/TO were transiently expressed in 10 × 15-cm dishes of 70% confluent HeLa cells. After 48 h of growth, the cell pellet was lysed for 20 min on ice in 5 ml of cell lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, and protease inhibitors cocktails). Cell lysates were split into 1.0 ml aliquots and clarified by centrifugation at 20,000 g in a microcentrifuge (5417R Microfuge; Eppendorf) for 20 min. The FLAG-tagged proteins were immunoprecipitated from the clarified lysate using 100 µl of anti-FLAG M2 affinity gel (Sigma-Aldrich) for 4 h at 4°C. The pellet was washed 10 times in 1 ml of cell lysis buffer, 10 times in 1 ml of high-salt buffer (50 mM Tris-HCl, pH 7.4, and 500 mM NaCl), and 10 times in TBS, and finally the proteins were eluted with 100 µl of 200 µg/ml FLAG-peptide in TBS containing 2 mM dithiothreitol. The eluted proteins were analyzed on 7.5–10% SDS-PAGE gels stained with Coomassie brilliant blue, and concentrations were estimated by comparison to a series of BSA standards in the range of 0.1–1 mg. The peak fractions were snap frozen in liquid nitrogen for storage at −80°C without dialysis.
Nucleotide binding and GEF assays
Nucleotide loading was performed as follows: 10 µg of GST-tagged Rab was incubated in 50 mM Hepes-NaOH, pH 6.8, 0.1 mg/ml BSA, 125 µM EDTA, 10 µM Mg-GDP, and 5 µCi [3H]-GDP (10 mCi/ml; 5,000 Ci/mmol) in a total volume of 200 µl for 15 min at 30°C. For standard GDP-releasing GEF assays, 100 µl of the loading reaction was mixed with 10 µl of 10 mM Mg-GTP and 10–100 nM GEF protein to be tested or a buffer control, and adjusted to 120 µl final volume with assay buffer. The GEF reaction occurred for 20 min at 30°C. After this, 2.5 µl was taken for a specific activity measurement; the remainder was split into two tubes, then incubated with 500 µl of ice-cold assay buffer containing 1 mM MgCl2 and 20 µl of packed glutathione-sepharose for 60 min at 4°C. After washing three times with 500 µl of ice-cold assay buffer, the sepharose was transferred to a vial containing 4 ml of scintillation fluid and counted. The amount of nucleotide exchange was calculated in pmoles of GDP released. For GTP-binding assays the following modifications were made: only unlabeled GDP was used in the loading reaction; in the GEF reaction, 0.5 µl of 10 mM GTP and 1 µCi [35S]-GTPγS (10 mCi/ml; 5,000 Ci/mmol) were used. The amount of nucleotide exchange was calculated in pmoles of GTP bound.
Fixed samples on glass slides were imaged using a 60× 1.35 NA oil immersion objective lens on a standard upright microscope equipped with a CoolSNAP HQ2 camera (Roper Industries) under the control of MetaMorph 7.5 software (MDS Analytical Technologies). Images were cropped in Photoshop CS3 (Adobe) or ImageJ and placed into Illustrator CS3 (Adobe) without performing any other contrast adjustments of image manipulations to produce the figures.
Protein samples for mass spectrometry were separated on 4–12% gradient NuPAGE gels (Invitrogen), then stained using a colloidal Coomassie blue stain. Gel lanes were typically cut into 12 slices, and then digested with trypsin using published methods (Wilm et al., 1996
). The resulting tryptic peptide mixtures in 0.05% trifluoroacetic acid were then analyzed by online liquid chromatography tandem mass spectrometry with a nanoAcquity UPLC (Waters) and Orbitrap XL ETD mass-spectrometer (Thermo Fisher Scientific) fitted with a nano-electrospray source (Thermo Fisher Scientific). Peptides were loaded on to a 5 cm × 180 µm BEH-C18 Symmetry trap column (part no. 186003514; Waters) in 0.1% formic acid at 15 µl/minute, and then resolved using a 25 cm × 75 µm BEH-C18 column (part no. 186003815; Waters) in 99–37.5% acetonitrile in 0.1% formic acid at a flow rate of 400 nl/min. The mass spectrometer was set to acquire a mass spectrometry survey scan in the Orbitrap (R = 30,000) and then perform tandem mass spectrometry on the top five ions in the linear quadrupole ion trap after fragmentation using collision ionization (30 ms, 35% energy). A 90-s rolling exclusion list with n
= 3 was used to prevent redundant analysis of the same ions. Maxquant and Mascot (Matrix Science) were then used to compile and search the raw data against the human International Protein Index database. Protein group and peptide lists were sorted and analyzed in Excel (Microsoft) and Maxquant (Cox and Mann, 2008
). Mass spectrometry and tandem mass spectrometry spectra were manually inspected using Xcalibur Qualbrowser (Thermo Fisher Scientific).
Sequence alignments of DENNs and Rabs were done with ClustalX (Chenna et al., 2003
) or MUSCLE (Edgar, 2004
), and the results were visualized and manipulated with Jalview (Waterhouse et al., 2009
). ClustalX was also used to produce a dendrogram illustrative of sequence relationships. Regions of intrinsic disorder were predicted using the metaPrDOS server (Ishida and Kinoshita, 2008
), and portions of them with energetic properties appropriate for forming protein–protein interactions were highlighted with ANCHOR (Mészáros et al., 2009
). Linear sequence motifs were browsed in the ELM database (Gould et al., 2010
Online supplemental material
Fig. S1 shows the pattern of localization for all human DENN domain proteins when transfected as EGFP-tagged constructs in Hela cells. Table S1 lists the DENN domain proteins identified in human, mouse, zebrafish, fruit fly, and nematode. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201008051/DC1