H]Mannose (60 Ci/mmol) and [14
C]isopentenyl pyrophosphate (I-P-P) (55 mCi/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). GDP-[3
H]mannose was prepared enzymatically as described previously (Rush et al. 1993
). YEp352 (yeast shuttle vector), strain YG932 (yeast rer2Δ
mutant), and the cDNA for yeast RER2
were gifts from Drs. M. Aebi (ETH, Zurich, Switzerland) and F. Fernandez (GlycoVaxyn, Schlieren, Switzerland). Vector PEX-T21 containing the E. coli uppS
sequence was a generous gift from Steffi Balada (GlycoVaxyn, Schlieren, Switzerland). Anti-CPY antiserum was purchased from Molecular Probes (Invitrogen, Eugene, OR). Undecaprenol and purified individual isoprenologues of (55)Dol, (75)Dol, and (95)Dol were obtained from Warzawa (Warsaw, Poland) and phosphorylated chemically as described by Danilov et al. (1989)
and Danilov and Chojnacki (1981)
H]Man-P-(75)Dol, and [3
H]Man-P-(95)Dol were prepared enzymatically using partially purified Man-P-Und synthase from M. luteus
and purified as described previously (Rush et al. 1993
Molecular cloning of cis-IPTases, construction of YEp352 shuttle vectors and expression in S. cerevisiae
The coding sequence of the E. coli uppS
gene was amplified by polymerase chain reaction (PCR) using the two primers (forward) 5′-TTCCCGGG
ATGTTGTCTGCTACTCAACCAC-3′ and (reverse) 5′-TTCAAGCTT
CAA TGATGATGATGATGATGGGCTGTTTCATCACCGGC-3′ with Xma1 and HindIII restriction sites (underlined) and E. coli
genomic DNA as template. The reverse primer is designed to introduce a His6
-tag at the carboxy terminus of the protein. The PCR product was digested with Xma1 and HindIII and ligated into similarly prepared YEp352, containing the putative yeast RER2 promoter (the 250 bp region immediately upstream of the yeast RER2
gene) described previously (Shridas et al. 2003
). The structure of the resulting plasmid with E. coli
uppS downstream of the putative yeast promoter sequence was verified by sequence analysis (Eurofins MWG Biotech, info@Eurofins.com). Yeast strains were transformed as described by Schiestl and Gietz (1989)
and transformants were identified by uracil selection.
Preparation of pEGFP-C1:UPPS
The DNA sequence encoding the E. coli UPPS gene was amplified by PCR using primers (forward) 5′-TTGAATTCGATGTTGTCTGCTACTCAACCA-3′ and (reverse) 5′-TTGTCGACTAGGCTGTTTCATCACCGGGC-3′ containing EcoR1 and SalI restriction sites (underlined). Following digestion with the appropriate restriction enzymes, the insert was purified and ligated into similarly digested pEGFP-C1 to produce an expression plasmid encoding the bacterial cis-IPTase, UPPS, containing an N-terminal GFP tag. The structure and sequence of the expected plasmid were verified by direct sequencing by Davis Sequencing (Davis, CA).
Yeast culture and preparation of yeast microsomes
Yeast strains SS328 (MATα ade2-101 ura3-52 his3Δ200 lys2-801
) and YG932 (rer2Δ
mutant) (MATα rer2Δ::kanMX4 ade2-101 ura3-52 his3Δ200 lys2-801
) were obtained from Dr. M. Aebi (ETH, Zurich, Switzerland). The strains were cultured at 30°C in 1% yeast extract (Becton, Dickinson and Co., Sparks, MD), 2% Bacto-Peptone (Becton, Dickinson and Co., Sparks, MD), and 2% glucose (yeast peptone dextrose, YPD). Yeast strains transformed with YEp352 (Hill et al. 1986
) and its derivatives were grown in 0.67% yeast nitrogen base, 50 mM sodium succinate pH 5.0, 2% glucose and all necessary auxotrophic requirements, except for uracil.
Yeast microsomes were prepared from logarithmically growing cell cultures by homogenization (tight-fitting Dounce homogenizer) following Lyticase (Sigma-Aldrich, St. Louis, MO) treatment as described previously (Fernandez et al. 2001
CHO cell culture, transfection with pEGFP-C1/uppS and preparation of crude microsomal fractions
CHO cells were maintained at 37°C in 5% CO2 in DMEM/F-12 50/50 with glutamine and 15 mM HEPES (Mediatech, Manassas, VA), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. CHO cells were transfected at 95% confluence using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Twenty-four hours following transfection, cell monolayers were scraped into 0.01 M Tris–HCl, pH 7.4, 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA) (buffer A) and homogenized with 20 passes with a tight-fitting Dounce homogenizer. The homogenates were centrifuged at 1000 × g, 10 min to remove unbroken cells and debris. Crude microsomes were collected by sedimentation at 100,000 × g, 10 min in a TL-100A tabletop ultracentrifuge and resuspended in buffer A.
Detection of UPPS in CHO cells by immunofluorescence
To localize GFP-tagged UPPS by immunofluorescence, CHO cells were grown on 12-mm coverslips to 30–45% confluence and transfected with pEGFP-C1/UPPS, as described above. After 24 h in culture, the coverslips were washed in PBS and fixed with cold (−
60°C) methanol for 6 min, followed by blocking with 5% FBS in Tris-buffered saline for 20 min at room temperature. The fixed coverslips were incubated with rabbit anticalnexin antibody (0.050 mL of 1:100 diluted) for 1 h and washed 3
× with blocking solution. After washing, the coverslips were probed with secondary antibody (Texas-Red conjugated sheep antirabbit IgG, Amersham) for 1 h and washed 3
× with blocking solution followed by PBS. Coverslips were mounted on glass slides with Vectashield mounting solution (Vector Laboratories, Burlingame, CA). The slides were viewed with a Nikon Eclipse E600 microscope.
In vitro assays of MPDS activity
Reaction mixtures for MPDS contained microsomes from yeast or CHO cells (0.25 mg protein), 50 mM Tris–HCl, pH 8, 5 mM AMP, 12.5 mM MgCl2
, 1 mM sodium orthovanadate, and 10 μM GDP-[3
H]Man (1000 dpm/pmol) in a total volume of 0.05 mL. Samples were incubated with GDP-[3
H]Man for 1 min, following a 5-min pre-incubation, at 30°C and the reaction terminated by the addition of 20 vols CHCl3
OH (2:1). The incorporation of [3
H]mannose into [3
H]Man-P-Dol was assayed as described previously (Waechter et al. 1976
). Where indicated, Dol-P (100 μM) was added as a dispersion in 1% CHAPS, with a final CHAPS concentration of 0.1%.
Metabolic labeling of S. cerevisiae
Cell cultures were labeled metabolically by incubation with [3
H]mannose (5 μCi/mL) at a cell density of 200 OD600
units/mL in ura selection medium containing 0.1% glucose and 25 mg/mL uracil. Following incubation for 15 min at 30°C, the cell cultures were quickly chilled by the addition of crushed ice (0.5
gm/mL). The cells were recovered by centrifugation (1000 × g
, 10 min), resuspended in 5 mL ice-cold PBS, and sedimented again. Incorporation of [3
H]mannose into [3
H-Man]glycoprotein was determined as described previously (Waechter et al. 1983
Assays for cis-IPTase activity in yeast and CHO microsomal fractions
Reaction mixtures for the assay of yeast cis
-IPTase contained 25 mM HEPES-NaOH, pH 8.5, 5 mM MgCl2
, 0.5 mM sodium orthovanadate, 0.1 mM F-P-P, microsomes (0.1–0.5 mg membrane protein), and 20 μM [14
C]I-P-P (121 dpm/pmol) in a total volume of 0.05 mL. Following incubation for 15 min at 30°C, the reactions were terminated by the addition of 20 vols CHCl3
OH (2:1). The enzymatic synthesis of labeled polyprenyl-P-P products was assayed basically as described previously (Shridas et al. 2003
Reaction mixtures assaying cis
-IPTase activity in CHO microsomes contained crude microsomes (30–40 μg protein), 25 mM Tris–HCl (pH 8.5), 5 mM MgCl2
, 1.25 mM DTT, 2.5 mM sodium orthovanadate, 10 μM squalestatin, 100 μM F-P-P, 0.35% Triton X-100, and 45 μM [1-14
C]I-P-P (55 mCi/mmol) in a total volume of 0.050 mL. After incubation at 37°C for 1 h, the reaction was terminated by the addition of 1 mL CHCl3
OH (2:1). The enzymatic synthesis of labeled polyprenyl-P-P products was then assayed basically as described previously (Crick et al. 1991
Characterization of products formed by cis-IPtase activities in microsomal fractions from yeast and CHO cells
To determine the chain length of the products formed in the yeast cis-IPTase reactions in vitro, the [14C]-labeled enzymatic products were dispersed by brief sonication (Kontes Micro Ultrasonic Cell Disruptor at 40% full power) in 1% Triton X-100 and dephosphorylated by overnight incubation (37°C) with calf brain microsomes (0.2 mg, in 50 mM Tris–Cl, pH 7.4, 2.5 mM EDTA, 0.5% Triton X-100 in a total volume of 0.1 mL). Following incubation, the [14C]-labeled products were recovered by organic extraction and Folch partition. The organic phase was dried under nitrogen, redissolved in hexane and chromatographed on a 1 gm column of Bio-Sil HA (Bio-Rad Laboratories, Richmond, CA) equilibrated in hexane. The Bio-Sil HA column was washed with 5 column volumes of hexane and then eluted with 1 mL portions of hexane/diethyl ether (9:1). The dephosphorylated [14C]polyprenols eluting in fractions 2–4 were pooled, dried under nitrogen, redissolved in a small volume of hexane, and resolved by chromatography on Baker Si-C18 TLC plates in acetone/water (98:2). The [14C]-polyprenols were detected by radiochromatoscanning with a Bioscan AR2000 Imaging Scanner (Bioscan, Washington, DC). The zones corresponding to polyprenol standards (obtained from Warszawa, Warsaw, Poland) were located by exposure to iodine vapors.
To determine the chain length of the products formed in the cis-IPTase reactions with CHO microsomes in vitro, the [14C]Poly-P-P products were converted to Poly-P by strong alkaline hydrolysis (70% methanol containing 3.6 M KOH, 2 h, 100°C). Following alkaline hydrolysis, the reactions were diluted with CHCl3 and H2O and partitioned, as described previously (Crick et al. 1991), and analyzed by TLC on Silica gel G TLC plates in CHCl3/CH3OH/H2O (75:25:4). The [14C]Poly-Ps and Poly-P standards were detected as described above.
Analysis of yeast cells and microsomal fractions for dolichol and Dol-P by NP-LC/MS methods
Normal phase LC-ESI/MS of lipids was performed using an Agilent 1200 Quaternary LC system coupled to a QSTAR XL quadrupole time-of-flight tandem mass spectrometer (Applied Biosystems, Foster City, CA). An Ascentis®
Si HPLC column (5 μm, 25 cm × 2.1 mm) was used. Mobile phase A consisted of chloroform/methanol/aqueous ammonium hydroxide (800:195:5, v/v/v). Mobile phase B consisted of chloroform/methanol/water/ aqueous ammonium hydroxide (60:34:5:0.5, v/v/v/v). Mobile phase C consisted of chloroform/methanol/water/aqueous ammonium hydroxide (45:45:9.5:0.5, v/v/v/v). The elution program consisted of the following: 100% mobile phase A was held isocratically for 2 min and then linearly increased to 100% mobile phase B over 14 min and held at 100% B for 11 min. The LC gradient was then changed to 100% mobile phase C over 3 min and held at 100% C for 3 min, and finally returned to 100% A over 0.5 min and held at 100% A for 5 min. The total LC flow rate was 300 μL/min. The postcolumn splitter diverted ~
10% of the LC flow to the ESI source of the Q-Star XL mass spectrometer, with MS settings as follows: IS = −
4500 V, CUR = 20 psi, GS1 = 20 psi, DP = −
55 V, and FP = −
150 V. Nitrogen was used as the collision gas for MS/MS experiments. Data acquisition and analysis were performed using the instrument’s Analyst QS software.
Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL) following precipitation of membrane proteins with deoxycholate and trichloroacetic acid according to the Pierce Biotechnology bulletin, “Eliminate interfering substances from samples for BCA protein assay.” Samples were analyzed for radioactivity by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation spectrometer following the addition of 0.5 mL 1% SDS and 4 mL Econosafe Economical Biodegradable Counting Cocktail (Research Products International, Corp., Mount Prospect, IL).