The transport assay measures transport of the VSV G-protein between the cis Golgi of the donor membranes and the medial Golgi of the acceptor membranes. This transfer requires cytosol and an ATP-regenerating system. The assay was almost exactly as described in Balch et al (1984)and Balch and Rothman (1985). The assay as described was used for studies of cell cycle-related control of transport
Stock solutions
lOx buffered salts (lOxB/S): 250mM HEPES-KOH (pH 7.0), 250mM KCl, 25mM MgOAc: either prepared as 2ml aliquots and frozen at -20°C for up to three months, or kept at 4°C for up to two months.
Buffered salts stocks: 500mM Hepes-KOH pH 7.0, IM KCl, lOOmM MgOAc; these were all kept at -20°C for up to a year.
ATP (Sigma): lOOmM in 250mM Hepes-KOH pH 7.0 and frozen at -20°C for up to a year; aliquots were not thawed above four times.
UTP (Sigma): lOOmM in water and frozen at -20°C for up to a year; aliquots were not thawed above four times.
Creatine phosphate (CP) (Boehringer Mannheim): 200mM in water and frozen at -20°C for up to a year; aliquots were not thawed above four times.
Creatine phosphokinase (CPK) (Boehringer Mannheim) was made up at 2mg/ml in 50% glycerol and kept at -20°C for up to a month.
DTT (Boehringer Mannheim): prepared for each use at l(X)mM in water.
Uridine diphosphate-N-acetyl-D-glucosamine [glucosamine-6-^H(N)-] (UDP-^H-GlcNAc): purchased from NEN at 5-25Ci/mmol and kept at -20°C.
Anti-VSV-G monoclonal was prepared by affinity isolation and stored indefinitely at -20°C. Once opened, the aliquots were stored at 4°C
Detergent stop buffer: 50mM Tris-HCl pH 7.5 (2M stock; room temperature), 250mM NaCl (2M stock; room temperature); ImM Na2EDTA pH 7.0 (SOOmM stock; room temperature)
1% Triton X-100, (20% w/v stock; 4°C);
1% Na cholate, (10% stock; -20°C): stored at room temperature
Washing buffer. 50mM Tris-HCl pH 7.5, (2M stock, room temperature): 250mM NaCl; 5mM Na2EDTA pH 7.0 (500mM stock, room temperature); 1% Triton X-1(X); stored at room
temperature
1 Desalting columns were prepared for cytosol. This was necessary to remove endogenous, unlabelled UDP-GlcNAc in the cytosol, which would otherwise compete with the labelled substance and reduce the transport signal below detectability. l(X)pl cytosol could be desalted over one column, and 2 0% more cytosol was desalted than was required, to allow for losses.
Each column consisted of a capless Eppendorf with a hole punched in the bottom with a syringe needle, packed with glasswool and filled with preswollen Bio-Gel P6 DG, and
supported in a Sarstedt 6ml polystyrene assay tube. The column was prepared during the
preparation of the assay by washing through 5ml of IxB/S with ImM DTT and ImM ATP. The column was centrifuged at 20(X) rpm for 2 minutes at 4°C in a Jouan benchtop
centrifuge. After the final spin, the collecting tube was changed and the newly-thawed
cytosol loaded. The cytosol collected in the subsequent spin was exchanged into assay buffer, and could remain on ice for a few minutes. There was normally 80-90% volume recovery from such a spin-column.
2 The assay mixture for ten assays was prepared as follows (such a mixture might be used for nine samples, since there were small dispensing losses). 50|il ^H-UDP-GlcNAc (in ethanolic solution) was dispensed into a borosilicate glass tube and dried under a stream of nitrogen. To this was added 50pl lOxB/S, 12.5p.l CP, 2.5|il CPK, 5pl ATP, 5pl UTP, 5pl DTT, lOpl pCoA and 260pl H^O. This was mixed by brief vortexing, and then 35pl mix was placed in borosilicate glass tubes (the transport assay cannot be performed in Eppendorf tubes). Note that this reaction mix volume permits of no additions; on occasions up to 5|il of other reagents was added to some assays. Then the volume of water in the reaction
premixture was reduced appropriately, and samples made up to 35|il with active agent or an appropriate control substance.
3 5|il desalted cytosol was added to the premixture (or sometimes less, according to the optimum performance established for each cytosol preparation). At this point, assays could be preincubated for some time, normally 20 minutes at 37°C, to allow effects upon the cytosol. At the end of any preincubation, the assays were returned to ice.
4 Membrane fractions were thawed and added to the assays. The volume of each added was normally 5|il, but could be less, particularly for non-salt-washed membranes. The membranes were thawed directly before addition to the assay, by holding in a 30°C or 37 °C waterbath for a few seconds, until almost completely thawed, and then transferring to ice. The thawed membranes were added to the transport assays with speed. The assays were mixed by gentle flicking, and never by vortexing.
5 For the transport incubation, non-salt-washed assays were covered and transferred directly to a 37°C waterbath and incubated for 60 minutes. Salt-washed assays were warmed gently by standing at room temperature for 10 minutes, then covered, transferred to a 30°C waterbath and incubated for 120 minutes. The lower temperature and room temperature warming was partly a concession to the reduced robustness of the membranes, and partly to avoid the increased background counts which apparently result from higher-temperature incubation.
6 1 0 minutes before the end of the incubation time, the immunoprecipitating complex was
prepared by mixing together anti-VSVG monoclonal and rabbit-anti-mouse IgG in appropriate ratio (in this work, normally 0.5|il anti-VSVG and 2pl rabbit anti-mouse per assay, with 10% more prepared than required, to account for dispensing losses). The
antibodies were incubated at 37°C for at least 2 minutes, and complex formation was shown by cloudiness of the suspension. Then 50pl of stop solution per assay was mixed with the antibodies by inversion, and the suspension added to the assays. The stopped assay was mixed by vortexing, and allowed to immunoprecipitate at 37°C for at least 45 minutes or at 4°C overnight (which resulted in slightly elevated recovery of counts).
7 The immunoprecipitates were collected as follows. A 50-100ml solution of 2.5% skim milk powder in wash buffer was made and assay filters were soaked for at least 5 minutes.
Meanwhile, the filter apparatus was set up with a mild vacuum line and a trap, and a heat lamp was positioned over a sheet of 3MM paper marked with the numbers of the (otherwise unmarked) filters. Wash buffer was placed in a beaker, and normally a multipipetter was used for washing.
For each assay, a filter circle was placed in the apparatus and washed with 3ml wash buffer, meanwhile 3ml more was added to the assay tube, and this was then poured over the drained filter. The tube was washed out with 3ml more buffer, then the filter was washed three times in 3ml wash buffer. The filter was then removed from the apparatus and placed on the 3MM paper. The filters were allowed to dry, and then loaded into 4ml scintillation vials, immersed in 4ml Beckman scintillation fluid, and counted.
duplicates. As a control for antibody specificity, a 'minus-donor' control was normally appended to each assay, being complete but for donor membranes, and treated exactly as the other samples; it was expected to show less than 5-10% of the positive counts of a
corresponding interphase assay. In some cases, particularly when comparing interphase and mitotic cytosol, counts were represented as a percentage of corresponding interphase counts.