Integration intermediates were generated by including SRP and column-washed rough microsomes (CRM) at respective final concentrations of 40nM and 8 equivalents (1 eq=50 A280 U /uL Walter and Blobel, 1983b) per 25uL translation. Hence, the length of a nascent polypeptide is dictated by the length of the truncated mRNA added to the translation, because the ribosome halts when it comes to the end of such a mRNA but does not dissociated because the truncated mRNA does not contain a stop codon. Furthermore, the nascent intermediate of defined length is formed using this experimental strategy.
Integration intermediates that are to be used in photocrosslinking studies, were generated by similar in vitro translation reactions as described above with a few modifications. The reaction volume were typically scaled up to 100uL per sample. The energy generating system that was added to these reactions contained all the amino acids except methionine and lysine, so that [35S]-methionine and the modified Lys could be incorporated into the polypeptides. The final concentration of [35S]-methionine was also increased to 2uCi/uL so that photoadducts could be detected on SDS gels. Eighteen pmol of the modified lysine (epsilonANB-Lys-tRNA) were also included in the reactions so that photoreactive probes were incorporated i the polypeptide chains. All translations were performed at 26°C for 50 min in the dark.
Photoreactions
The translations were halted by placing the samples on ice for 10 minutes prior to the exposure with ultraviolet radiation. In some experiments, puromycin was added to a final concentration of 2mM and incubated for 30 minutes at 26°C either prior to or after photolysis. The samples were normally photolyzed at 0°C for 15 minutes using a 500-W mercury arc lamp (Oriel) at a distance of 12 cm through a filter combination (Oriel 59855 and 66236) that provides a 300-400 nm bandpass.
After photolysis, the microsomal membranes were isolated by sedimentation through a sucrose cushion [0.5M sucrose/100mM KOAc (pH 7.5)/25mM Hepes (pH 7.5)/5mM Mg(OAc)2] at 20 psi for 2 minutes in an A-100 30°C rotor (Beckman Instruments). In some samples, the ribosomes were stripped off the membranes by incubation (4°C, 30 minutes) with 0.5M KOAc (pH 7.5)/25mM EDTA prior to pelting through a sucrose cushion. The microsomal pellets were usually processed further either by alkaline extraction, lectin binding or by immunoprecipitation
Alkaline Extraction
The standard operational criterium to determine whether a protein is integrated into microsomal membranes is to assess its solubility in alkaline buffers (Fujiki et al., 1982). Hence, a protein is considered inserted into the bilayer if the protein is insoluble in alkaline buffers.
Samples that were to be analyzed by alkaline extractions were subjected to alkaline conditions either prior to to after photolysis. Typically, Na2CO3 (pH 11.5) was added to each sample so that a final concentration of 100mM was obtained and then incubated on ice for 10 minutes. The samples were sedimented through a cushion containing 0.25M sucrose/100mM Na2CO3 at 30 psi for 10 minutes in a Beckman airfuge. Both the supernatant and membrane pellet fractions were often analyzed directly by SDS-PAGE or processed further by lectin binding or by immunoprecipitation.
Lectin Binding
To determine whether a crosslinked product was glycoprotein, the sample was analyzed for binding to concanavalin A. The membranes of a photocrosslinking experiment were isolated by sedimentation through a sucrose cushion as described above, and the pellet fractions were solubilized by boiling the samples in 40uL of 1% (w/v) SDS, 30mM DTT, 100mM Tris-HCl (pH 7.5) for 10 minutes. The samples were then diluted to 400uL with buffer C [70mM Tris (pH 7.5), 5mM CaCl2, 1mM MgCl2, 1.2% (v/v) Triton X-100]. Forty uL of 2X diluted Concanavalin A-Sepharose (Sigma) in buffer C containing 15% (w/v) bovine serum albumin (BSA) were added to individual samples and incubated overnight at 4°C with rocking. On the following day, the samples were spun in a microfuge for 10 seconds and the "supernatant" containing the unbound materials was precipitated with an equal volume of cold 25% (w/v) TCA. The precipitates were washed with acidic acetone and dried under vacuum as described earlier. The "resin" fraction containing the bound materials were washed three times with 700uL of buffer C and vortexed between each wash. Both fractions were analyzed by SDS-PAGE electrophoresis, and the dried gels were visualized using a phosphorimager.
Immunoprecipitation
Prior to immunoprecipitation, the membranes were isolated by sedimentation as described earlier. For immunoprecipitation using antibodies specific for Sec61 alpha (a generous gift of Dr. S. High), each membrane pellet was solubilized in 50uL of 0.25% (w/v) SDS/100mM Tris-HCl (pH 7.5) by heating at 50°C for 30 minutes. The samples were thoroughly mixed by pipeting, transferred to new 1.5mL microfuge tubes, and then diluted to 500uL with buffer D [140mM NaCl/10mM Tris HCl )pH 7.5)/1mM EDTA /1.5% (v/v) Triton X-100]. Individual samples were precleared by adding 40uL of protein A-sepharose (Sigma) and rocked at room temperature for 1 hour. The samples were then spun down in a microcentrifuge and individual supernatants were transferred to a new microfuge tubes. Four uL of Sec61alpha antisera were added to each sample and rocked at 4°C overnight. On the next day, 30uL of 2X-diluted Protein A-Sepharose in buffer D that contained 15% (w/v) BSA were added to each sample and the incubation was allowed to continue for another 2 hours at 4°C with rocking. The immunoprecipitated materials were recovered by sedimentation, and washed three times with buffer D with vortexing between each wash. Immunoprecipitated materials were separated by SDS-PAGE, and the dried gels were visualized using a Bio-Rad GS-250 phosophorimager.
For immunoprecipitations of TRAM using affinity-purified rabbit antibodies raised against its residue C-terminal peptide (alphaTRAM; a generous gift of Drs. Kent Matlock and Peter Walter), each microsomal membrane pellet was solubilized with 50uL of 1% (w/v) SDS/100mM Tris HCl (pH 7.5) after incubation at 50°C for 30 minutes. Each sample was diluted to 500uL with buffer E [150mM NaCl/50mM Tris-HCl (pH 7.5)/0.2% (w/v) SDS/1% (v/v) Triton-X-100], and gently rocked overnight at 4°C after the addition of 3 uL of alphaTRAM and 30 uL of 2X-diluted Protein A-Sepharose in buffer E that contained 15% (w/v) BSA. On the following day, the samples were recovered by sedimentation and washed three times with buffer E. Immunoprecipitated material was separated by SDS-PAGE, and the radioactivity in dried gels was detected using a phosphorimager.
