JCS049460 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Schematic representation of the recombinant plasmids used in this study. pGEM4-b5-ops28 (Brambillasca et al., 2006) contains under the SP6 promoter the sequence coding for: cyt b5 cytosolic, catalytic domain (dark grey), its TMD (black) and a 28 residue lumenal domain (light grey), consisting of the seven C-terminal residues of cytb5 plus the opsin tag. The latter contains the N-glycosylation consensus sequence (asterisk) that is used after translocation into MR. The total length of the translocated C-terminal domain is of 28 residues. pGEM4-b5-Syb2-ops28 (Brambillasca et al., 2006) codes for a chimera, in which the TMD of pGEM4-b5-ops28 is replaced with the one of Syb2 (striped box). pGEX-GST-b5-ops28 and pGEX-GST-b5-ops85 contain, under the IPTG inducible promoter Ptac, the coding sequence for GST, followed by a thrombin cleavage sequence (white box), and the sequence coding for either b5-ops28 (the same as in pGEM4-b5-ops28) or b5-ops85, a construct in which the C-terminal domain, that contains two copies of the entire N-terminal luminal sequence of bovine opsin, has been extended to 85 residues (Brambillasca et al., 2006). pGEX-GST-b5 (Ceppi et al., 2005) and pGEX-GST-b5-ΔTMD code for GST-fusion proteins containing the unmodified rabbit b5 or a truncated version thereof, without the TMD (residues 1-104).

• Supplemental Figure S2 -

  Fig. S2. Estimation of the protein to lipid ratio in PC liposomes after transmembrane insertion of b5-ops28 or b5-ops85. (A) Quantification and analysis of purity of b5-ops28 and b5-ops85 produced in bacteria and cleaved with thrombin in PBS. Example of a Coomassie-blue-stained gel containing samples of known amounts of BSA (3.2 µg, 1.6 µg, 800 ng, 400 ng, 200 ng; in lanes 1 to 5), 10 µl (lanes 6 and 8) and 5 µl (lane 7 and 9) of each of the indicated purified proteins. Faint bands of low molecular mass are degradation products of the two proteins, as demonstrated by positivity for anti-b5 antibodies (not shown). The very faint bands indicated by the asterisks are due to GST (28 kDa) and undigested fusion protein (43 kDa). (B) Comparison of translocation efficiency estimated by the percent glycosylation or by comparison of band intensities of intact protein and PF. In vitro translated b5-ops28 was incubated in the presence or absence of rat liver RM or liposomes, as indicated. After translocation, 1 µl of each sample was immunoprecipitated, whereas the remaining 9 µl were subjected to PK digestion before immunoprecipitation (+PK samples). After western blotting, the membrane was exposed to a phosphorimager screen and band intensities were quantified. Examination of the intact protein shows a glycosylation efficiency of 47.5% (g-b5). This sets the intensity of the glycosylated PF (g-PF) as a standard corresponding to 47.5% translocation, from which the amount of translocated, nonglycosylated protein into RM is calculated to be 11.2%, for a total of 59% translocated, as indicated under lane 4. The corresponding value for translocation into liposomes was 46% in this experiment, confirming the similar efficiency of translocation of b5 into liposomes and RM reported in our previous work (Brambillasca et al., 2005; Brambillasca et al., 2006). The percentage of translocation was also calculated from the ratio between the radioactivity (normalized for loading) associated with the PFs and with the intact protein, considering their differing methionine content (3 and 5 residues, respectively). This procedure led to a calculated 41% translocation, corresponding to an insertion efficiency underestimated by a factor of 0.7, when compared with the value based on glycosylation. This factor was used to correct the percentage insertion into liposomes estimated for the non-radioactive bacterially expressed protein by immunoblotting. (C) Analysis of bacterially expressed b5-ops28 and b5-ops85 translocation. 22.5 pmol b5-ops28 or b5-ops85 produced in bacteria and eluted in PBS were incubated with PC liposomes (12 nmol PC), in parallel with the in vitro translated b5-ops28 incubation with RM (described in B). Samples were treated as described for B, and bands were revealed by immunoblotting with anti-opsin mAb. Note that, although equal molar amounts of the two proteins were loaded, the intensity of the b5-ops85 signal is stronger than that of b5-ops28, because in the former construct the opsin epitope is duplicated.

• Supplemental Figure S3 -

  Fig. S3. DIA inhibits the translocation of another TA protein, PTP-1B, that follows the unassisted pathway of insertion. In vitro translated PTP1B-ops-35 (Brambillasca et al., 2006) was incubated with or without 5mM DIA for 30 minutes at 32°C as indicated. 20 mM GSH was added either after (GSH-post) or during (GSH-co) the incubation with DIA. After translocation into PC liposomes the samples were tested for insertion by protease protection.

• Supplemental Figure S4 -

  Fig. S4. Characterization of inhibitory effects of NEM and DIA on b5-ops28 transmembrane integration. (A) The effect of NEM is not reversed by addition of fresh lysate. After incubation with NEM and quenching with DTT, or incubation with NEM and DTT together, as indicated, the samples were diluted in two volumes of fresh RRL (final lysate protein concentration 90 mg/ml, lanes 1-6) or in TB (lanes 7-12). Samples were then subjected to translocation into liposomes and to the protease protection assay. For each treatment, samples with no liposomes were run to reveal the presence of protease-resistant material (asterisks) generated by the NEM treatment. The relative percentage of translocation efficiency shown below is normalized to the untreated sample (lanes 2, 8). (B) The decreased insertion of b5-ops28 into liposomes caused by NEM and DIA is not due to its inclusion in large aggregates. After incubation with NEM or DIA as described in the legend to Figure 5, samples of 40∼l of in vitro translated b5-ops28 were ultracentrifuged for 1 hour at 60,000 r.p.m. in a TLA100.3 rotor (Beckman Instruments). After resuspension of the pellets in 40∼l of TB, equal volumes of pellet (P) and supernatant (S) were loaded onto SDS-PA gels.

• Supplemental Figure S5 -

  Fig. S5. Pull-down assays from RRL in the presence or absence of DIA. Incubation of RRL with GSH-Sepharose in the presence of 5 mM DIA (lanes 6-10) was carried out as described in the Materials and Methods and in the legend to Fig. 4. Total (T), unbound (U) and bound (B) proteins were analyzed by western blotting with anti-TRC40 antibodies. Lanes 1-5 show the same gel as in Fig. 4 (pull down in the absence of DIA) for comparison. In the presence of DIA, the amount of bound TRC40 is slightly increased.