Figure 1.

P58 selectively associates with misfolded VSV-G in vivo. (A) Immunoblot of GFP-tagged VSV-G^ts045 recovered in complex with FLAG–P58 from lysates of FLAG–P58-expressing CHO cells that had been maintained at the permissive temperature (32°C) or the non-permissive temperature (41°C) for 12 h before harvest or switched back to the permissive temperature for the last hour before harvest (41–>32°C). Where indicated, the cells were exposed to the proteasome inhibitor MG132 for 3 h before harvest. The middle panel reports on the amount of VSV-G^ts045–GFP in the cell lysates (1.5% of the input) and the lower panel on the recovery of FLAG–P58 in the immunoprecipitate. (B) Autoradiograph of immunopurified VSV-G^ts045–GFP from transfected CHO cells metabolically labelled for 30 min at the permissive (32°C) or non-permissive temperature (41°C) in the absence or presence of brefeldin A (BFA). Where indicated, the immunopurified proteins were digested by an endoglycosidase that does not react with Golgi-modified N-linked saccharides (Endo H). The slower migrating Endo H-resistant (Endo H^R) and faster migrating Endo H-sensitive (Endo H^S) forms of VSV-G–GFP are indicated. (C) Autoradiograph of proteins immunopurified from CHO cells co-expressing FLAG–P58 and VSV-G^ts045–GFP. The cells were briefly radiolabelled (30 min) in the presence of BFA at the permissive (32°C) or non-permissive temperature (41°C) and where indicated, chased with unlabelled media for 30 min in the continued presence of BFA, before lysis and immunoprecipitation with anti-FLAG antibody or anti-GFP serum. (D) Same as (C) except that cells were pulse labelled in the presence of BFA at the non-permissive temperature (41°C) and a cold chase was performed for 1 h at the permissive (32°C, lane 4) or non-permissive temperature (41°C, lane 5) in the continued presence of BFA. The lysate of cells lacking FLAG–P58 (CHO parental, lanes 1 and 6) serves as a specificity control for the recovery of VSV-G^ts045–GFP in complex with FLAG–P58.