Fig. 1.
Cysteine engineering to assess selective H transcomplementation on a homodimer/heterotetramer level. (A) Schematic of the MeV H tetramer; the locations of independent complementation groups and naturally present disulfide bonds are indicated. (B) Illustration of the cysteine-engineering strategy. (C) Coimmunoprecipitation of epitope-tagged surface-exposed H dimers confirms that only H homodimers with paired thiol moieties reach the cell surface. Surface proteins were biotinylated, and total cell lysates were subjected to α-Flag immunoprecipitation (first IP), followed by reprecipitation (re-P) of the plasma membrane fraction with immobilized streptavidin. A previously described H variant carrying an endoplasmic reticulum-retention signal (H-ERFlag (15)] served as specificity control for the streptavidin reprecipitation. Immunoblots were developed with α-Flag or α-HA antibodies, respectively.