Figure 3.
Split-Ub monitors the interaction between Sec63p and Sec62p in vivo. (A) Immunoblot analysis of cells expressing Sec63-Cub-Dha together with an empty plasmid (lane a) or together with Nub-, Nua-, or Nug-Sec62p (lanes b, c, and d, respectively) or Nub-, Nua-, or Nug-Bos1p (lanes e, f, and g, respectively). The nitrocellulose membrane was probed with the anti-ha antibody that recognizes the uncleaved Cub fusion and the cleaved Dha. (B) Growth assay of the interaction between Sec63p and Sec62p based on split-Ub and a short-lived Ura3p (RUra3p) as a reporter. Sec63CRUp-containing cells bearing either the UBR1 gene or a UBR1 deletion were transformed with an empty plasmid or Nub-, Nua-, or Nug-Sec62p. Cells were pregrown in selective media containing uracil. Cells (103 or 102) were spotted on selective plates lacking uracil and also lacking leucine and tryptophan to select for the presence of the Cub- and Nub-constructs.