Fig. 4

Complementation of rer2-deficient yeast with human DHDDS transcripts. a (left panel). Yeast strains in which the RER2 (rer2DR), ALG14 (alg14DR), ALG13 (alg13DR) and ALG7 (alg7DR) genes are under the control of the TET repressor were cultivated, alongside a parental strain, in the indicated concentrations of doxycycline and inhibition of growth with respect to that seen in the absence of doxycycline was calculated. (Right panel) Subsequent to cultivation of alg14DR and rer2DR cells in either the absence or presence of 1.0 and 0.3 μg/mL doxycycline, respectively, as described above, cell extracts were submitted to SDS-PAGE and Western Blot using an antibody directed to carboxypeptidase Y (CPY). The migration positions of different CPY glycoforms are indicated to the left of the image. b (upper panel) The rer2DR strain was transformed with wild type RER2, wild type DHDDS, as well as the DHDDS(W64X) and DHDDS(K42E) variants and the empty vector. Cell growth was measured (OD 600_nm) in duplicate transformants. (Lower panel) CPY glycosylation was examined in one of these duplicate cultures