Figure 2.

Functional Complementation of the Yeast Dolichol (rer2Δ) Mutant by AtCPT7.

(A) The full-length (AtCPT7 FL) or three truncated (AtCPT7 Δ34N, -Δ7-67N, and -Δ76N) open reading frames of AtCPT7 were introduced into the yeast rer2Δ mutant, and serially diluted transformed cells were grown at the indicated temperatures. As positive and negative controls, the mutant was transformed with the native RER2 gene or the expression vector (EV) alone, respectively. Note that growth of the rer2Δ mutant is fully restored only when two of the truncated versions of AtCPT7 (-Δ34N and -Δ7-67N) or the native RER2 gene are expressed. The rer2Δ mutant strains containing AtCPT7 ∆7-67N and -∆76N were plated separately and are therefore presented as an individual group.

(B) Total polyisoprenoid content of the yeast strains indicated above was determined and a representative HPLC/UV chromatogram of this analysis is presented. Note the accumulation of dolichols (D-11 and D-12) in the rer2Δ mutant cells containing two of the truncated forms of AtCPT7 and dolichols composed of mainly D-15 and D-16 in cells containing the native RER2 gene.

(C) Restoration of N-glycosylation in the rer2Δ mutant by AtCPT7. Microsomal proteins from the various rer2Δ mutant strains above were resolved by SDS-PAGE and analyzed by immunoblotting using antibodies specific for CPY. The positions of mature CPY (mCPY) and its hypoglycosylated forms lacking between one and four (−1 to −4) N-linked oligosaccharide chains are indicated.