Figure 7. Examination of the deglycosylating role of human Engase1p in Png1p-deficient yeast cells.

A. Obtention of the S. cerevisiae ams1Δ and ams1Δpng1Δ strains, deficient in the vacuolar mannosidase, Ams1p, and their transfection with an empty vector or a vector encoding Hist-tagged human Engase1p, is described in Materials and Methods. Cells were grown to mid log phase and fOS were extracted, purified and derivatised with 2-AP. Derivatised fOS (corresponding to 146, 92, 166 and 88 DU_{600 nm} for the ams1Δ, ams1Δ^hENGASE, ams1Δpng1Δ and ams1Δpng1Δ^hENGASE strains, respectively) were resolved by HPLC and detected using an on line fluorescence detector. Arrows indicate the elution times of fOS containing either a single (1) or two (2) residues of N-acetylglucosamine at their reducing termini. Insets. The ams1Δ (upper panel) and ams1Δpng1Δ (lower panel) strains transfected with the empty vector (− hEngase1p) or the vector encoding Hist-tagged human Engase1p (+ hEngase1p) were grown to mid log phase and cell extracts were probed with an anti Hist tag antibody after SDS PAGE by Western blot. Hist-tagged hEngase1p (*) has a molecular mass of 80 kDa. The migration position of 100 and 75 kDa markers are indicated to the left of the blots. B. After correction for the different quantities of starting material used for each yeast strain the areas under the HPLC peaks corresponding to Man₇GlcNAc₂ and Man₇GlcNAc (M₇GN_1-2) were summed, as were those for Man₈GlcNAc₂ and Man₈GlcNAc (M₈GN_1-2). C. Wild type cells were transfected with the vector encoding Hist-tagged human Engase1p, grown to mid log phase, converted into spheroplasts, and permeabilised as described in Materials and Methods. After centrifugation, cytosol and membrane fractions were assayed for Engase1p (ENG), cytosolic α-glucosidase (αGlc) and vacuolar carboxypeptidase Y (CPY) activities which have been expressed as a percentage of the sum of the cytosol and membrane values. This experiment was performed once.