FIGURE 7.

Unique regions of SFR2 are required for activity. A, thin layer chromatogram of lipids extracted from microsomes purified from yeast producing MGDG synthase (MGD1) alone or MGD1 and SFR2 constructs. B, thin layer chromatogram of lipid extracts of glycosyl transfer assays under optimal conditions with MGDG (substrate) after 1 h. Chromatograms in A and B are stained for sugars and locations of substrates and products (DGDG, TGDG, and TeGDG) are indicated. C, immunoblots of yeast microsomes used in A loaded with equal total protein and detected using a mixture of antisera specific to the N or C terminus of SFR2. D, immunoblots of equivalent protein levels of yeast microsomes digested or mock-digested with trypsin (Tryp.) before or after denaturation (denat.) with heat and detergent as indicated at top. Detection was with antisera recognizing the C terminus of SFR2. E, immunoblots of yeast expressing SFR2 or mutant constructs separated by blue native-PAGE detected using a mixture of antisera recognizing the N or C terminus of SFR2. White spaces separate lanes taken from distinct exposures of the same immunoblot. F, immunoblots of equal culture volumes of yeast producing SFR2 or 27N as indicated at left extracted with reagents indicated above before separation into soluble, S, and insoluble, P, fractions. Detection is by a mixture of antisera specific to the N or C terminus of SFR2 and representative of three repeats.