FIGURE 1.

Proteinase K assay to determine the orientation of the N- and C terminus of Tgl3p. A, scheme of Tgl3p showing the patatin domain, the conserved lipase motif, and the acyltransferase motif. For detection of the N terminus and C terminus, an Myc tag and an HA tag were introduced by PCR. B, relative amounts of TG in tgl3Δ and a tgl3Δ strains carrying untagged TGL3, the double-tagged version of TGL3, and N-terminal HA or C-terminal HA-tagged TGL3 are shown. Cells grown to the stationary phase were analyzed. Results are the average from at least three independent experiments with S.D. values (error bars) as indicated. C and D, proteinase K protection assays were performed as described under “Experimental Procedures” with isolated LD from a strain carrying the N-terminal Myc-tagged and C-terminal HA-tagged variant of Tgl3p. Samples were taken at time points from 10 s to 20 min, and 10 μg of LD protein was loaded on each lane. Western blot analysis was performed with antibodies directed against the Myc and HA epitope, respectively. The sample −PK was taken before the addition of PK. The sample +Tx +PK was taken after solubilization of LD with 1% Triton X-100 for 20 min on ice. E and F, the effect of PK treatment on Erg6p and Ayr1p, two LD resident proteins, was investigated with the respective polyclonal antibodies over the time interval from 10 s to 20 min.