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. 1998 Sep 15;95(19):11181–11186. doi: 10.1073/pnas.95.19.11181

Figure 4.

Figure 4

PI-Sec14p vesiculates TGN membranes. (A) Golgi membranes (0.25 mg/ml) were incubated (60 min at 37°C or 20°C, as indicated) with varying concentrations of Sec14p charged with either PI, PC, or PG. Inset, Sucrose gradient profile of released VSV-G protein after incubation with PI-Sec14p (1 mg/ml). (B) PC- and PG-charged Sec14p prevent the vesiculation caused by PI-Sec14p (1 mg/ml) or F0–40AS (5 mg/ml). Vesicle generation reactions were carried out in the absence of nucleotides with either PI-Sec14p or F0–40AS and the indicated concentrations of PC-Sec14p. Inset, Incubations were carried out as in A with either PI-Sec14p (1 mg/ml) or F0–40AS (5 mg/ml) in the presence or absence of PG-Sec14p (3 mg/ml). (C) The addition of PC-containing liposomes to PI-Sec14p (1 mg/ml) or to F0–40AS (5 mg/ml) abolishes their capacity to vesiculate Golgi membranes, as expected from the replacement of PI in PITP by PC. Vesicle release assays were carried out with PI-Sec14p (1 mg/ml) or F0–40AS fraction (5 mg/ml) and the indicated concentrations of PC-containing liposomes. (D) F40–100AS abolishes the PI-Sec14p-mediated vesiculation of TGN membranes. Incubations for vesicle generation were carried out with PI-Sec14p (1 mg/ml) and various amounts of F40–100AS. (A–D) Each point represents the average (±SD) from three determinations using different Sec14p preparations.