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. 2000 Jul 25;97(16):8889–8891. doi: 10.1073/pnas.160269997

Figure 1.

Figure 1

Ypt7p is needed for the vacuole association of Vam7p after priming. (A) Vam7p is released from the vacuole upon extraction of Ypt7p. BJ3505 vacuoles (30 μg) were incubated for 60 min at 27°C in the presence or absence of Gdi1p and 0.5 mM Mg-ATP and an ATP regenerating system (13) where indicated and were reisolated (4 min, 8,000 × g, 4°C) as in Materials and Methods. The pooled supernatant of reaction and wash and the reisolated vacuoles were each mixed in sample buffer and were analyzed by SDS/PAGE and immunoblotting. Immunoblots were decorated with antibodies to Vam7p. (B) After priming, Ypt7p is required for Vam7p association with vacuoles. BJ3505 vacuoles (30 μg) were incubated for 60 min at 27°C. Gdi1p or antibodies to the indicated proteins were added to the reaction where indicated. Proteins from the pellet fraction were analyzed by SDS/PAGE and immunoblot with antibodies to Vam7p, Vti1p, and Ypt7p. (C) Vam7p release is not triggered by dilution. Vacuoles (30 μg) were incubated in 150 μl (lanes 1, 2, 5, and 6) or 1.5 ml (lanes 3, 4, 7, and 8) as in A, and the pellet and supernatant fractions were analyzed by immunoblot with antibodies to Vam7p.