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. 1997 Jun 30;137(7):1469–1482. doi: 10.1083/jcb.137.7.1469

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Gap1p is transported from the Golgi to the vacuole in sec13-1 mutants. (A) Wild-type (CKY520), sec13-1 (CKY521), and sec13-1 pep4Δ (CKY467) strains were grown at 24°C to exponential phase. Cultures were labeled with [³⁵S]methionine and [³⁵S]cysteine for 10 min and chased by the addition of an excess of unlabeled methionine and cysteine. Gap1p-HA was immunoprecipitated from labeled extracts, resolved by SDS-PAGE, and was quantified using a phosphorimager. (B) Gap1p activity was assayed by the rate of [¹⁴C]citrulline uptake in wild-type (CKY443), sec13-1 (CKY444), sec13-1 pep12Δ::TRP1 (CKY455), sec13-1 end3-1 (CKY456), and sec13-1 pep4Δ::LEU2 (CKY457) strains. (C) Cell lysates from wild-type (CKY465), sec13-1 (CKY466), and sec13-1 pep12Δ::TRP1 (CKY468) strains were fractionated on density gradients of 20%–60% sucrose containing 10 mM EDTA. Fractions containing Pma1p were pooled, and Gap1p-HA in these fractions was detected by Western blotting with anti-HA antibody. The relative amount of plasma membrane in each extract was determined by quantitation of Pma1p. For A–C, all strains were grown in ammonia medium.

Gap1p is transported from the Golgi to the vacuole in sec13-1 mutants. (A) Wild-type (CKY520), sec13-1 (CKY521), and sec13-1 pep4Δ (CKY467) strains were grown at 24°C to exponential phase. Cultures were labeled with [³⁵S]methionine and [³⁵S]cysteine for 10 min and chased by the addition of an excess of unlabeled methionine and cysteine. Gap1p-HA was immunoprecipitated from labeled extracts, resolved by SDS-PAGE, and was quantified using a phosphorimager. (B) Gap1p activity was assayed by the rate of [¹⁴C]citrulline uptake in wild-type (CKY443), sec13-1 (CKY444), sec13-1 pep12Δ::TRP1 (CKY455), sec13-1 end3-1 (CKY456), and sec13-1 pep4Δ::LEU2 (CKY457) strains. (C) Cell lysates from wild-type (CKY465), sec13-1 (CKY466), and sec13-1 pep12Δ::TRP1 (CKY468) strains were fractionated on density gradients of 20%–60% sucrose containing 10 mM EDTA. Fractions containing Pma1p were pooled, and Gap1p-HA in these fractions was detected by Western blotting with anti-HA antibody. The relative amount of plasma membrane in each extract was determined by quantitation of Pma1p. For A–C, all strains were grown in ammonia medium.