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. 1997 May 5;137(3):609–618. doi: 10.1083/jcb.137.3.609

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The membrane accumulation phenotype of the K12R API ts mutant is thermally reversible. (A) K12R API accumulates in the pellet fraction at nonpermissive temperature. Spheroplasts were labeled for 10 min at 38°C followed by nonradioactive chase. Aliquots were removed at the indicated chase times and separated into supernatant (sup) and pellet fractions. Samples were immunoprecipitated with antiserum to API, and the radiolabeled signals were quantitated. The percent radiolabeled precursor at a given chase point represents the ratio of precursor from each supernatant or pellet fraction to the total API combined in both fractions. (B) Protease accessibility of K12R API in the supernatant and pellet fractions. Spheroplasts were labeled for 5 min, chased for 30 min at 38°C, and separated into a supernatant and pellet fraction after differential osmotic lysis. The supernatant and pellet fractions were subjected to proteinase K and Triton X-100 as indicated and immunoprecipitated with antiserum to API (left). An aliquot of the recovered pellet fraction was also immunoprecipitated with antisera to the vacuolar marker CPY and the cytosolic marker PGK before protease treatment (right). The percent of marker proteins recovered in the pellet fraction was calculated as described for Fig. 3. (C) Accessibility of K12R API in the pellet fraction to cross-linking with Sulfo-NHS-biotin. Labeled spheroplasts were fractionated exactly as in B. The pellet fraction was cross-linked with Sulfo-NHS-biotin (Biotin-X) in the presence or absence of Triton X-100. API and CPY were recovered by immunoprecipitation followed by precipitation with avidin agarose beads. (D) Thermal reversibility of the K12R membrane-accumulation phenotype. The K12R API mutant was pulse-labeled for 10 min, chased for 30 min at 38°C, and then shifted to 30°C (top). Samples were removed at the indicated times during the shift period at 30°C and lysed with glass beads. The resulting cell extracts were immunoprecipitated with antiserum to API and resolved by SDS-PAGE. The double-headed arrow in the top panel marks the 20–40-min window of time when mature API increases from 10 to 56% during the 30°C shift. The K12R API strain was also pulse labeled, chased, and incubated all at 38°C (middle) or 30°C (bottom) in this experiment.

The membrane accumulation phenotype of the K12R API ts mutant is thermally reversible. (A) K12R API accumulates in the pellet fraction at nonpermissive temperature. Spheroplasts were labeled for 10 min at 38°C followed by nonradioactive chase. Aliquots were removed at the indicated chase times and separated into supernatant (sup) and pellet fractions. Samples were immunoprecipitated with antiserum to API, and the radiolabeled signals were quantitated. The percent radiolabeled precursor at a given chase point represents the ratio of precursor from each supernatant or pellet fraction to the total API combined in both fractions. (B) Protease accessibility of K12R API in the supernatant and pellet fractions. Spheroplasts were labeled for 5 min, chased for 30 min at 38°C, and separated into a supernatant and pellet fraction after differential osmotic lysis. The supernatant and pellet fractions were subjected to proteinase K and Triton X-100 as indicated and immunoprecipitated with antiserum to API (left). An aliquot of the recovered pellet fraction was also immunoprecipitated with antisera to the vacuolar marker CPY and the cytosolic marker PGK before protease treatment (right). The percent of marker proteins recovered in the pellet fraction was calculated as described for Fig. 3. (C) Accessibility of K12R API in the pellet fraction to cross-linking with Sulfo-NHS-biotin. Labeled spheroplasts were fractionated exactly as in B. The pellet fraction was cross-linked with Sulfo-NHS-biotin (Biotin-X) in the presence or absence of Triton X-100. API and CPY were recovered by immunoprecipitation followed by precipitation with avidin agarose beads. (D) Thermal reversibility of the K12R membrane-accumulation phenotype. The K12R API mutant was pulse-labeled for 10 min, chased for 30 min at 38°C, and then shifted to 30°C (top). Samples were removed at the indicated times during the shift period at 30°C and lysed with glass beads. The resulting cell extracts were immunoprecipitated with antiserum to API and resolved by SDS-PAGE. The double-headed arrow in the top panel marks the 20–40-min window of time when mature API increases from 10 to 56% during the 30°C shift. The K12R API strain was also pulse labeled, chased, and incubated all at 38°C (middle) or 30°C (bottom) in this experiment.