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. 1997 Aug 11;138(3):517–529. doi: 10.1083/jcb.138.3.517

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vps33^tsfvam3^tsf double mutant cells display synthetic vacuolar protein sorting defects. (A) LBY317 (vps33Δ) cells harboring complementing VPS33 plasmid (pVPS33.415) or vps33^tsf plasmid (VPS33-8.415) were incubated at either 26°C or 38°C for 5 min, and then labeled with [³⁵S]cysteine/methionine for 10 min. Chase was initiated by the addition of nonradioactive cysteine and methionine and incubation was continued for 30 min. Cells were spheroplasted and separated into intracellular (I) and extracellular (E) fractions. CPY was immunoprecipitated from each fraction, resolved by SDS-PAGE, and viewed by autoradiography. (B) vam3^tsf (TDY7 + pVPS33.416 and pVAM3-6.414) and vps33^t ^sf (pTDY7 + pVAM3.414 and pVPS33-8.416) single mutant cells, and vam3^tsfvps33^tsf (TDY7 + pVAM3-6.414 and pVPS33-8.416) double mutant cells were incubated at 26°C for 5 min, labeled with [³⁵S]cysteine/methionine for 10 min, and chased for 30 min. CPY was recovered by immunoprecipitation, separated by SDS-PAGE, and followed by autoradiography. The positions of both Golgi-modified precursor (p2) and mature (m) CPY are indicated.