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

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Vacuolar protein sorting in vam3^tsf mutant cells. TDY1 (vam3Δ) cells transformed with either complementing plasmid (pVAM3.414) or plasmid containing a temperature-sensitive for function (tsf) allele of vam3 (pVAM3-6.414) were converted to spheroplasts, and then incubated at either permissive (26°C) or nonpermissive (38°C) temperature for 5 min. Cultures were labeled with [³⁵S]cysteine/methionine for 10 min, and then chased for an additional 45 min at the indicated temperature. The cultures were separated into intracellular (I) and extracellular (E) fractions, and the vacuolar proteins CPY, PrA, CPS, and ALP were immunoprecipitated from each fraction, resolved by SDS-PAGE, and followed by autoradiography. CPS samples were treated with endoglycosidase H before electrophoresis. The positions of Golgi-modified precursor (p2, pro) and mature vacuolar (m) proteins are indicated.