Fig. 6. Yeast chm deletions show delayed processing and partial secretion of CPY and produce a Class E vps multi-lamellar structure. (A) Parental yeast (+; BY4741), derivatives generated by deletion of the indicated CHM open reading frames, and the structurally unrelated class E vps sorting mutant, vps4, were pulse-chase labeled as spheroplasts. Intracellular (I) and extracellular (E) fractions were immunoprecipitated with CPY monoclonal antibody (Molecular Probes), resolved by SDS-PAGE, and visualized by fluorography. Proenzyme (pro, 69 kDa) and mature (m, 61 kDa) forms of CPY are indicated. Note that all mutants missort CPY and that the extent of missorting is not increased with a double chm mutant (chm1 snf7). (B-D) Transmission electron micrographs of parental (B) and chm1 mutant cells (C,D). An example of a class E structure (C, arrow) is shown magnified (D). A vacuole in each cell is indicated (v). (E) The frequency (%) of cells with one or more of these multi-lamellar class E vps structures is tabulated for each chm mutant. n, number of cells scored.