The ultracytochemical localization of X-prolyl-dipeptidyl (amino)peptidase (DPP) activity was studied in a late exponential culture of a haploid (alpha) wild-type strain of Saccharomyces cerevisiae and its pep4-3 mutant. Yeast cells were fixed for 20 min in cold 1% glutaraldehyde buffered with 50 mM TES buffer to pH 7.0 and then incubated for 80 min with 1.2 mM L-alanyl-L-proline-4-methoxy-2-naphthylamide (Ala-Pro-MNA) or Lys-Pro-MNA as cytochemical substrates plus 0.06% hexazonium p-rosaniline (HPR) buffered with 160 mM cacodylate to pH 7.0. The osmiophilic azoindoxyl complex was formed by coupling HPR with MNA liberated by DPP activity and was then osmicated during an overnight post-fixation of cells in cold 1% OsO4. In the wild-type strain, conspicuous deposits of DPP reaction product were observed in vacuolar membranes. When compared with the parent strain, the pep4-3 mutant cells were enriched in endoplasmic reticulum (ER), cytoplasmic lipoprotein, and microcompartments: membranous vesicles and microglobules. In the mutant, DPP reaction product was found in about 50% of non-vacuolated cells at the following sites: the nuclear envelope, polar layers of ER sheets and of membranous vesicles (diameter, 40-90 nm), the surface or the lumen of these vesicles, the cytoplasmic membrane (under some bud scars) and the periplasmic space. The largest amount of reaction product was found in microglobules (diameter, 20-50 nm) that were mainly observed in the cytoplasmic matrix but were also present in nuclei (nucleoli) and mitochondria. These microglobules had a single-line boundary and appeared to be composed of lipoprotein. The surface ultrastructure of sectioned microglobules in the cytoplasmic matrix was similar to that of the coated vesicles found in mammalian cells. Only sparse amounts of DPP reaction product were seen in budding yeast. In all pep4-3 cells with electron-lucent vacuoles, the reaction product was confined to the vacuolar membranes (i.e. homologous to the ER), microglobules and the periplasmic space. Polysaccharides with free vic-groups were shown by the cytochemical reaction to be present on the surface of ER membranes, in microglobules, in the periplasmic space and in the cell wall. Our cytochemical results indicate that microglobules participate in the exocytosis of both DPP and glycoproteins, and reveal new features of vacuolar morphogenesis in yeast.

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Vorísek J

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