Oleaginous yeast Cryptococcus curvatus exhibits interplay between biosynthesis of intracellular sugars and lipids

Patterns of the biosynthesis ofmajor metabolites of the oleaginous yeast Cryptococcus curvatus NRRL Y-1511 were investigated during cultivation on sugar-based media. When lactose or sucrose was employed as substrate under nitrogen-limited conditions, the yeast strain accumulated high quantities of intra-cellular total sugars (ITS) at the beginning of fermentation (up to 68% w/w), with ITS values progressively decreasing to 20%, w/w, at the end of the fermentation. Decrease in ITS content and consumption of extracellular lactose led to a subsequent rise in lipid accumulation, reaching 29.8% in dry cell weight at 80 g/L of initial lactose concentration. Lactose was a more favorable substrate for lipid production than sucrose. In nitrogen-excess conditions, ITS were produced in significant quantities despite the continuous presence of nitrogen in the medium. Growth on lactose was not followed by secretion of extra-cellular b-galactosidase. High quantities of extra-cellular invertase were observed during growth on sucrose. The composition of ITS was highly influenced by the sugar used as substrate. Cellular lipids contained mainly palmitic and to lesser extent linoleic and stearic acids. This is the first report in the literature that demonstrates the interplay between the biosynthesis of intra-cellular total sugars and lipid synthesis for oleaginous yeast strains.

Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.