Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)‐anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide‐containing, mild base‐resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI‐anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI‐anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI‐anchored proteins.

We have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)-gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2-mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditions.

SummaryThe function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c 1 (apo c 1 ) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apo c 1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by twodimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative č ě š Š š sˇČ eˇ oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.

Glycosylphosphatidylinositol (GPI)-anchoring represents a mechanism for attaching proteins to the cell surface that is used among all eukaryotes. A common core structure, EthN-P-Man 3 -GlcN-PI, is synthesized by sequential transfer of sugars and ethanolamine-P to PI and is highly conserved between organisms. We have screened for natural compounds that inhibit GPIanchoring in yeast and have identified a terpenoid lactone, YW3548, that specifically blocks the addition of the third mannose to the intermediate structure Man 2 -GlcN-acylPI. Consistent with the block in GPI synthesis, YW3548 prevents the incorporation of [ 3 H]myo-inositol into proteins, transport of GPIanchored proteins to the Golgi and is toxic. The compound inhibits the same step of GPI synthesis in mammalian cells, but has no significant activity in protozoa. These results suggest that despite the conserved core structure, the GPI biosynthetic machinery may be different enough between mammalian and protozoa to represent a target for anti-protozoan chemotherapy.

Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle.