Yeast phosphatidylinositol transfer protein (Sec14p) is required for the production of secretory vesicles from the Golgi. This requirement can be relieved by inactivation of the cytosine 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis, indicating that Sec14p is an essential component of a regulatory pathway linking phospholipid metabolism with vesicle trafficking (the Sec14p pathway). Sac1p (refs 7 and 8) is an integral membrane protein related to inositol-5-phosphatases such as synaptojanin, a protein found in rat brain. Here we show that defects in Sac1p also relieve the requirement for Sec14p by altering phospholipid metabolism so as to expand the pool of diacylglycerol (DAG) in the Golgi. Moreover, although short-chain DAG improves secretory function in strains with a temperature-sensitive Sec14p, expression of diacylglycerol kinase from Escherichia coli further impairs it. The essential function of Sec14p may therefore be to maintain a sufficient pool of DAG in the Golgi to support the production of secretory vesicles.

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi‐derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP‐choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP‐choline pathway‐mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand‐binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi‐derived transport vesicle biogenesis.

We generated highly immunogenic virus-like particles that are based on the capsid protein VP1 of the hamster polyomavirus (HaPV-VP1) and harbor inserted foreign epitopes. The HaPV-VP1 regions spanning amino acids 81-88 (position 1), 222/223 (2), 244-246 (3), and 289-294 (4) were predicted to be surface exposed. An epitope of the pre-S1 region of the hepatitis B virus (designated S1; amino acid sequence DPAFR) was introduced into the predicted positions of VP1. All VP1/S1 fusion proteins were expressed in yeast and generated virus-like particles. Immunoassays using the S1-specific monoclonal antibody MA18/7 and immunization of C57Bl6 mice with different VP1/S1 constructs showed a pronounced reactivity and a strong S1-specific antibody response for particles carrying the insert in position 1, 2, 1+2, and 1+3. Our results suggest that HaPV-VP1 represents a highly flexible carrier moiety for the insertion of foreign sequences offering a broad range of potential uses, especially in vaccine development.

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Objectives: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. Methods: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. Results: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. Conclusion: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.