Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe

Abstract

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/ Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31 + or erg32 + did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in ergD mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6D but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the ergD mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins. INTRODUCTIONSterols are essential structural and regulatory components of eukaryotic cell membranes. Mammals, plants and fungi produce similar sterols, which differ in the number and location of double bonds and methyl side chains. Ergosterol is the end product of the sterol biosynthetic pathway and is the major sterol in yeasts (Fig. 1a). Like cholesterol in mammalian cells, it is responsible for membrane fluidity and permeability (Parks et al., 1995).Ergosterol synthesis and metabolism have been well defined in the budding yeast Saccharomyces cerevisiae (Daum et al., 1998). Multiple genetic and biochemical studies have culminated in the virtually complete elucidation of the pathway leading to ergosterol (Lees et al., 1995;Parks et al., 1995). The enzymic reactions have been largely defined by identification of erg mutants defective in ergosterol biosynthesis, and by their complementation based on sterol auxotrophy or altered sterol composition. Most genes involved in the early part of the pathway to lanosterol are essential for growth, because yeasts require sterols and no sterol molecule is synthesized up to this point. In contrast, mutations in the steps from zymosterol to ergosterol (Fig. 1b) are not essential for growth because the intermediates produced can partially substitute for ergosterol. The latter part of the ergosterol biosynthetic pathway is not linear in the sense of consecutive reactions, because the enzymes converting lanosterol to ergosterol do not show a strict substrate preference. Thus, null mutants impaired in the steps from zymosterol to ergosterol accumulate char...

CUE Domain-Containing Protein Vps901 Is Required for Vacuolar Protein Transport inSchizosaccharomyces pombe

“…2D). 25) In contrast, Vps10-GFP (F 1425 , 1426 A) was not transported to the vacuolar membrane in the vps901Á cells, but was instead localized to dot structures (Fig. 2D).…”

“…In the vps5Á cells, degraded Vps10-GFP was detected. 25) In contrast, degraded Vps10-GFP was not detected in the vps901Á or WT cells by Western blotting (Fig. 2C).…”

“…In 2006, Vps10-GFP was constructed previously and is found in the Golgi in WT cells and in vacuolar membrane in cells lacking retromer complex proteins, Vps5p, Vps17p, Vps26p, Vps29p, and Vps35p. 25) Degraded Vps10-GFP has been detected in these vps mutants. 25) The Vps10-GFP fusion protein was transfected into WT, vps901Á and vps5Á cells.…”

“…25) Degraded Vps10-GFP has been detected in these vps mutants. 25) The Vps10-GFP fusion protein was transfected into WT, vps901Á and vps5Á cells. In the vps5Á cells, degraded Vps10-GFP was detected.…”

“…These results indicate that Vps901p is required for anterograde transport of Vps10p from the Golgi to the endosomes. 25) Next we investigated the transport of another vacuolar marker protein, S. pombe CPS, in vps901Á cells. We used ubiquitin-conjugated fusion protein Ub-GFPSpCPS, constructed previously, which is delivered from the Golgi apparatus to the vacuole lumen via an MVB sorting pathway in WT S. pombe cells.…”