Specific Sterols Required for the Internalization Step of Endocytosis in Yeast

Abstract

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.

Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p Requirement

“…An ergosterol requirement for vacuole biogenesis had been previously shown (Munn et al, 1999;Kato and Wickner, 2001;Hongay et al, 2002). Several erg mutants have fragmented vacuoles and when purified are defective for homotypic fusion (Kato and Wickner, 2001).…”

“…However, we cannot rule out that accumulation of other sterol intermediates such as fecosterol or a reduction of zymosterol might be the driving force behind our observations. Indeed, specific sterols are necessary at multiple steps of endocytosis (Munn et al, 1999;Heese-Peck et al, 2002). These studies showed the rate of receptor-mediated internalization could be correlated to structural differences of ergosterol intermediates with mutants in B-ring desaturases most strongly delayed.…”

Defects in Structural Integrity of Ergosterol and the Cdc50p-Drs2p Putative Phospholipid Translocase Cause Accumulation of Endocytic Membranes, onto Which Actin Patches Are Assembled in Yeast

“…Total sterols were isolated as previously described (Munn et al, 1999). Cholesterol, as an internal control, was added to total isolated sterols, which were then dried under continuous nitrogen gas and stored at -20°C.…”

Pag1p, a Novel Protein Associated with Protein Kinase Cbk1p, Is Required for Cell Morphogenesis and Proliferation inSaccharomyces cerevisiae

“…Media was separated from the cells by centrifugation. Cells were resuspended in water and split in two; one half was assayed directly, representing the cell surface fraction, and the other half was lysed by freeze-thawing in the presence of 1% Triton X-100 (Munn et al, 1999). Chitinase activity was assayed essentially as described (Kuranda and Robbins, 1991).…”