Specific sterols required for the internalization step of endocytosis in yeast

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in t he internalization step of endocytosis in a gene (ERG2) encoding a C-8 ster ol isomerase that acts in the late part of the ergosterol biosynthetic path way. Ln the absence of Erg2p, yeast cells accumulate sterols structurally d ifferent from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in mo re detail, several erg mutants (erg2 Delta, erg6 Delta, and erg2 Delta erg6 Delta) were made. Analysis of fluid phase and receptor-mediated endocytosi s indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory path way were not strongly affected in the erg Delta mutants. The severity of th e endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the erg Delta mutants. Desaturation of th e B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalizati on at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or a t C-5,6 and C-8,9, allowed internalization.