Characterization of the submitochondrial compartments: study of the site of synthesis of dolichol and dolichol-linked sugars

Abstract

The fractionation of mitochondrial membranes on discontinuous sucrose gradient leads to the obtaining of free outer membranes, free inner membranes and two distinct membrane contact site populations characterized as follows. Only outer membrane contact sites and inner membrane contact sites bind hexokinase. Outer membranes and outer membrane contact sites are cholesterol-rich fractions. The endogenous dolichol content is twice fold higher in outer membranes and outer membrane contact sites than in inner membranes and inner membrane contact sites, only the biosynthesis of dolichol in inner membrane contact sites is not stimulated by addition of exogenous [14C]-IPP and FPP. The glycosylation of endogenous dolichol from labeled nucleotide-sugars (UDP-GlcNAc, GDP-Man and UDP-Glc) leads to the synthesis of dolichol-pyrophosphoryl-sugars and dolichol-monophosphoryl-sugars with the rate of synthesis proportional to the dolichol content of each submitochondrial fraction.