Transport of Newly Synthesized Sterol to the Sterol-Enriched Plasma Membrane Occurs via Nonvesicular Equilibration

Abstract

The mechanism by which newly synthesized sterols are transported from their site of synthesis, the endoplasmic reticulum (ER), to the sterol-enriched plasma membrane (PM) is not fully understood. Studies in mammalian cells suggest that newly synthesized cholesterol is transported to the PM in Golgi-bypassing vesicles and/or via a nonvesicular process. Using the yeast Saccharomyces cerevisiae as a model system, we now rule out an essential role for known vesicular transport pathways in transporting the major yeast sterol, ergosterol, from its site of synthesis to the PM. We use a cyclodextrin-based sterol capture assay to show that transport of newly synthesized ergosterol to the PM is unaltered in cells defective in Sec18p, a protein required for almost all intracellular vesicular trafficking events; we also show that transport is not blocked in cells that are defective in formation of transport vesicles at the ER or in vesicle fusion with the PM. Our data suggest instead that transport occurs by equilibration (t(1/2) approximately 10-15 min) of ER and PM ergosterol pools via a bidirectional, nonvesicular process that is saturated in wild-type exponentially growing yeast. To reconcile an equilibration process with the high ergosterol concentration of the PM relative to ER, we note that a large fraction of PM ergosterol is found condensed with sphingolipids in membrane rafts that coexist with free sterol. We propose that the concentration of free sterol is similar in the PM and ER and that only free (nonraft) sterol molecules have access to a nonvesicular transport pathway that connects the two organelles. This is the first description of biosynthetic sterol transport in yeast.

Nonvesicular Lipid Transfer from the Endoplasmic Reticulum

“…Both vesicular and nonvesicular lipid transport mediate intracellular lipid trafficking from the ER. Yet, inhibition of vesicular transport by pharmacological and genetic manipulations such as BFA or colchicine treatment, ATP depletion, and specific sec mutants (such as Sec18 mutant) has no effect on intracellular transport of certain lipid species (Kaplan and Simoni 1985b;Urbani and Simoni 1990;Heino et al 2000;Baumann et al 2005). Furthermore, imaging and biochemical studies using fluorescent lipid analogs, such as DHE (dehydroergosterol; a fluorescent cholesterol analog) and radioactive labels lipids, respectively, support the involvement of nonvesicular transport in intracellular lipid trafficking (Maxfield and Mondal 2006).…”

“…The transport of sterols from the ER is unaffected by drugs or genetic mutations that block vesicular transport, and is thought to be mediated mainly by spontaneous lipid transfer and by certain LTPs (Urbani and Simoni 1990;Heino et al 2000;Maxfield and Wustner 2002;Baumann et al 2005). Although sterols are highly hydrophobic lipids their spontaneous movement between certain membranes could be very rapid (a half-time of 1 min or less) (Dawidowicz 1987), and can thereby support substantial sterol transport between specific membranes.…”

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

“…Previous studies have identified the PM-associated ER as sites of phosphatidylinositol (PI) metabolism, nonvesicular transfer of sterols, and Ca 2þ level regulation (Li and Prinz 2004;Baumann et al 2005;Carrasco and Meyer 2011;Stefan et al 2011). EM analysis has revealed that 20% -45% of the cytoplasmic surface of the PM in budding yeast is within tethering distance of the ER and ribosome excluded (Fig.…”

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

“…Previous studies have shown that cholesterol and related sterols move by vesicular and non-vesicular pathways between the plasma membrane and intracellular compartments (Baumann et al 2005;Hao et al 2002;Kaplan and Simoni 1985;Li and Prinz 2004;Wüstner et al 2002). Employing the intrinsically fluorescent sterol dehydroergosterol (DHE), it was shown that the endocytic recycling compartment (ERC) is a major sterol storage compartment under basal conditions in non-polarized cells including macrophages Wüstner et al 2005).…”