Review
Mechanisms of sterol uptake and transport in yeast

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Abstract

Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in understanding how sterols are transported between different membranes. Baker's yeast is a particularly attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of the fungal ergosterol can be provided by structurally related sterols, including the mammalian cholesterol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells.

Article from a special issue on Steroids and Microorganisms.

Introduction

Sterols constitute an important class of lipids, which harbor multiple essential functions ranging from signal transduction to protein lipidation. The precise structure and concentration of sterols within eukaryotic membranes affect many membrane associated functions, including vesicle formation and protein sorting, endocytosis, homotypic membrane fusion, the activity of membrane embedded enzymes, the lateral aggregation between proteins and lipids, and the ion permeability of the membrane barrier [1], [2], [3], [4], [5]. Due to their concentration-dependent modulation of these functions, the distribution of sterols between different membranes needs to be tightly controlled. The lowest concentration of the free sterol is typically found in the endoplasmic reticulum (ER), where cholesterol levels are maintained at around 5 mol.% of total lipids [6]. The sterol concentration then appears to increase along the membranes of the secretory pathway to reach a maximum at the plasma membrane, which harbors 90% of the free sterol pool of the cell and where free sterols account for ∼30 mol.% of total lipids [7], [8], [9], [10], [11], [12].

Sterols are synthesized and mature in the ER by a cascade of coupled enzymatic reactions. The final product is cholesterol in case of animal cells, stigmasterol, sitosterol, and campesterol in case of plants and ergosterol in fungal cells [13]. While these mature sterols slightly differ in structure between the different kingdoms of life, their basic functions appear to be conserved. It is interesting to note that these kingdom-specific changes in sterol structure are frequently accompanied by corresponding structural changes in the major sphingolipids synthesized, indicating that complex formation between sterols and sphingolipids is one of the conserved properties of these lipids [14], [15], [16]. In addition to the free membrane embedded sterol, sterols are also converted to a storage form by esterification with long chain fatty acids. The resulting steryl esters are then stored in lipid droplets from where the free sterol is liberated by the action of specific lipases [17]. Balance between synthesis, transport, esterification of the free sterol and hydrolysis of steryl esters has to be tightly regulated to maintain sterol homeostasis [18]. To prevent the toxic accumulation of free sterols at its site of synthesis, the ER membrane, the free sterol has to be efficiently transported to the plasma membrane [19]. This transport pathway is still incompletely understood but involves both vesicular and non-vesicular components because it is ATP-dependent, but only partially sensitive to brefeldin A, which disrupts vesicular transport at the level of the Golgi apparatus [12], [20], [21]. The proposition that sterol transport between the ER and the plasma membrane involves both vesicular as well as non-vesicular components is also consistent with the observation that lipid transport continues in yeast mutants that have a conditional block of vesicular transport between the two compartments [22].

In this review, we will summarize the approaches that were taken and the progress made over the past couple of years to understand how sterols are transported between different intracellular membranes, how this lipid transport may be regulated, and how these membranes may establish their characteristic sterol concentration – focusing on studies using yeast as a genetically tractable model organism.

Section snippets

Uptake and export of sterols by mammalian cells

Mammalian cells take up cholesterol by receptor-mediated endocytosis of low-density lipoproteins (LDLs) containing cholesteryl esters [23]. LDLs are then delivered to late endosomes or lysosomes where cholesteryl esters are hydrolyzed by an acidic lipase. The liberated free cholesterol is recycled to the plasma membrane or transported to the ER where it is esterified and stored in lipid droplets. The transport of the free sterol from the plasma membrane and the endocytic compartment back to the

Sterol transport in yeast

Yeast is a powerful genetic model organism to study basic cellular processes that are conserved in eukaryotic cells [33]. Saccharomyces cerevisiae was adopted early on to study the structure to function relation of different sterols by Bloch and colleagues and subsequently to identify the genes that participate in sterol synthesis and in its regulation [13], [34], [35], [36]. As in mammalian cells, ergosterol, the mature sterol made by yeast, is synthesized by ER-localized enzymes and is then

Sterol uptake and transport back to the ER

Yeast cells do not take up free sterols from the environment if cultivated in the presence of oxygen, i.e., under aerobic conditions [64]. The molecular mechanisms underlying this aerobic sterol-exclusion are not well understood, but could be caused by properties of the cell wall that might prevent exogenous sterols from reaching the plasma membrane [65]. Because the synthesis of sterols requires molecular oxygen, S. cerevisiae becomes sterol auxotroph when grown under anaerobic conditions and

Sterol sensing and uptake by other fungal species

Unlike baker's yeast, which is a facultative anaerobe, Schizosaccharomyces pombe is an obligate aerobe that adapts to changes in oxygen availability by regulating sterol levels through the activation of hypoxic transcription factors. These are homologues of the human sterol regulatory element binding proteins (SREBP, Sre1) and Scap (SREBP cleavage-activating protein, Scp1) and sense the sterol content in the ER to coordinate the expression of sterol biosynthetic enzymes with oxygen levels [87],

Genetic screens for sterol uptake and transport mutants

To identify components important for sterol uptake and transport, a number of genetic screens were performed over the last couple of years. Taking advantage of the fact that sterol uptake and transport are essential for cell growth under anaerobic conditions, we screened the yeast deletion mutant collection for mutants that fail to grow under anaerobic conditions. This allowed us to identify 17 mutants, which affect sterol uptake and/or trafficking as assessed by genetic and biochemical

Sterol acetylation, a quality-control step that controls sterol efflux

Mammalian cells can reduce their intracellular cholesterol load by delivering free cholesterol for lipidation of ApoA-1 and the maturation of high density lipoprotein (HDL) particles. This reverse cholesterol transport pathway requires the ABC transporters ABCA1 and ABCG1 and defects in ABCA1 cause Tangier disease and the early onset of atherosclerosis [32]. No comparable pathway for the excretion of sterols has been described in yeast. Since yeast is a unicellular organism, it is questionable

Perspectives

The identification of this novel acetylation cycle indicates that sterols may be subject to others, as yet unidentified modifications that may confer novel functions to these lipids. One of these modifications includes the glycosylation of sterols, which is important for pexophagy in the methylotrophic yeast Pichia pastoris [121]. Clearly sterols have evolved with the eukaryotic cell and have taken over a surprising number of diverse cellular functions and new ones are likely to be uncovered in

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