Increased Phospholipid Flux Bypasses Overlapping Essential Requirements for the Yeast Sac1p Phosphoinositide Phosphatase and ER-PM Membrane Contact Sites.

In budding yeast cells, much of the inner surface of the plasma membrane (PM) is covered with the endoplasmic reticulum (ER). This association is mediated by seven ER membrane proteins that confer cortical ER-PM association at membrane contact sites (MCSs). Several of these membrane "tether" proteins are known to physically interact with the phosphoinositide phosphatase Sac1p. However, it is unclear how or if these interactions are necessary for their interdependent functions. We find that SAC1 inactivation in cells lacking the homologous synaptojanin-like genes INP52 and INP53 results in a significant increase in cortical ER-PM MCSs. We show in sac1Δ, sac1tsinp52Δ inp53Δ, or Δ-super-tether (Δ-s-tether) cells lacking all seven ER-PM tethering genes that phospholipid biosynthesis is disrupted and phosphoinositide distribution is altered. Furthermore, SAC1 deletion in Δ-s-tether cells results in lethality, indicating a functional overlap between SAC1 and ER-PM tethering genes. Transcriptomic profiling indicates that SAC1 inactivation in either Δ-s-tether or inp52Δ inp53Δ cells induces an ER membrane stress response and elicits phosphoinositide-dependent changes in expression of autophagy genes. In addition, by isolating high-copy suppressors that rescue sac1Δ Δ-s-tether lethality, we find that key phospholipid biosynthesis genes bypass the overlapping function of SAC1 and ER-PM tethers and that overexpression of the phosphatidylserine/phosphatidylinositol-4-phosphate transfer protein Osh6 also provides limited suppression. Combined with lipidomic analysis and determinations of intracellular phospholipid distributions, these results suggest that Sac1p and ER phospholipid flux controls lipid distribution to drive Osh6p-dependent phosphatidylserine/phosphatidylinositol-4-phosphate counter-exchange at ER-PM MCSs.

ER-PM membrane contact site regulation by yeast ORPs and membrane stress pathways.

In yeast, at least seven proteins (Ice2p, Ist2p, Scs2/22p, Tcb1-Tcb3p) affect cortical endoplasmic reticulum (ER) tethering and contact with the plasma membrane (PM). In Δ-super-tether (Δ-s-tether) cells that lack these tethers, cortical ER-PM association is all but gone. Yeast OSBP homologue (Osh) proteins are also implicated in membrane contact site (MCS) assembly, perhaps as subunits for multicomponent tethers, though their function at MCSs involves intermembrane lipid transfer. Paradoxically, when analyzed by fluorescence and electron microscopy, the elimination of the OSH gene family does not reduce cortical ER-PM association but dramatically increases it. In response to the inactivation of all Osh proteins, the yeast E-Syt (extended-synaptotagmin) homologue Tcb3p is post-transcriptionally upregulated thereby generating additional Tcb3p-dependent ER-PM MCSs for recruiting more cortical ER to the PM. Although the elimination of OSH genes and the deletion of ER-PM tether genes have divergent effects on cortical ER-PM association, both elicit the Environmental Stress Response (ESR). Through comparisons of transcriptomic profiles of cells lacking OSH genes or ER-PM tethers, changes in ESR expression are partially manifested through the induction of the HOG (high-osmolarity glycerol) PM stress pathway or the ER-specific UPR (unfolded protein response) pathway, respectively. Defects in either UPR or HOG pathways also increase ER-PM MCSs, and expression of extra "artificial ER-PM membrane staples" rescues growth of UPR mutants challenged with lethal ER stress. Transcriptome analysis of OSH and Δ-s-tether mutants also revealed dysregulation of inositol-dependent phospholipid gene expression, and the combined lethality of osh4Δ and Δ-s-tether mutations is suppressed by overexpression of the phosphatidic acid biosynthetic gene, DGK1. These findings establish that the Tcb3p tether is induced by ER and PM stresses and ER-PM MCSs augment responses to membrane stresses, which are integrated through the broader ESR pathway.

Sticking With It: ER-PM Membrane Contact Sites as a Coordinating Nexus for Regulating Lipids and Proteins at the Cell Cortex.

Membrane contact sites between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM) provide a direct conduit for small molecule transfer and signaling between the two largest membranes of the cell. Contact is established through ER integral membrane proteins that physically tether the two membranes together, though the general mechanism is remarkably non-specific given the diversity of different tethering proteins. Primary tethers including VAMP-associated proteins (VAPs), Anoctamin/TMEM16/Ist2p homologs, and extended synaptotagmins (E-Syts), are largely conserved in most eukaryotes and are both necessary and sufficient for establishing ER-PM association. In addition, other species-specific ER-PM tether proteins impart unique functional attributes to both membranes at the cell cortex. This review distils recent functional and structural findings about conserved and species-specific tethers that form ER-PM contact sites, with an emphasis on their roles in the coordinate regulation of lipid metabolism, cellular structure, and responses to membrane stress.

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization.

Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott-Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange.

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM-ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM-ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions.

Arv1 regulates PM and ER membrane structure and homeostasis but is dispensable for intracellular sterol transport.

The pan-eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild-type and Arv1-deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport-coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild-type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C-terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail-anchored proteins involved in membrane homoeostasis.

Vesicle trafficking from a lipid perspective: Lipid regulation of exocytosis in Saccharomyces cerevisiae.

The protein cargo transported by specific types of vesicles largely defines the different secretory trafficking pathways operating within cells. However, mole per mole the most abundant cargo contained within transport vesicles is not protein, but lipid. Taking a "lipid-centric" point-of-view, we examine the importance of lipid signaling, membrane lipid organization and lipid metabolism for vesicle transport during exocytosis in budding yeast. In fact, the essential requirement for some exocytosis regulatory proteins can be bypassed by making simple manipulations of the lipids involved. During polarized exocytosis the sequential steps required to generate post-Golgi vesicles and target them to the plasma membrane (PM) involves the interplay of several types of lipids that are coordinately linked through PI4P metabolism and signaling. In turn, PI4P levels are regulated by PI4P kinases, the Sac1p PI4P phosphatase and the yeast Osh proteins, which are homologs of mammalian oxysterol-binding protein (OSBP). Together these regulators integrate the transitional steps required for vesicle maturation directly through changes in lipid composition and organization.

A detour for yeast oxysterol binding proteins.

Oxysterol binding protein-related proteins, including the yeast proteins encoded by the OSH gene family (OSH1-OSH7), are implicated in the non-vesicular transfer of sterols between intracellular membranes and the plasma membrane. In light of recent studies, we revisited the proposal that Osh proteins are sterol transfer proteins and present new models consistent with known Osh protein functions. These models focus on the role of Osh proteins as sterol-dependent regulators of phosphoinositide and sphingolipid pathways. In contrast to their posited role as non-vesicular sterol transfer proteins, we propose that Osh proteins coordinate lipid signaling and membrane reorganization with the assembly of tethering complexes to promote molecular exchanges at membrane contact sites.

The sterol-binding protein Kes1/Osh4p is a regulator of polarized exocytosis.

Oxysterol-binding protein (OSBP)-related protein Kes1/ Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed complexes with the small GTPases Cdc42p, Rho1p and Sec4p, and the exocyst complex subunit Sec6p, which was also required for Osh4p association with vesicles. Although Osh4p directly affected polarized exocytosis, its role in sterol trafficking was less clear. Contrary to what is predicted for a sterol-transfer protein, inhibition of sterol binding by the Osh4p Y97F mutation did not cause its inactivation. Rather, OSH4(Y97F) is a gain-of-function mutation that causes dominant lethality. We propose that in response to sterol binding and release Osh4p promotes efficient exocytosis through the co-ordinate regulation of Sac1p, a phosphoinositide 4-phosphate (PI4P) phosphatase, and the exocyst complex. These results support a model in which Osh4p acts as a sterol-dependent regulator of polarized vesicle transport, as opposed to being a sterol-transfer protein.

Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM.

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol-binding protein (Osh1-7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1(ts)) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well as by extracting sterols from intact cells with methyl-ß-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-ß-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non-vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.

Yeast oxysterol-binding proteins: sterol transporters or regulators of cell polarization?

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) are a conserved family of soluble cytoplasmic proteins that can bind sterols, translocate between membrane compartments, and affect sterol trafficking. These properties make ORPs attractive candidates for lipid transfer proteins (LTPs) that directly mediate nonvesicular sterol transfer to the plasma membrane. To test whether yeast ORPs (the Osh proteins) are sterol LTPs, we studied endoplasmic reticulum (ER)-to-plasma membrane (PM) sterol transport in OSH deletion mutants lacking one, several, or all Osh proteins. In conditional OSH mutants, ER-PM ergosterol transport slowed approximately 20-fold compared with cells expressing a full complement of Osh proteins. Although this initial finding suggested that Osh proteins act as sterol LTPs, the situation is far more complex. Osh proteins have established roles in Rho small GTPase signaling. Osh proteins reinforce cell polarization and they specifically affect the localization of proteins involved in polarized cell growth such as septins, and the GTPases Cdc42p, Rho1p, and Sec4p. In addition, Osh proteins are required for a specific pathway of polarized secretion to sites of membrane growth, suggesting that this is how Osh proteins affect Cdc42p- and Rho1p-dependent polarization. Our findings suggest that Osh proteins integrate sterol trafficking and sterol-dependent cell signaling with the control of cell polarization.

Genome-wide analysis of sterol-lipid storage and trafficking in Saccharomyces cerevisiae.

The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (Saccharomyces cerevisiae) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis.

Homologues of oxysterol-binding proteins affect Cdc42p- and Rho1p-mediated cell polarization in Saccharomyces cerevisiae.

Polarized cell growth requires the establishment of an axis of growth along which secretion can be targeted to a specific site on the cell cortex. How polarity establishment and secretion are choreographed is not fully understood, though Rho GTPase- and Rab GTPase-mediated signaling is required. Superimposed on this regulation are the functions of specific lipids and their cognate binding proteins. In a screen for Saccharomyces cerevisiae genes that interact with Rho family CDC42 to promote polarity establishment, we identified KES1/OSH4, which encodes a homologue of mammalian oxysterol-binding protein (OSBP). Other yeast OSH genes (OSBP homologues) had comparable genetic interactions with CDC42, implicating OSH genes in the regulation of CDC42-dependent polarity establishment. We found that the OSH gene family (OSH1-OSH7) promotes cell polarization by maintaining the proper localization of septins, the Rho GTPases Cdc42p and Rho1p, and the Rab GTPase Sec4p. Disruption of all OSH gene function caused specific defects in polarized exocytosis, indicating that the Osh proteins are collectively required for a secretory pathway implicated in the maintenance of polarized growth.

PhLP3 modulates CCT-mediated actin and tubulin folding via ternary complexes with substrates.

Many ATP-dependent molecular chaperones, including Hsp70, Hsp90, and the chaperonins GroEL/Hsp60, require cofactor proteins to regulate their ATPase activities and thus folding functions in vivo. One conspicuous exception has been the eukaryotic chaperonin CCT, for which no regulator of its ATPase activity, other than non-native substrate proteins, is known. We identify the evolutionarily conserved PhLP3 (phosducin-like protein 3) as a modulator of CCT function in vitro and in vivo. PhLP3 binds CCT, spanning the cylindrical chaperonin cavity and contacting at least two subunits. When present in a ternary complex with CCT and an actin or tubulin substrate, PhLP3 significantly diminishes the chaperonin ATPase activity, and accordingly, excess PhLP3 perturbs actin or tubulin folding in vitro. Most interestingly, however, the Saccharomyces cerevisiae PhLP3 homologue is required for proper actin and tubulin function. This cellular role of PhLP3 is most apparent in a strain that also lacks prefoldin, a chaperone that facilitates CCT-mediated actin and tubulin folding. We propose that the antagonistic actions of PhLP3 and prefoldin serve to modulate CCT activity and play a key role in establishing a functional cytoskeleton in vivo.

Transport of newly synthesized sterol to the sterol-enriched plasma membrane occurs via nonvesicular equilibration.

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.

A role for yeast oxysterol-binding protein homologs in endocytosis and in the maintenance of intracellular sterol-lipid distribution.

The seven yeast OSH genes (OSH1-OSH7) encode a family of orthologs of the mammalian oxysterol-binding protein (OSBP). The OSH genes share at least one essential overlapping function, potentially linked to the regulation of secretory trafficking and membrane lipid composition. To investigate the essential roles of the OSH genes, we constructed conditional OSH mutants and analyzed their cellular defects. Elimination of all OSH function altered intracellular sterol-lipid distribution, caused vacuolar fragmentation, and resulted in an accumulation of lipid droplets in the cytoplasm and within vacuolar fragments. Gradual depletion of Osh proteins also caused cell budding defects and abnormal cell wall deposition. In OSH mutant cells endocytosis was severely impaired, but protein transport to the vacuole and the plasma membrane was largely unaffected. Other mutants affecting sterol-lipid function and distribution, namely erg2Delta and arv1Delta, shared similar defects. These findings suggested that OSH genes, through effects on intracellular sterol distribution, establish a plasma membrane lipid composition that promotes endocytosis.

Creating precise GFP fusions in plasmids using yeast homologous recombination.

Using a combination of primer amplification, homologous recombination, and yeast genetics, we established a method for creating precise promoter and protein fusions in genes originating from organisms other than yeast. One major advantage of this new method is its versatility. Fusions can be produced within a target gene without constraints regarding the site of insertion. Thus, fusions can be generated within a target sequence exactly at the site desired, and all sequences upstream and downstream of the insertion site were preserved. To illustrate the general applicability of this technique, we fused the gene encoding GFP to a Caenorhabditis elegans homologue of the dishevelled gene, dsh-2. This approach is not restricted to GFP fusions but can be utilized to create fusions between almost any two sequences regardless of the source. Therefore, this method provides a flexible alternative to other PCR-mediated techniques.