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

Isolation and characterization of a gut-specific acid phosphatase in the nematode caenorhabditis elegans

Bibliography: p. 98-106

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

<div><p>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 <i>sec4-Q79L</i> 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.</p></div

GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation in vitro.

<p>All assays contained 1.5 μM actin (99% pyrene-labeled) polymerized at 30°C in the presence 75 nM Arp2/3 complex. <b>A.</b> Actin polymerization was induced upon addition of 75 nM bacterially expressed GST-Las17p, but this activation was inhibited by the addition of 75 nM full-length Sla1p (expression and purified as a GST fusion protein). <b>B.</b> Time-course (left) showing concentration-dependent effects of GTPγS-Sec4p (fused to GST) on Sla1p inhibition of Las17p, in which a 1, 2, 5, 10, or 20 X molar excess of GTPγS-Sec4p was added (relative to Las17p, Arp2/3, and Sla1p) into each actin polymerization reaction. Based on the accompanying graph, the bar graph (right) shows calculated rates of actin polymerization with increasing concentrations of GTPγS-Sec4p (rates were calculated from slopes of the linear segment of curves corresponding to half maximal polymerization). <b>C.</b> GTPγS versus GDP nucleotide dependence of Sec4p for activating actin polymerization in the presence of Sla1p and Las17p (Sec4p was added in 10 X molar excess as indicated). <b>D.</b> In contrast to the effect of GTPγS-Sec4p, GTPγS- or GDP-bound Ypt1p fails to counteract Sla1p inhibition of Las17p (Sec4p and Ypt1p were added in 10 X molar excess). <b>E.</b> In the absence of Las17p, GTPγS-Sec4p had negligible actin nucleation activity (as indicated, Sec4p was added in 1 and 10 X molar excess, and 10 X molar excess of Ypt1p was added). <b>F.</b> Affinity-purified GST fusion proteins (1 μg) used in actin polymerization assays, separated by SDS-PAGE and stained with Coomassie. All plots shown represent averages of 6–10 independent trials.</p

Physical interaction of Sec4p with actin patch subunits.

<p><b>A.</b> Top panel: representative in vitro binding assay showing in vitro transcribed and translated ³⁵S-Las17p binding to bacterially expressed GST-Sec4p purified and immobilized on beads prior to SDS-PAGE and autoradiography. Average percentage of input Las17p interacting with GDP- or GTPγS-bound GST-Sec4p as shown (<i>n</i> = 3). Bottom panel: SDS-PAG showing equal amounts (1 μg) of GST-Sec4p, GST, and GST-Ypt1p preloaded with GDP or GTPγS prior to ³⁵S-Las17p addition. <b>B.</b> BiFC assays for cells expressing Sla2p-YFP^N (CBY4625) or YFP^N-Sec4p (CBY4629) when mated with cells expressing Las17p-YFP^C (CBY4660), Sla2p-YFP^C (CBY4661), or Abp1p-YFP^C (CBY4632). Fluorescence at the cell cortex (arrowheads) indicates in vivo interactions at actin patches (bar = 5 μm). <b>C.</b> Competition of BiFC binding following overnight P^<i>GAL</i>-<i>LAS17</i> induction or 6 h P^<i>GAL</i>-<i>SEC4</i> induction with galactose (Gal), compared to no induction in glucose (Glc) medium, in cells expressing YFP^N-Sec4p and Las17p-YFP^C (CBY4638). In all cells observed (including controls), non-specific cytoplasmic fluorescence increased after transfer to galactose-containing medium. <b>D.</b> Bar graphs quantifying reductions in BiFC particles within cells corresponding to images shown in panels <b>B</b> and <b>C</b> (<i>n</i> > 100 cells).</p

Reciprocal effects of endocytosis on polarized exocytosis.

<p>Sec4p motility is dependent on actin patch assembly. <b>A.</b> Representative tracings from three-dimensional time-lapse confocal microscopy showing GFP-Sec4p movement after its transport into photobleached zones at the bud cortex in <i>las17-1</i>^<i>ts</i> (CBY4356), <i>las17-13</i>^<i>ts</i> (CBY4357), <i>rvs167Δ</i> (CBY4733), <i>bbc1Δ</i> (CBY4373), and <i>sla2/end4-1</i>^<i>ts</i> (CBY4452) endocytosis-defective cells, relative to WT (CBY4741). Temperature-conditional mutations were incubated at 37°C for 2 h, whereas motility in deletion mutants was assessed at 23°C. On each axis, 0.5 μm intervals are indicated. The bar graph quantifies GFP-Sec4p particle motility at the PM for each strain (<i>n</i> > 30 particles). <b>B.</b> Images of GFP-Sec4p localization at sites of polarized growth (asterisk) in WT (BY4741), <i>las17Δ</i> (CBY1024), and <i>las17-14</i> (CBY4358) cells. Under these conditions, GFP-Sec4p was not detected on any membrane in <i>las17Δ</i> cells grown at 30°C (as shown) or in <i>las17-14</i> cells incubated at 37°C for 2 h (bar = 2 μm). <b>C.</b> Immunoblots assaying Bgl2p polarized exocytosis showing defective Bgl2p internalization in <i>sla2Δ</i> (DDY1980), <i>las17Δ</i> (DDY1709), <i>las17-13</i> (CBY4357), and <i>las17-1</i> (CBY4356) endocytosis mutants, compared to the <i>sec6-4</i> exocytosis-defective control (NY17) and congenic WT strains (BY4741 and DDY130). Bgl2p exocytosis was not defective in <i>rvs167Δ</i> cells (CBY4372). The same blots were probed for tubulin (Tub2p) or actin (Act1p) as internal loading controls.</p