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

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

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

Spatial and temporal co-localization of Sec4p with actin patch subunits during actin patch assembly.

<p><b>A–C.</b> Images of wild-type cells (WT; BY4741) showing the co-localization (arrowheads) of newly transported GFP-Sec4p particles after photobleaching with Sla1p-, Las17-, and Abp1-RFP (bar = 2 μm). Duplicate examples of kymographs are shown comparing the relative timing of GFP-Sec4p co-localization with each actin patch subunit. For each kymograph shown, green (GFP-Sec4p) and red (RFP fusions) arrowheads indicate maximum voxel fluorescence intensities. <b>D.</b> Bar graph reporting average differences in time for the maximum fluorescence of each RFP-marked actin patch subunit relative to GFP-Sec4p (<i>n</i> = 22 particles/strain; kymographs from ≥ 11 independent cells). In all graphs, data is shown as mean values with error bars representing standard error of the mean (S.E.M).</p

Mutations in <i>SEC4</i> and <i>SEC2</i> disrupt actin patch assembly and proper endocytic internalization.

<p><b>A</b>. Kymographs show coincident localization of single Sla1p-RFP and GFP-Abp1p particles during actin patch assembly in buds from WT (BY4741), <i>sec2-41</i> (CBY4710), and <i>sec4-8</i> (CBY4711) cells incubated at 37°C for 60 min. <b>B.</b> Scatterplots quantifying increased average lifetime of individual Sla1p-RFP and GFP-Abp1p particles in <i>sec4-8</i> and <i>sec2-41</i> cells relative to WT, with modest lifetime increases in <i>sec6-4</i> (CBY4712) cells and no change detected in <i>msb3Δ msb4Δ</i> cells (CBY1981). <b>C.</b> Two representative tracings of GFP-Abp1p particles moving at the cell cortex in WT, <i>sec4-8</i>, and <i>sec2-41</i> cells at 37°C, as tracked using confocal video microscopy (green and red dots mark the first and last positions of the particles, respectively); tracings are oriented so that the bud cortex is up and the cell interior is down. Differences between positions (black dots) are 1 s and total elapsed times are shown above each tracing, and, below, the average velocities for GFP-Abp1p particles are plotted in the bar graph (<i>n</i> > 50 tracings). <b>D.</b> Scatterplots show total numbers of GFP-Abp1p and Sla1p-RFP particles in buds of <i>sec6-4</i>, <i>sec4-8</i>, and <i>sec2-41</i> cells relative to WT after incubation at 37°C, and in buds of <i>msb3Δ msb4Δ</i> and WT cells (<i>n</i> > 20 buds). <b>E.</b> Representative actin patch internalization defects observed in <i>sec4-8</i>, <i>sec2-41</i>, and <i>sla2Δ</i> (CBY4863) cells as shown by Abp1p-GFP “comet tails” (inserts), compared to Abp1p-GFP spots in WT (bar = 2 μm). Unless stated otherwise, for all plots, statistical differences relative to congenic WT control strains are shown by single, double, and triple asterisks indicating <i>p</i> < 0.05, 0.0015, and 0.0001, respectively.</p

Slow growth of Δ-s-tether cells is rescued by expression of an artificial ER-PM tether or choline.

<p><b>A.</b> The “ER-PM staple" has a modular architecture consisting of an N-terminal GFP, an ER anchor comprising two transmembrane domains and a lumenal loop from herpes virus (MVH68) mK3 E3 ubiquitin ligase, two helices from mitofusin 2 that are predicted to adopt an antiparallel arrangement about 9 nm long, and the polybasic domain from RitC that targets the PM. <b>B.</b> Tenfold serial dilutions of WT (SEY6210) and Δ-s-tether (CBY5838) cells, transformed with either the vector control (YCplac111) or a plasmid expressing the artificial staple (pCB1185), spotted on solid growth medium, and incubated for 2 d at 30 °C. <b>C.</b> DIC images of WT and Δ-s-tether cells and the corresponding spinning disc confocal fluorescence microscopy images showing the colocalization of RFP-ER (pCB1024) and the GFP-marked artificial staple (pCB1185) at three different optical focal planes. Scale bar = 5 μm. <b>D.</b> Quantification of the staple distribution within mother and buds and at cER versus internal cytoplasmic ER. <b>E.</b> Choline-dependent growth of Δ-s-tether cells. WT, Δtether (ANDY198), and Δ-s-tether (CBY5838) cells were streaked onto solid growth medium supplemented with 1 mM choline chloride, as indicated, and incubated for 3 d at 30 °C. <b>F.</b> Quantification of ER-RFP localization in WT and Δ-s-tether cells, with and without 1 mM choline, represented as a ratio of the length of PM-associated ER per circumference of PM in each cell (<i>n</i> > 50 cells; error bars represent SEM). <b>G</b>. Lipid composition of WT, Δtether, and Δ-s-tether cells represented as a normalized mole percentage relative to WT (set to 1.0). The data represent the mean ± SEM derived from the analysis of five independent samples. Numerical data presented in this figure may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003864#pbio.2003864.s003" target="_blank">S1 Data</a>. Δ-s-tether, Δ-super-tether; cER, cortical ER; DAG, diacylglycerol; DIC, differential interference contrast; ER, endoplasmic reticulum; GFP, green fluorescent protein; IPC, inositol-phosphoceramide; MIPC, mannosylinositol phosphoceramide; mmPE, dimethyl PE; mPE, monomethyl PE; PA, phosphatidic acid; PC, phosphatidylcholine; PCe, ether phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PM, plasma membrane; PS, phosphatidylserine; RFP, red fluorescent protein, RitC; C-terminal polybasic region from mammalian Rit1; WT, wild type.</p

Functional interactions between ER-PM tethers and PI4P regulators.

<p><b>A.</b><i>OSH4</i> deletion in Δ-s-tether cells results in synthetic lethality. WT (SEY6210), Δtether (ANDY198), Δ-s-tether (CBY5838), <i>osh4</i>Δ Δtether (CBY5940), and <i>osh4</i>Δ Δ-s-tether cells (CBY5988) were transformed with an episomal copy of the <i>SCS2</i> tether gene (+ [<i>SCS2</i>]; pCB1183) and streaked onto selective solid media with and without choline supplementation. The presence of the <i>SCS2</i> gene provides an ER-PM tether that confers robust growth, even in the absence of all other tether genes. On growth medium selecting against the <i>SCS2</i> plasmid (− [<i>SCS2</i>]), <i>osh4</i>Δ Δ-s-tether cells were inviable with or without choline. <b>B.</b> <i>OSH6</i> expression suppresses the synthetic lethality of <i>osh4</i>Δ in Δ-s-tether cells. WT and <i>osh4</i>Δ Δ-s-tether cells containing an episomal copy of <i>SCS2</i> were transformed with either the high-copy vector control (YEplac181), <i>OSH4</i> (pCB598), or <i>OSH6</i> (pCB1266) and streaked onto solid growth media. On a medium selecting against the <i>SCS2</i> plasmid, <i>OSH4</i> or <i>OSH6</i> expression suppressed <i>osh4</i>Δ Δ-s-tether synthetic lethality, whereas vector control did not. <b>C.</b> Representative images of WT, Δtether, and Δ-s-tether cells by DIC with corresponding fluorescence microscopy showing the localization of the PI4P sensor GFP-2xPH^<i>OSH2</i> (pTL511). Scale bar = 2 μm. <b>D.</b> Bar graphs quantifying the number of GFP-2xPH^<i>OSH2</i> fluorescent Golgi spots (lower and upper boundaries of boxes correspond to data quartiles; the white bar indicates the median; lines represent the range of spots/cell) and the percentage of GFP-2xPH^<i>OSH2</i> fluorescent mothers detected in WT, Δtether, and Δ-s-tether cells. <b>E.</b> <i>SAC1</i> deletion in Δ-s-tether cells results in a synthetic lethal interaction. WT, Δtether, <i>sac1</i>Δ Δtether (CBY6142), Δ-s-tether, and <i>sac1</i>Δ Δ-s-tether cells (CBY6146) were transformed with an episomal copy of <i>SCS2</i> and streaked onto selective solid media with and without choline supplementation. On a medium that selects against the <i>SCS2</i> plasmid, <i>sac1</i>Δ Δ-s-tether cells were inviable whether or not choline was added. Numerical data presented in this figure may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003864#pbio.2003864.s003" target="_blank">S1 Data</a>. Δ-s-tether, Δ-super-tether; DIC, differential interference contrast; ER, endoplasmic reticulum; PI4P, phosphatidylinositol-4-phosphate; PM, plasma membrane; WT, wild type.</p

Alterations in ergosterol pools and dynamics at the PM in Δ-s-tether cells.

<p><b>A.</b> Sensitivity of Δ-s-tether cells to nystatin. Tenfold serial dilutions of WT (SEY6210), <i>osh4</i>Δ (HAB821), Δtether (ANDY198), and Δ-s-tether (CBY5838) cultures spotted onto solid rich medium containing no nystatin, 1.25 μM (+) nystatin, or 2.5 μM (++) nystatin and incubated for 3 d at 30 °C. <b>B.</b> Tenfold serial dilutions of WT, Δtether, and Δ-s-tether, <i>lem3</i>Δ (CBY5194) cultures were spotted onto solid rich media containing no drug, 5 μM duramycin, or 60 μM edelfosine and incubated for 2 d at 25 °C and 30 °C. The <i>lem3</i>Δ strain is known to be duramycin-sensitive and was used as a positive control. <b>C.</b> Tenfold serial dilutions of WT, Δtether, Δ-s-tether, and <i>osh3</i>Δ (JRY6202) cultures were spotted onto solid rich media containing no drug or 0.5 μg/mL myriocin and incubated for 2 d at 30 °C. The <i>osh3</i>Δ strain is known to be myriocin resistant and was used as a positive control. <b>D.</b> Assay to measure the proportion of cellular ergosterol that is extracted by MβCD. The PM of a yeast cell is shown, with outer (green) and inner (blue) leaflets delineated. Incubation of cells with MβCD on ice results in extraction of ergosterol from the outer leaflet. The sample is centrifuged to recover MβCD-ergosterol complexes in the supernatant. Ergosterol is extracted from the cell pellet and supernatant with hexane/isopropanol and quantified by HPLC (UV detection). <b>E.</b> The MβCD-accessible pool of ergosterol (quantified as in panel D) is about 20-fold greater in Δ-s-tether cells versus WT cells, and partially restored to WT levels in cells expressing the “ER-PM staple.” The statistical significance of the difference between the measurement of WT cells and each of the different Δ-s-tether samples is <i>p</i> < 0.0001, and between the Δ-s-tether samples is <i>p</i> = 0.0205 (*) and 0.436 (ns). <b>F.</b> Assay to measure transport of newly synthesized ergosterol from the ER to the MβCD-accessible pool. Cells are pulse-labeled with [³H]methyl-methionine to generate [³H]ergosterol in the ER, and chased as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003864#pbio.2003864.g003" target="_blank">Fig 3</a>. After a 30 min chase period, energy poisons are added and cells are placed on ice and incubated with MβCD. The ratio of the specific radioactivity of ergosterol in MβCD-ergosterol complexes versus that of the cell homogenate (RSR) provides a measure of transport. <b>G.</b> Transport of newly synthesized ergosterol from the ER to the MβCD-accessible pool. The bar chart shows RSR values for the different samples. The dotted line indicates the average RSR (about 0.82, averaged over both WT and Δ-s-tether samples) after 30 min of chase for the PM fraction, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003864#pbio.2003864.g003" target="_blank">Fig 3</a>. The statistical significance was determined by one-way ANOVA (***<i>p</i> = 0.0003, **<i>p</i> = 0.0027, *<i>p</i> = 0.043). Numerical data presented in this figure may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003864#pbio.2003864.s003" target="_blank">S1 Data</a>. Δ-s-tether, Δ-super-tether; ER, endoplasmic reticulum; HPLC, high-performance liquid chromatography; MβCD, methyl-β-cyclodextrin; ns, not significant; PM, plasma membrane; RSR, relative specific radioactivity; UV, ultraviolet; WT, wild type.</p

Sterol depletion induces both ER-PM MCS formation and Tcb3 tether expression.

<p><b>A.</b> Electron micrographs of WT (CBY858) and <i>erg9</i>Δ P^<i>MET3</i>-<i>ERG9</i> (CBY745) cells before (− Met) and after (+ Met) methionine repression of P^<i>MET3</i>-<i>ERG9</i> synthesis of sterols (methionine was also added to WT). Inserts correspond to magnifications of boxed regions at the cell cortex showing PM-associated ER (arrowheads); cER is highlighted in magenta. Scale bar = 2 μm. <b>B.</b> Corresponding to panel <b>A</b>, quantification of cER length expressed as a percentage of the total circumference of the PM in each cell section counted (<i>n</i> = 25 cells for each strain; error bars show SD; <i>p</i> = 7.6 × 10⁻²⁵ for the difference between WT and <i>erg9</i>Δ P^<i>MET3</i>-<i>ERG9</i> (+ Met)). <b>C.</b> WT (CBY5836) and <i>erg9</i>Δ (CBY5834) cells with integrated <i>TCB3-</i>GFP and P^<i>MET3</i>-<i>ERG9</i> constructs in the presence of methionine, which represses <i>ERG9</i> expression and sterol synthesis in <i>erg9</i>Δ cells. Scale bar = 2 μm. <b>D.</b> Corresponding to panel <b>C</b>, representative immunoblots probed with anti-GFP and anti-actin antibodies showing Tcb3-GFP levels in WT and sterol-depleted <i>erg9</i>Δ P^<i>MET3</i>-<i>ERG9</i> cells, as compared to the actin (Act1) control. Relative to WT, Tcb3-GFP levels increased 5.6 ± 1.6 (mean ± SD; <i>n</i> = 5)-fold in sterol-depleted <i>erg9</i>Δ P^<i>MET3</i>-<i>ERG9</i>. <b>E</b>. Continuous Tcb3-GFP and ER-RFP fluorescence along the PM (arrowheads) dissipated with the addition of exogenous cholesterol to sterol-depleted <i>hem1</i>Δ <i>erg9</i>Δ P^<i>MET3</i>-<i>ERG9</i> cells (CBY5995 and CBY5842 pCB1024, respectively). Intense ER-RFP nuclear fluorescence (arrows) also diminished after cholesterol addition. The normal discontinuous dashed line of Tcb3-GFP and ER-RFP around the cell cortex was unaffected in sterol-prototrophic <i>hem1</i>Δ cells (CBY5993 and CBY5844 pCB1024, respectively); scale bar = 2 μm. <b>F.</b> Quantification of contiguous association between cER and the PM after cholesterol addition in <i>hem1</i>Δ, <i>TCB3</i>-GFP <i>hem1</i>Δ cells, and sterol-depleted <i>hem1</i>Δ <i>erg9</i>Δ P^<i>MET3</i>-<i>ERG9</i> cells and <i>TCB3</i>-GFP <i>hem1</i>Δ <i>erg9</i>Δ P^<i>MET3</i>-<i>ERG9</i> cells. Following cholesterol addition to sterol-depleted <i>hem1</i>Δ <i>erg9</i>Δ P^<i>MET3</i>-<i>ERG9</i> cells, reductions in cortical Tcb3-GFP localization were detected 1 h after cholesterol addition, with reductions in cER-RFP lagging slightly behind (<i>n</i> > 100). Numerical data presented in this figure may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003864#pbio.2003864.s003" target="_blank">S1 Data</a>. cER, cortical ER; ER, endoplasmic reticulum; <i>ERG9</i>, squalene synthase; GFP, green fluorescent protein; MCS, membrane contact site; PM, plasma membrane; RFP, red fluorescent protein; Tcb, tricalbin; WT, wild type.</p