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Vol. 17, Issue 7, 3009-3020, July 2006

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Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

Johan-Owen De Craene^*, Jeff Coleman^*, Paula Estrada de Martin, Marc Pypaert^*, Scott Anderson, John R. Yates, III, Susan Ferro-Novick, and Peter Novick^*

*Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510; and Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037

Submitted January 27, 2006; Revised April 6, 2006; Accepted April 10, 2006
Monitoring Editor: Akihiko Nakano

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1 in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1 cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The endoplasmic reticulum (ER) is a large, elaborate structure that is typically spread throughout the cytoplasm of eukaryotic cells (Terasaki and Jaffe, 1991; Greenfield and High, 1999). In higher eukaryotic cells, the ER has classically been divided into three distinct regions: the nuclear envelope; the rough ER, characterized by the presence of ribosomes and a cisternal morphology; and the smooth ER, a polygonal network of membrane tubules that seems to emanate from the cisternae of the rough ER (Palade, 1956). These morphologically distinct membrane structures define a continuous lumen, indicating that they are all part of one organelle (Terasaki et al., 1996). In the yeast Saccharomyces cerevisiae, the ER is composed of the nuclear envelope and a meshwork of membrane tubules closely apposed to the cell cortex with only a few cytoplasmic tubules connecting the cortical ER to the nuclear envelope (Preuss et al., 1991; Prinz et al., 2000).

In all cells, the ER structure is very dynamic. The smooth ER can increase in volume, as in hepatocytes (Remmer and Merker, 1963), or undergo structural rearrangements of three types: a new tubule can stem from an existing tubule and fuse with another one to form new alveolus, an existing alveolus can shrink in a process akin to ring closures, and a tubule can slide along another tubule (Lee and Chen, 1988; Prinz et al., 2000). The reorganization and maintenance of the ER in higher eukaryotes depends on microtubules (Terasaki et al., 1986; Terasaki, 1990), whereas in yeast, ER reorganization depends on actin (Prinz et al., 2000).

In yeast, actin also plays a defining role in the inheritance of the ER between mother and daughter cell, a process that occurs before nuclear inheritance and that is independent of microtubules (Fehrenbacher et al., 2002; Estrada et al., 2003). Indeed, early in the cell cycle, an ER tubule emanating from the mother cell is pulled along actin cables into the nascent bud by the type V myosin Myo4 and its adaptor protein She3 (Du et al., 2001; Fehrenbacher et al., 2002; Estrada et al., 2003). Once the tubule is in the bud, it anchors at the bud tip (Fehrenbacher et al., 2002), a process that requires at least three subunits of the exocyst complex, Sec3, Sec5, and Sec8 (Wiederkehr et al., 2003, 2004; Reinke et al., 2004). The exocyst is an octameric complex found at sites of secretion, such as the bud tip, where it acts to tether secretory vesicles to the plasma membrane in preparation for exocytic fusion. After anchoring at the bud tip, the ER spreads along the cortex of the bud by an unknown mechanism.

A key question concerns the mechanism by which the ER forms and maintains its meshwork structure. In this study, we provide compelling evidence for a role of Rtn1, an integral ER membrane protein, in structuring the ER. This protein belongs to the Rtn family of proteins that is ubiquitously found in eukaryotes and is characterized by a reticulon domain consisting of two transmembrane domains flanking a hydrophilic loop (Oertle et al., 2003). In mammalian cells, four RTN genes are present and several splice variants for each have been identified. In yeast, only one paralog has been found and named Rtn2 with no known function (Oertle et al., 2003). RTN genes are differentially expressed in different cell types (Roebroek et al., 1998; Moreira et al., 1999; GrandPre et al., 2000). All homologues of Rtn1 are localized on the ER, yet they have no identified function there (Van de Velde et al., 1994; Chen et al., 2000; Hamada et al., 2002). A small fraction of Rtn4, also called Nogo, is also localized at the plasma membrane where it is involved in the negative regulation of neuronal growth (Chen et al., 2000; GrandPre et al., 2000; Prinjha et al., 2000). We also show that Rtn1, through its conserved hydrophilic loop, interacts with Sec6p, a subunit of the exocyst.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Yeast Strains, Plasmids, and Growth Conditions
S. cerevisiae strains used in this study are listed in Table 1. Cells were grown in either YP (rich medium) or SC (complete medium) with the appropriate amino acids omitted to ensure plasmid maintenance and the mentioned carbon source to a final concentration of 2%. Standard techniques were used for sporulation and tetrad analysis of yeast strains (Sherman, 1991). Yeast cells were transformed using the modified lithium acetate method (Gietz et al., 1992).

To tag Sec61 with GFP, we used plasmid SFNB1018 (LEU2 marker) linearized by PmlI and Ssh1 by SFNB1068 (LEU2 marker) linearized by EcoRI (Wiederkehr et al., 2003). To tag Sec7p with GFP, plasmid SFNB798 (pUSE-URA3) was linearized by SpeI (Seron et al., 1998).

Plasmid pNRB1264 was constructed by digesting plasmid HDEL-DsRed (a gift of the Glick laboratory, University of Chicago, Chicago, IL) with HindIII and NdeI, and this last site was blunted by Klenow. The fragment was inserted in pRS305 (LEU2 marker) between sites SmaI and HindIII and linearized by EcoRV. Plasmid pNRB1265 was constructed by digesting plasmid Atp9-DsRed (Mozdy et al., 2000) with ApaI and SacI. The fragment was inserted in pRS303 (HIS3 marker) between sites KpnI and SacI and linearized by HindIII.

Isolation of rtn1 Mutant
The yeast MATa haploid deletion library from Research Genetics (ResGen/Invitrogen, Carlsbad, CA) was screened to identify genes displaying a defect in the inheritance of the cortical ER. The transformation of this library was performed as described in Estrada et al. (2003) in 96-well blocks. The cells were transformed with plasmid SFNB1000 (HMG1-GFP CEN LEU2; Estrada et al., 2003). A total of 4080 mutants were screened.

Fluorescence Microscopy
To visualize the ER, cells expressing GFP or DsRed fusion proteins were grown overnight in SC medium at 25°C to optical density (OD)₆₀₀ of 0.3. 1 ml of culture was pelleted and resuspended in 50 µl of growth medium. Then, 3 µl of cells was mixed with an equal volume of growth medium containing 0.6% of low melting agarose.

Epifluorescence microscopy was performed using a Zeiss Axioplan2 microscope with a 63x oil immersion objective. Images were collected with an Orca ER digital camera (Hamamatsu, Bridgewater, NJ) and the OpenLab 4.02 imaging software (Improvision, Lexington, MA).

Confocal microscopy was performed using a laser scanning microscope (LSM 510; Carl Zeiss MicroImaging, Thornwood, NY) with a 100x oil immersion objective. All images were processed for display using Photoshop and Illustrator from Adobe Systems (Mountain View, CA). Three dimensional (3D) rendering was performed using Imaris from BitPlane (St. Paul, MN).

Electron Microscopy
For ER analysis, yeast cells were harvested, resuspended, and incubated for 20 min at room temperature (RT) in 1.5% KMnO₄. After five washes with distilled water, cells were stained with 2% uranyl acetate for 4 h at RT in the dark.

For vesicle density, cells were fixed in 3% glutaraldehyde with 0.1 M sodium cacodylate buffer, pH 6.8, overnight. After two washes in 50 mM phosphate buffer, pH 7, cells were resuspended in 2 ml of 0.25 mg/ml Zymolyase 100T (Seigaku Corporation, Tokyo, Japan) for 25 min. After two washes in 0.1 M sodium cacodylate, cells were resuspended in 0.5 ml of cold 2% OsO₄ in 0.1 M sodium cacodylate for 1 h on ice. After three washes with water, cells were resuspended in 2% uranyl acetate for 1 h at RT and washed five times with water.

All samples were dehydrated by incubation with 50, 70, 95 (2 times for 5 min each), and 100% ethanol (2 times for 15 min each). The cells were then washed briefly in acetone before embedding in Spurr resin (Electron Microscopy Science, Fort Washington, PA). For polymerization, the cell pellets in Spurr were cured for 48 h at 80°C. Ultrathin (60-nm) sections were cut on a Reichert ultramicrotome and collected on Formvar- and carbon-coated grids. The samples were poststained with 2% uranyl acetate and lead citrate and examined on a Philips Tecnai 12 electron microscope.

Subcellular Fractionation
The ER and plasma membrane were fractionated on a linear sucrose density gradient in the presence of EDTA as described previously (Roberg et al., 1997). In brief, cells were grown to an OD₆₀₀ of 1.0 in YP glucose, harvested and resuspended in 0.5 ml of STE buffer [10% (wt/wt) sucrose, 10 mM EDTA, and 10 mM Tris-HCl, pH 7.5] containing a protease inhibitor cocktail (10 µM antipain, 30 µM leupeptine, 30 µM chymostatin, 1 µM pepstatin A, 1 µM phenylmethylsulfonyl fluoride [PMSF], and 1 µg/ml aprotinin). Cells were then lysed by vortexing with glass beads. After lysis, 1 ml of STE buffer was added, and the lysate was cleared by centrifugation at 500 x g for 10 min. Then, 15 OD₆₀₀ units (300 µl of the lysate) were layered on top of a 20–60% linear sucrose gradient (5 ml) prepared in TE (10 mM Tris-HCl, pH 7.5, and 1 mM EDTA). Samples were then centrifuged at 100,000 x g for 18 h at 4°C using a SW50.1 rotor (Beckman Coulter, Fullerton, CA). Fractions (440 µl each) were collected from the top of the gradient, and proteins were precipitated with 500 µl of 50% trichloroacetic acid (TCA), and pelleted by centrifugation at 14,000 rpm for 5 min at 4°C. Protein pellets were resuspended in 66 µl of Tris base and 99 µl of 2x SDS sample buffer. Then, 30 µl of each fraction was resolved by SDS-PAGE and subjected to Western blot analysis. The antibodies were subsequently detected by enhanced chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom), and the bands were quantified using BioImage software (Yale University, New Haven, CT). The data are plotted as a percentage of the maximum of antigenic material.

Membrane Extraction Experiment
Cells expressing Rtn1-3xHA were used for preparation of microsomes as described in Gunthrie and Fink (1991). Cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl) and complete protease inhibitors (Roche Diagnostics, Indianapolis, IN). Equal volumes of lysate were treated either with 0.5 M NaCl, 0.1 M Na₂CO₃, 1% TritonX-100, or 0.1% SDS to a finale volume of 100 µl and incubated for 30 min on ice. The treated lysates were centrifuged for 1 h at 100,000 x g. The supernatant fractions were mixed to equal volume of 2x loading buffer, and pellets were resuspended in 200 µl of loading buffer. Ten microliters of each sample was loaded on a 10% SDS-PAGE and after Western blotting was probed with anti-hemagglutinin (HA) (BabCO, Richmond, CA).

Tandem Affinity Purification (TAP) Tag Purification of Exocyst Proteins
Exocyst proteins were C-terminally fused to a TAP tag (NY2463; Sec10p-TAP LEU2). Fusion proteins were isolated using the method described in Puig et al. (2001) with some modifications. Instead of using a French press, 2 liters of cells (grown at 25°C to OD₆₀₀ 1.5) were lysed in a buffer of 20 mM PIPES, pH 6.8, 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, 10 µM antipain, 20 µM aprotinin, 20 µM chymostatin, 20 µM leupeptin, 20 µM pepstatin A, and 10 mM -mercaptoethanol (-ME) using a Bead Beater (Biospec Products, Bartlesville, OK). The 30-ml chamber was filled halfway with 0.5-mm glass beads (Biospec Products) and run four times for 1 min on ice. NP-40 (IGEPAL CA-630; Sigma-Aldrich, St. Louis, MO) was added [0.5% (vol/vol)], and the lysate was incubated at 4°C for 15 min and then centrifuged at 30,000 x g for 15 min. The method for protein isolation in Puig et al. (2001) was followed except 20 mM PIPES, pH 6.8, was substituted for Tris-HCl in all buffers, the TEV protease incubation was allowed to proceed overnight at 4°C, and the concentration of EGTA in the elution buffer was increased to 10 mM.

Mass Spectrometry
Fifty percent of the exocyst TAP tag purification was precipitated using 20% (vol/vol) TCA, washed five times with 100% acetone (–20°C), and sent to the Proteomic Mass Spectrometry Laboratory at the Scripps Research Institute (La Jolla, CA). Samples were suspended in 8 M urea, 100 mM Tris, pH 8.5, reduced with 100 mM Tris(2-carboxyethyl)phosphine (Pierce, Rockford, IL), and cysteines were alkylated with 55 mM iodoacetamide. Then, Lys-C was used to digest the proteins for 4 h at 37°C at a concentration of 1 µg/100 µl. CaCl₂ was added to ensure tryptic specificity at 1 mM, and trypsin was used to digest the samples further at 1 µg/100 µl. The digests were then analyzed by micro-liquid chromatography (µLC)/µLC-tandem mass spectrometry (MS)/MS using an LCQ Deca ion trap mass spectrometer (Thermo Electron, Waltham, MA). Multidimensional chromatography was performed online using the following salt steps of 500 mM ammonium acetate: 10, 25, 35, 50, 65%, 80, and 100% (MacCoss et al., 2002). Tandem mass spectra were collected in data-dependent manner by collecting one full MS scan (m/z range = 400–1600) followed by MS/MS spectra of the three most abundant precursor ions. The collection of resulting spectra was then searched against a database of yeast open reading frames obtained from Saccharomyces Genome Database (release date August 27, 2004) using the SEQUEST algorithm (Eng et al., 1994). Peptide identifications were organized and filtered using the DTASelect program (Tabb et al., 2002).

Expression of Exocyst Proteins in Escherichia coli
Exocyst genes were PCR-amplified and cloned using the pET-46 Ek/LIC cloning kit (strains NRB1266–1273; Novagen, Madison, WI). Constructs were transformed into Rosetta 2 cells (Novagen) and grown in Terrific Broth (Sambrook et al., 1997) at 25°C to an OD₆₀₀ of 1. Cultures were chilled on ice for 30 min and then induced using 0.1 mM isopropyl--D-thiogalactopyranoside (IPTG) for 16 h at 15°C. The cultures were pelleted, resuspended in a buffer of 1x phosphate-buffered saline (PBS), 160 mM NaCl, 15 mM imidazole, and 1 mM PMSF, and lysed on ice using a Branson Sonifier 450 sonicator (4 times for 1 min each). Triton X-100 was added [1% (vol/vol)], and the lysates were incubated at 4°C for 30 min and then centrifuged at 30,000 x g for 15 min. Fusion proteins were isolated using Ni-NTA agarose (QIAGEN, Valencia, CA) for 1.5 h at 4°C. The resin was washed with 1x PBS, 160 mM NaCl, 1% Triton X-100, and 25 mM imidazole, and fusion proteins were eluted with the same buffer, except the imidazole concentration was raised to 400 mM.

In Vitro Binding Assays for Rtn1p
RTN1 was PCR amplified from the yeast strain NY1210 using primers that created a BamHI and SmaI site at the N and C terminus, respectively, and cloned into pGEX4T-1 (NRB1274; Promega, Madison, WI). A deletion series of Rtn1p was constructed using PCR on the full-length clone with primers that allowed for cloning into the BamHI and XhoI sites of pGEX4T-1. The first construct contains the small N terminus and the first predicted transmembrane domain (amino acids 1–58; NRB1275). The second construct includes the N terminus and the entire reticulon domain (aa 1–168; NRB1276). The third construct contains only the reticulon domain (aa 27–168; NRB1277). The fourth construct includes the hydrophilic loop and the second predicted transmembrane domain (aa 59–168; NRB1278). The fifth construct contains only the hydrophilic loop (aa 59–142; NRB1279). The sixth construct contains the C terminus of the protein (aa 169–295; NRB1280). As a control, the Snc1p coding sequence was amplified from pADH-cSNC1 (Gerst et al., 1992) and cloned into pGEX4T-1 using the same restriction enzymes (NRB1281).

Glutathione S-transferase (GST) fusion proteins were induced at OD₆₀₀ 1.0 with 1 mM IPTG for 1.5 h at 37°C. Cultures were pelleted and resuspended in a buffer of 1x PBS, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5 mM PMSF and lysed on ice using a sonicator (4 times for 1 min each). Triton X-100 was added [1% (vol/vol)], and the lysates were incubated at 4°C for 30 min and then centrifuged at 30,000 x g for 15 min. Glutathione beads (GE Healthcare) were incubated with soluble proteins for 1.5 h at 4°C followed by five washes.

Approximately equal amounts (100 nM) of soluble exocyst proteins were mixed with either GST or GST-Rtn1 protein immobilized on beads in a buffer of 20 mM PIPES, pH 6.8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5 mM PMSF, and 10 mM -ME. Reactions were incubated at RT for 2 h and then washed five times. Proteins were boiled off beads and run on a 7% gel, transferred to membrane, and detected using a His monoclonal antibody (mAb) (Novagen). The same protocol was used for binding Sec6p to the deletion series of Rtn1p.

Overproduction of Rtn1p and Sec6p
To overproduce Rtn1p, the gene was PCR amplified from NRB1274 and inserted into a vector (pNB530) containing the GAL1 promoter and an ADH1 terminator using BamHI and SmaI. The gene encoding GST was cloned upstream in frame as a BamHI fragment (NRB1283 URA3). The same methodology was used for GST (NRB1282 URA3) and GST-Snc1p (NRB1292 LEU2), which both act as negative controls in this experiment, except SNC1 was cloned into pNB529 using the restriction sites BamHI and PstI. The plasmids were integrated into the yeast genome, and the strains were crossed to wild type or a strain overexpressing Sec6p to create diploids.

Strains overproducing either GST alone, GST-Rtn1p alone, GST and Sec6p, GST-Rtn1p and Sec6p, or GST-Snc1p and Sec6p were grown 16 h at 25°C to an OD₆₀₀ of 1.0 in 50-ml cultures of YP containing 2% galactose. Proteins were lysed in the same buffers used for TAP purification, and glutathione beads were incubated with soluble proteins for 2 h at 4°C followed by five washes. Proteins were boiled off the beads and loaded on a 7% polyacrylamide gel, transferred to a membrane, and a Sec6p antibody (Potenza et al., 1992) was used for detection.

To overproduce the loop of Rtn1p, the sequence expressed in NRB1279 was PCR amplified and cloned into a vector containing the GAL1 promoter (pNB530) and a 2µ vector (pRS426 URA3; Mumberg et al., 1995) containing the GPD1 promoter using BamHI and SmaI. The sequence encoding GST was cloned upstream of the loop, in frame as a BamHI fragment creating plasmids NRB1284 URA3 and NRB1286 URA3, respectively. As a control, GST was cloned after the GPD1 promoter by itself (NRB1285 URA3).

Generation of Glycosylation Sites in Rtn1p
Glycosylation sites were created in the hydrophilic loop and C terminus of Rtn1p to address the topology of the protein. RTN1, along with 516 base pairs of its own promoter and a single C-terminal HA tag, was amplified and cloned into a vector (pRS306; Sikorski and Hieter, 1989) using the KpnI and SacII sites. The ADH1 terminator was added using SacI and SacII, and PstI was used to integrate the plasmid (NRB1287 URA3) into the genome of an rtn1 strain (NY 2651) at the URA3 gene (NY2671). PCR mutagenesis was used to create glycosylation sites by changing the Rtn1p amino acids 94, 116, 125, and 215 to a serine, asparagine, asparagine, and serine, respectively (NRB1288–1291). These plasmids were also integrated at URA3 in an rtn1 strain using PstI (NY2672–2675, respectively). Soluble protein from a 50-ml culture was isolated in a buffer of 20 mM PIPES, pH 6.8, 150 mM NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1% Triton X-100, 0.2 mM PMSF, 10 µM antipain, 20 µM aprotinin, 20 µM chymostatin, 20 µM leupeptin, 20 µM pepstatin A, and 10 mM -ME, and concanavalin A (ConA)-Sepharose 4B (Sigma-Aldrich) was added for 1 h at 4°C. The beads were washed in the same buffer, and proteins were eluted by boiling, loaded on a 10% polyacrylamide gel, transferred to membrane, and detected using either an antibody to the HA tag or carboxypeptidase Y.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
rtn1 Affects the Structure of the Cortical ER
To identify new mutants affected in cortical ER structure and inheritance, we screened the yeast deletion library as described in Estrada et al. (2003) using the ER marker Hmg1-GFP. We selected the rtn1 mutant for further analysis because the ER was clearly aberrant in this strain. Although it did not have an ER inheritance defect as shown by characterization using two other ER markers, Sec61-GFP and Ssh1-GFP, both subunits of the translocon in yeast, the structure of the ER was dramatically altered in this mutant. In almost 100% of the wild-type cells, both markers show a very even punctate distribution along the cell cortex as well as continuous labeling of the perinuclear ER (Figure 1, A and B). The punctate pattern reflects a cross section view of the cortical network of ER tubules. In contrast, the cortical ER staining in the rtn1 mutant seemed to be more cisternal than reticular, as indicated by the continuous stretches of fluorescence, and separated by large cortical regions with little or no fluorescence (Figure 1A). Indeed <5% of the cells examined showed a wild-type structure of the cortical ER (Figure 1B). Although the cortical ER is contiguous with the nuclear envelope, there was no change evident in the distribution of the fluorescence around the nucleus.

View larger version (94K): [in this window] [in a new window]   Figure 1. Rtn1p is required for the structure of the ER. (A) Wild type (NY2647, NY2649) and rtn1 mutant (NY2648, NY2650) cells expressing the ER marker Sec61-GFP or Ssh1-GFP were grown at 25°C in SC medium. The cells were examined using an epifluorescence microscope. Arrows point to cortical regions devoid of fluorescence. (B) Quantification of the altered cortical ER phenotype was done by scoring at least 300 cells with wild-type or altered morphology based on the presence of large gaps in fluorescence at the cell cortex and a more cisternal appearance of the cortical ER in an rtn1 mutant. The graph shows the percentage of cells having wild-type ER in each strain. (C) Thin section electron microscopy micrographs of wild-type (NY1210) and rtn1 (NY2651) cells grown in YPD and fixed with KMnO4 and embedded in Spurr. Arrows point to ER membrane stretches. Magnification, 4700x.

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We also performed a 3D optical reconstruction of the cortical ER in wild-type and rtn1 cells using Sec61-GFP as an ER marker. As expected, in wild-type cells, Sec61-GFP localizes to a reticulated structure at the cell cortex that corresponds to the cortical ER as well as to a spherical structure in the cell that corresponds to the nucleus (Supplemental Figure S1). In contrast, in the rtn1 mutant, the nuclear structure is not affected, but the cortical ER seems more cisternal with large areas of the cortex devoid of ER (Supplemental Figure S2).

Previous work by Prinz et al. (2000) has shown that mutants affecting ER-to-Golgi transport in yeast result in a more cisternal ER morphology. More recently, Wakana et al. (2005) have shown that overexpression of RTN3 results in a block of the retrograde transport of ERGIC-53 in HeLa cells. Furthermore, Geng et al. (2005) have shown that Rtn1 interacts with Yip3, a membrane protein that interacts with the ER-to-Golgi Rab GTPase Ypt1 in S. cerevisiae (Schmitt et al., 1986; Bacon et al., 1989). Nonetheless, in that study, they did not observe alterations in trafficking of several biosynthetic markers in an rtn1 mutant. We have confirmed this by comparing the secretion of invertase in an rtn1 mutant to a wild type strain and found no difference (our unpublished data). This is consistent with the normal generation time of an rtn1 mutant. In total, these results suggest a role for Rtn1p in the maintenance of the cortical ER structure but not in ER membrane proliferation.

Effect of rtn1 on Other Organelles
Because the rtn1 mutant has a strong effect on the structure of the cortical ER, we looked at the structure of other organelles in the cell. We used Sec7-GFP as a late Golgi marker, which in wild type occurs as small punctae distributed throughout the cell (Seron et al., 1998). In the rtn1 mutant, we could not see any alteration in size, number, or distribution of the Sec7-GFP punctae compared with the wild type (Supplemental Figure S3A).

We next assessed the role of Rtn1p in the morphology of the vacuole using the vacuolar ATPase subunit Vph1-GFP (Urbanowski and Piper, 1999). In wild-type cells, the vacuole often occurs as a multilobed structure. In the rtn1 mutant, we could not see any alteration in size or distribution of the vacuole compared with the wild type (Supplemental Figure S3B).

Finally, we assessed the role of Rtn1p in the morphology of mitochondria using the mitochondrial ATPase subunit Atp9-DsRed as a marker. In wild-type cells, the mitochondria form a tubular structure that is apposed to the plasma membrane (Mozdy et al., 2000). In the rtn1 mutant, we could not see any alteration in shape or distribution of mitochondria compared with wild type (Supplemental Figure S3C). These results indicate that Rtn1p is specifically involved in the structure of the ER.

Rtn1p Is Enriched at the Cell Cortex
Previous studies have shown that homologues of Rtn1p in mammalian cells or in Caenorhabditis elegans cells are localized to the ER with only a small fraction at the plasma membrane in the case of the mammalian Rtn4 or Nogo (reviewed in Oertle et al., 2003). To localize Rtn1p in yeast, we fused GFP to its carboxy terminus and expressed this construct as the sole copy of RTN1. As shown in Figure 2, Rtn1-GFP was prominently localized at the cell cortex and in cytoplasmic tubules, but it was only marginally detected in the perinuclear region. This localization is consistent with an association of Rtn1-GFP with the cortical ER. However, it was important to establish that the chimeric protein was functional. Having no direct assay for function, we have relied on the fact that if Rtn1-GFP were mislocalized or otherwise nonfunctional, the ER would show a similar structure to that observed in an rtn1 mutant using Sec61-GFP as an ER marker. Because we have not observed any such alteration of the ER structure in strains expressing Rtn1-GFP, we conclude that the GFP tag does not significantly affect Rtn1p function. Geng et al. (2005) have reported a similar localization of Rtn1p. To quantify the enrichment of Rtn1-GFP at the cortex of the cell versus the perinuclear region, we performed a ratiometric analysis as described in Wang et al. (2002). We determined that there is a twofold enrichment of Rtn1-GFP at the cell cortex compared with the perinuclear region.

View larger version (42K): [in this window] [in a new window]   Figure 2. Rtn1-GFP is enriched at the cortical ER. Wild-type cells (NY2658) expressing ER markers Rtn1-GFP and HDEL-DsRed were grown at 25°C in SC medium. The cells were examined using a confocal microscope.

Rtn1p Is an Integral ER Membrane Protein
Our results so far strongly suggest that Rtn1p is a cortical ER protein. But to definitively rule out a plasma membrane localization of Rtn1-GFP, we fractionated a wild-type lysate on a sucrose density gradient and compared the distribution of Rtn1-GFP with that of markers for the ER and plasma membrane (Figure 3A). We found that Rtn1p fractionates as a single peak that overlaps with the Sec61p peak, a marker for the ER, and is resolved from the Pma1p peak, a plasma membrane marker. This confirms that Rtn1p is an ER-associated protein.

View larger version (33K): [in this window] [in a new window]   Figure 3. Rtn1p is an ER integral membrane protein. (A) Wild-type cells (NY2658) expressing Rtn1-GFP were grown at 25°C in YPD. The lysate was loaded on top of a sucrose density gradient containing EDTA. Rtn1-GFP is represented by the solid black line, the ER marker Sec61p by the solid gray line, and the plasma membrane marker Pma1p by the dashed black line. (B) Wild-type cells (NY2659) expressing Rtn1-3HA were grown at 25°C in SC medium with. Cell lysates were incubated with reagents as indicated and separated into a pellet fraction P and a supernatant fraction S. The fractions were immunoblotted with anti-HA antibodies.

Overproduction of Rtn1p Alters the Distribution of Sec61-GFP
We have so far addressed the consequence of the loss of Rtn1p function, but it would also be interesting to determine whether the overproduction of Rtn1p has a demonstrable phenotype. To this effect, we transformed a wild-type strain expressing the ER marker Sec61-GFP with an intergrating plasmid containing the GAL1 promoter upstream of either GST or GST-Rtn1p. As shown in Figure 4, A and B, when the strain overproducing GST was grown on galactose-containing medium, 70% of the cells showed a wild-type structure for the ER. But when GST-Rtn1p was overproduced in the same strain, we observed round and bright Sec61-GFP–containing structures in the cytoplasm. These structures differ in appearance from karmellae, the multilayered ER membrane surrounding the nucleus that occurs in response to overproduction of Hmg1p (Wright et al., 1988).

View larger version (50K): [in this window] [in a new window]   Figure 4. Rtn1 overproduction alters ER structure. (A) Wild-type strain expressing the ER marker Sec61-GFP and either GST (NY2660), GST-Rtn1 (NY2661) under the control of the GAL1 were grown at 25°C in SC galactose medium. The cells were examined using an epifluorescence microscope. Arrows point to fluorescence patches. (B) Quantification of the altered cortical ER phenotype was done by scoring at least 300 cells with wild-type or altered morphology based on the presence of large puncta or the presence of large gaps in fluorescence at the cell cortex. The graph shows the percentage of cells having either ER structure.

View larger version (82K): [in this window] [in a new window]   Figure 5. TAP tag isolation of the intact exocyst complex. The exocyst complex was purified from 2 liters of culture using a C-terminal TAP tag on Sec10p. Twenty percent of the sample was run on a 7% polyacrylamide gel and silver stained. The eight exocyst subunits are identified with arrows. Proteins isolated from an untagged strain are shown for comparison.

Tandem mass spectrometry was performed on the purified exocyst preparation and on the control sample from the untagged strain to identify any novel proteins that were coisolated with the complex (Eng et al., 1994). One-half of the above-mentioned purification was precipitated and sent to the Proteomic Mass Spectrometry Laboratory at the Scripps Research Institute. Peptides present in the mixture were identified through a search of the protein database using the algorithm SEQUEST. An array of 135 proteins was identified in the tagged sample as opposed to 105 in the untagged control. All eight exocyst subunits were identified with the percent of coverage being higher (31–36%) for the smaller proteins (Exo70 and Exo84), and in the range of 17–26% for the other six proteins (Table 2). Most of the background proteins (44%) were found to be ribosomal in origin. Sixteen additional proteins specific to the exocyst isolation were noted and are available on the Yeast Resource Center’s public data repository Web page (http://www.yeastrc.org/pdr/). Among the 16 proteins specific to the exocyst TAP tag purification was Rtn1p. Mass spectrometry identified 13.2% coverage of the 34-kDa protein, approximately one-half the coverage identified for most exocyst subunits.

Approximately equal amounts of soluble exocyst proteins and either GST or GST-Rtn1 immobilized on glutathione beads was incubated together for 2 h. The beads were washed, and bound proteins were eluted, loaded on gels, transferred to membranes, and probed with anti-His antibody. Figure 6A shows that Sec6p is the only exocyst subunit that detectably binds Rtn1p. This result also implies that there is a direct interaction between the exocyst complex and Rtn1p. By comparison to a lane loaded with 30% of the amount of Sec6 input, we conclude that 15% of Sec6 binds to GST-Rtn1p under the conditions used. None of the exocyst proteins were found to bind to GST alone.

View larger version (30K): [in this window] [in a new window]   Figure 6. (A) Rtn1p binds to the Sec6p subunit of the exocyst. Soluble recombinant exocyst proteins tagged with 6xHis were incubated with recombinant GST or GST-Rtn1p immobilized on glutathione beads. Approximately 15% of the available Sec6p binds to Rtn1p based in comparison with the 30% input loaded on Western blots. (B) Sec6p binds to the hydrophilic loop of Rtn1p. A deletion series of the Rtn1 protein was constructed and expressed in E. coli. The proteins, immobilized on glutathione beads, were incubated with recombinant 6xHis-Sec6. The hydrophilic loop between the two transmembrane domains (TMD) of Rtn1 was capable of binding 30% of the available Sec6 protein. (C) As a control, GST-Snc1p was included in the binding assay. Again, the Rtn1 loop was sufficient to bind 30% of the available Sec6p, whereas no binding was observed between Sec6p and GST-Snc1p or GST alone.

Overexpression of Sec6p and Rtn1p
We next tested whether Rtn1p binds to Sec6p in yeast. Diploid strains were constructed using the GAL1 promoter to overexpress either GST alone, GST-Rtn1p alone, GST and Sec6p, GST-Rtn1p and Sec6p, or GST-Snc1p and Sec6p as a control. If a direct interaction between Sec6p and Rtn1p occurs in vivo, it should be exaggerated when the levels of both these proteins are increased. Glutathione beads were used to isolate GST, GST-Snc1p, or GST-Rtn1p from a culture grown overnight in 2% galactose. Figure 7 shows a Western blot detecting Sec6p in the five strains. The amount of Sec6p is much higher when driven by the GAL1 promoter, and therefore not as readily detected in the strains expressing endogenous levels. When Rtn1p and Sec6p were cooverexpressed, 0.5% of the Sec6p copurified with Rtn1p. The band seems to run slightly higher in the last lane due to the coincidence that Sec6p is similar in size to the large amount of Rtn1p isolated, causing the Sec6p band to slightly shift. No copurification of Sec6p with Snc1p was identified under these conditions. This finding, along with the mass spectrometry result, suggests that the exocyst and Rtn1p can interact in vivo.

View larger version (25K): [in this window] [in a new window]   Figure 7. Coprecipitation of Rtn1p and Sec6p when both proteins are overexpressed. The GAL1 promoter was used to overexpress GST, GST-Snc1p, GST-Rtn1p, Sec6p, or combinations of these proteins. GST, GST-Snc1p, or GST-Rtn1p was isolated on glutathione bead, and Sec6p was detected on a Western blot using a Sec6p antibody. The amount of Sec6p is much lower when not driven by the GAL1 promoter, and therefore not as readily detected in samples with endogenous levels. When both Rtn1p and Sec6p are overexpressed in the same strain, 0.5% of the available Sec6p (I) is purified (P) with Rtn1p. This band is slightly higher because Sec6p is similar in size to the large amount of Rtn1p isolated, thus causing a slight shift in mobility. No copurification between GST or GST-Snc1 and Sec6 was observed.

All four mutagenized constructs, as well as wild-type RTN1, were introduced by transformation into an rtn1 strain. Soluble protein was isolated and bound to ConA-Sepharose. Unbound protein (0.25% of total volume) was loaded beside protein (2.5%) that bound to ConA beads on a polyacrylamide gel and a C-terminal HA tag was used to detect Rtn1p. Not only did none of the engineered glycosylation sites cause a shift in mobility of Rtn1p, none of them caused Rtn1p to bind to the ConA beads (Figure 8). As a control, a parallel filter was immunoblotted using an antibody directed against carboxypeptidase Y, a protein that is known to be glycosylated. Carboxypeptidase Y was efficiently bound by the ConA beads. Although this evidence does not conclusively prove that these domains of Rtn1p are exposed to the cytosol, they do suggest that they are not exposed to the lumen of the ER and therefore are likely to be appropriately oriented to interact with the exocyst.

View larger version (25K): [in this window] [in a new window]   Figure 8. Orientation of the Rtn1 loop. Three different asparagine-linked glycosylation sites were created in the loop by mutating amino acids 94, 116, and 125 to a serine, asparagine, and asparagines, respectively. A fourth glycosylation site was added near the C terminus of the protein by mutating amino acid 215 to a serine. Soluble protein was isolated and unbound protein (I; 0.25% of total volume) was loaded beside protein (P; 2.5%) that bound to ConA beads. A C-terminal HA tag was used to detect Rtn1p. None of the engineered glycosylation sites caused a shift in mobility, and likewise none of them caused Rtn1p to bind to ConA beads. This negative result suggests these domains are not exposed to the lumen and therefore likely oriented to the cytosol.

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View larger version (67K): [in this window] [in a new window]   Figure 9. Rtn1p loop overproduction affects ER structure. (A) Wild-type strain expressing the ER marker Sec61-GFP and either GST (NY2660) or GST-loop (NY2662) under the control of the GAL1 was grown at 25°C in SC galactose medium. The cells were examined using an epifluorescence microscope. Arrows point to fluorescence patches. (B) Quantification of the altered cortical ER phenotype was done by scoring at least 300 cells with wild-type or altered morphology based on the presence of large puncta or the presence of large gaps in fluorescence at the cell cortex. The graph shows the percentage of cells having either ER structure.

As a more sensitive test of secretory function, we examined thin sections of the same strains by electron microscopy. By quantitative analysis of the number of vesicles in buds of both strains, we determined that in the strain overproducing GST, the vesicle density was 0.82 vesicles/µm², whereas overproduction of GST-loop led to a density of 2.84 vesicles/µm² (Figure 10). This suggests a very mild but significant increase.

View larger version (98K): [in this window] [in a new window]   Figure 10. Rtn1p loop overproduction interferes with secretion. Thin section EM micrographs of wild type transformed either with a 2µ plasmid bearing GST or GST-loop under the control of the GPD1 promoter (NY2678 and NY2679). Cells were grown on SC medium and fixed with glutaraldehyde and stained with OsO4 and embedded in Spurr. Arrows point to vesicles. Magnification, 6500x.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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Prinz et al. (2000) have observed that mutations that inhibit ER-to-Golgi transport result in the formation of a largely cisternal ER. Such mutations also result in dramatic expansion of the surface area of the ER as membrane destined for export accumulates in the ER (Novick et al., 1980). In contrast, rtn1 cells exhibit no significant increase in the surface area of the ER, despite the dramatic change in ER organization. This is consistent with the observation that there is no intracellular accumulation of the secreted protein invertase (our unpublished data) or any slowing in the growth rate of rtn1 cells, as would be expected of a mutation affecting ER export. Therefore, it is likely that the change in ER organization observed in rtn1 cells reflects a direct role for Rtn1p, rather than an indirect effect of a transport block.

Rtn proteins are defined by the presence of a reticulon domain, consisting of two transmembrane domains linked by a conserved hydrophilic loop (Oertle et al., 2003). All members analyzed to date localize exclusively to the ER with the sole exception of the Rtn4 protein, which is also partially localized to the plasma membrane. The localization of Rtn1-GFP that we report here and that Geng et al. (2005) have recently reported is consistent with this pattern. Interestingly, Rtn1-GFP is enriched in the cortical ER relative to the nuclear envelope. This enrichment could either reflect the partial exclusion of Rtn1-GFP from the nuclear envelope or the partial retention of Rtn1-GFP at the cell cortex. Additional studies will be needed to resolve these two possibilities.

We have also shown that Rtn1p, like its mammalian homologues, behaves as an integral membrane protein in extraction studies. Furthermore, we show that the hydrophilic loop connecting the two transmembrane domains is most likely cytoplasmic in its orientation, because it does not seem to be accessible to the glycosylation machinery in the lumen of the ER. This orientation is opposite to that proposed for its mammalian homologues (GrandPre et al., 2000). However, that proposal was based on the orientation of the small pool of Rtn4 that reaches the plasma membrane, which may differ in orientation from the bulk of Rtn4p that remains in the ER. More recently, Voeltz et al. (2006) have shown in COS cells that the NogoC loop as well as its hydrophilic N terminus faces the cytoplasm, in agreement with our topology for Rtn1p in yeast.

The identification of Rtn1p as a binding partner of the exocyst is very interesting in light of the documented role of the exocyst in ER inheritance. Previous work has shown that at least three subunits of the exocyst, Sec3p, Sec5p, and Sec8p, are involved in ER inheritance (Wiederkehr et al., 2003, 2004; Reinke et al., 2004). In sec3 cells, an ER tubule often migrates into the bud, but it fails to be retained at the bud tip, as it is in wild-type cells (Wiederkehr et al., 2003). This phenotype has suggested a model in which the exocyst serves as an anchor for the ER at the bud tip early in the cell cycle; however, the mechanism by which the ER becomes attached to this anchor has been unclear. One suggestion has been that the translocon serves as the link to the exocyst, because it was also shown that the translocon subunit Sec61p interacts with the exocyst (Toikkanen et al., 2003; Lipschutz et al., 2003). Our binding data suggest that Rtn1p might also serve as a receptor for the exocyst on the ER. The cytoplasmic orientation of the hydrophilic loop of Rtn1p is consistent with our identification of this loop as a binding partner for the Sec6p subunit of the exocyst. In addition, the apposition of the ER to the plasma membrane in yeast is also consistent with an interaction of the exocyst with Rtn1p. The elongated shape of the exocyst (Hsu et al., 1998; Dong et al., 2005) would allow it to bridge the small gap between the two membranes. Nonetheless, we did not observe a clear ER inheritance defect in rtn1 cells, suggesting either that Rtn1p is redundant with the translocon in this function or that the Rtn1–exocyst interaction serves a function other than anchoring the ER at the bud tip. The exocyst might signal to Rtn1p to direct the formation of reticular ER along the cortex of the bud from the ER tubules that have attached to the bud tip. Thus, in an rtn1 mutant, the ER would still migrate into the bud and spread along the cortex, but it would not be able to form a reticulum.

It is noteworthy that in mammalian tissues, cell function dictates the structure of the ER. Cell types that specialize in protein secretion have a greater amount of rough ER, which is typically cisternal in structure. In contrast, cells types that are involved in hormone synthesis, fat metabolism, calcium storage, and detoxification have more smooth ER, which is typically reticulated in structure. Thus, the morphology of this organelle is regulated by cell function (reviewed in Baumann and Walz, 2001). In some cell types, this regulation is dynamic. In hepatocytes for instance, the ER is more cisternal in unchallenged cells but upon drug challenge, the reticulated portion increases (Remmer and Merker, 1963). It will be important to determine whether any or all of the Rtn proteins in mammalian cells act to regulate the structure of the ER and whether the differential expression of the four Rtn proteins and their many isoforms (Roebroek et al., 1998; GrandPre et al., 2000) determines the structure of the ER in different cell types.

	ACKNOWLEDGMENTS

 
We thank C. Chalouni for help with confocal microscopy and A. Hartley for the 3D reconstructions. We also like thank Prof. A. Merz for help with ratiometrics. This work was supported by National Institutes of Health Grants GM-73892 and GM35370 (to P. N.) and CA-46128 (to S.F.-N.).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-01-0080) on April 19, 2006.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Peter Novick ( peter.novick{at}yale.edu)

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