rtn1Δ 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 rtn1Δ
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 (, 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 rtn1Δ
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 (A). Indeed <5% of the cells examined showed a wild-type structure of the cortical ER (B). Although the cortical ER is contiguous with the nuclear envelope, there was no change evident in the distribution of the fluorescence around the nucleus.
Figure 1. Rtn1p is required for the structure of the ER. (A) Wild type (NY2647, NY2649) and rtn1Δ 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 (more ...)
To better ascertain exactly how the cortical ER was altered, rtn1Δ cells were examined by thin section electron microscopy (EM) after permanganate treatment to highlight the ER. Using a double lattice grid, we measured the average length of cortical ER membrane segments in wild-type and in rtn1Δ cells (C). In wild-type cells, the average length of an ER membrane segment is 0.6 ± 0.2 μm, whereas in the rtn1Δ cells the average length is 1.7 ± 1.3 μm. The ER segments in the mutant are thus threefold longer than in the wild-type, but also more variable, confirming that in an rtn1Δ mutant, the structure of the cortical ER is altered. This modification of the ER does not reflect a change in the overall surface area of the cortical ER. Indeed, the average surface of the ER in rtn1Δ cells is 1.6 ± 0.7 μm2/μm3, which is not significantly different from the 1.5 ± 0.6 μm2/μm3 measured in wild type. Other membrane-bound organelles were normal in appearance.
We also performed a 3D optical reconstruction of the cortical ER in wild-type and rtn1Δ 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 rtn1Δ 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
(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 rtn1Δ
mutant. We have confirmed this by comparing the secretion of invertase in an rtn1Δ
mutant to a wild type strain and found no difference (our unpublished data). This is consistent with the normal generation time of an rtn1Δ
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 rtn1Δ on Other Organelles
Because the rtn1Δ
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 rtn1Δ
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 rtn1Δ
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 rtn1Δ
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 , 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 rtn1Δ
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.
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.
Further proof of Rtn1-GFP functionality was obtained when we performed a 3D reconstruction of Rtn1-GFP localization (Supplemental Figure S4). This showed that Rtn1p is localized in a reticulated structure very similar to that observed with Sec61-GFP in wild-type cells (Supplemental Figure S1), supporting our hypothesis that Rtn1p is on the cortical ER. We also observe tubules emanating from this cortical structure oriented toward the center of the cell where the unlabeled nucleus resides.
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 (A). 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.
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, (more ...)
We then set out to determine whether Rtn1p is loosely associated with or tightly bound to the ER membrane, as are its mammalian homologues (Senden et al., 1994
; Van de Velde et al., 1994
). The region that identifies a protein as a member of the Rtn family is called the reticulon, a domain defined by two large (>30 aa) transmembrane domains separated by a conserved, hydrophilic loop of ~60 amino acids (Oertle et al., 2003
). In Rtn1p, the two transmembrane domains are poorly defined because they are predicted to be only weakly hydrophobic and the loop is ~80 amino acids. To assess whether Rtn1p is indeed an integral membrane protein, we performed an extraction study on a total yeast lysate using high salt, high pH, nonanionic detergent and anionic detergent (B). Rtn1p remained predominantly in the pellet in the presence of 0.5 M NaCl or 0.1 M Na2
but shifted to the supernatant in the presence of either detergent, indicating that although it has nonconventional transmembrane domains, it is an integral membrane protein.
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 , 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
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 (more ...)
TAP Tag Purification of the Exocyst and Mass Spectrometry
In an independent study we sought to identify novel proteins that interact with the exocyst complex. The Sec10p subunit was tagged with a C-terminal TAP tag, and this construct was expressed as the sole copy of SEC10
. Sec10-TAP was purified from a 2-liter culture of using a variation of the published methods (Puig et al., 2001
). In preliminary trials, we performed the isolation over a pH range and found pH 6.8 to be optimal for maintaining the octameric complex intact. This is consistent with our earlier observations regarding immunoisolation of the exocyst (Terbush et al., 1996
). Twenty percent of the purification was loaded on an SDS gel and after silver staining, eight bands were identified above the background bands that were present in a mock isolation from an untagged strain (). Four of the bands were confirmed as exocyst subunits by Western blot using Sec6p, Sec8p, Sec10p, and Sec15p polyclonal antibodies (Salminen and Novick, 1989
; Bowser et al., 1992
; Potenza et al., 1992
; our unpublished data). Antibodies to the other four subunits were not available.
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 (more ...)
The yield from 2 liters of culture was ~2–4 μg of intact exocyst complex based on a comparison with known amounts of bovine serum albumin protein. Therefore, each subunit comprises 0.06% of the 600 mg of soluble protein used in the purification. A similar abundance was calculated when the exocyst was purified using a 3xmyc tag (Terbush et al., 1996
). Based on band intensity of the silver stain gel (), all of the subunits except Sec3p seem to be approximately stoichiometric. The reduced amount of Sec3p may be a result of degradation, as was observed by Terbush et al. (1996)
, or it may partially dissociate during purification.
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 (). 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.
Results of mass spectrometry on the exocyst complex purification
In Vitro Binding Assays for Rtn1
To determine whether there was a direct interaction between the exocyst complex and Rtn1 and to define the interacting subunit, all eight exocyst subunits were separately expressed in bacteria, and purified using a 6xHis tag. Expression levels varied, with Sec6p, Exo70p, and Exo84p being purified in higher quantities than the other five subunits from the same volume of culture (our unpublished data). Rtn1p was N-terminally tagged with GST, expressed in bacteria and purified with glutathione beads. As a control, the GST protein was also purified.
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. A 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.
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 (more ...)
To determine which domain of Rtn1p binds to Sec6p, six different sections of the protein were used in an in vitro binding assay. Two transmembrane domains in Rtn1p are predicted according to the Saccharomyces
Genome Database (http://www.yeastgenome.org/
). These two domains, surrounding an 84-amino acid hydrophilic loop, constitute the reticulon domain. Recombinant proteins containing the reticulon domain were found to bind Sec6p, whereas proteins with only the N or C terminus did not (B). When the reticulon domain was broken down even further, the hydrophilic loop between the transmembrane domains was found to be sufficient to bind Sec6p. Snc1p, a small transmembrane domain protein involved in fusion of secretory vesicles, was expressed as a GST fusion protein and used as a control. No binding between Sec6p and Snc1p was observed under the conditions used (C).
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. 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.
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 (more ...)
Generation of Glycosylation Sites in Rtn1
If the hydrophilic loop within the reticulon domain of Rtn1p interacts with the exocyst, as our binding experiments imply, then we would predict that loop to be oriented toward the cytosol rather than toward the lumen of the ER, because the exocyst is a cytosolic complex. To determine the orientation of the yeast reticulon loop, three different asparagine-linked glycosylation sites were added to the Rtn1p loop at amino acids 92, 116, and 125. A fourth glycosylation site was added near the C terminus of the protein at amino acid 213. If any of these regions were exposed to the lumen of the ER, we would expect them to be glycosylated. Such glycosylation would be revealed by a shift in mobility on a Western blot using a C-terminal HA tag on Rtn1p and by precipitation using ConA-Sepharose beads, which bind specifically to mannosyl and glucosyl residues of polysaccharides and glycoproteins in the presence of calcium and manganese ions.
All four mutagenized constructs, as well as wild-type RTN1, were introduced by transformation into an rtn1Δ 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 (). 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.
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 (more ...)
The Rtn1p Loop Is Involved in Organizing the Structure of the ER
So far, we have shown that the loop of Rtn1p is cytoplasmic and interacts with the exocyst subunit Sec6p. To assess the function of this loop in ER organization, we transformed a wild-type strain expressing Sec61-GFP with an integrative plasmid carrying the GAL1 promoter to overproduce either GST or GST-loop. As shown in A, overproduction of GST alone did not strongly affect the perinuclear and cortical localization of Sec61-GFP (B). We did note an increase in the percentage of cells showing a gap in the cortical ER that correlated with growth in galactose; however, in general, the ER structure was very similar to that observed when cells are grown on glucose. Alternatively, the overproduction of the loop resulted in a significant increase in the number of cells having an altered ER structure, similar to that observed in rtn1Δ cells (A), yet not as dramatic. Indeed, 50% of the cells had a wild-type ER structure and 40% had a structure reminiscent of the rtn1Δ mutant (B). This supports the possibility that this loop is involved in organizing the structure of the ER.
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 (more ...)
Rtn1p Loop Overproduction Leads to an Accumulation of Secretory Vesicles
Last, we addressed the question as to whether this interaction between the loop of Rtn1p and Sec6p has any effect on the role of Sec6p in secretion. We compared the percentage of invertase secreted by wild-type cells transformed with a 2μ plasmid bearing GST under the strong GPD1 promoter to wild type transformed with a plasmid bearing the GST-loop in a parallel construct. We measured no significant difference in the secretion of invertase between the wild type overproducing GST and the wild type overproducing GST-loop (our unpublished data). This indicated that no major effect on secretion resulted from the overproduction of GST-loop. We did notice that overproduction of the GST-loop resulted in slower growth compared with GST overproduction.
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 (). This suggests a very mild but significant increase.
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 (more ...)
Finally, we assessed the effect of the overproduction of GST or GST-loop on the cut-off temperature of all late acting sec mutants as well as one mutant affecting each of the earlier steps of the secretory pathway. As shown in , mutants sec2-41, sec6-4, sec8-9, sec5-24, and sec15-1 are the most strongly affected by the overproduction of the loop. Alternatively, sec1-1 and more surprisingly sec3-2 are only weakly affected, if at all. We could not assess the effect on sec9-4 because its cut-off temperature is 30°C. In all, we did observe a genetic interaction between the late-acting sec mutants and Rtn1 loop. These results support our hypothesis that the hydrophilic loop of Rtn1p interacts with Sec6p.
Rtn1 loop overproduction decreases the cut-off temperature of most sec mutants