Correspondence to: David I. Meyer, Department of Biological Chemistry, University of California, Los Angeles School of Medicine, Los Angeles, CA 90024-1737., dimeyer{at}ucla.edu (E-mail), (310) 206-3122 (phone), (310) 206-5197 (fax)
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Abstract |
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Expression of the canine 180-kD ribosome receptor (p180) in yeast cells resulted in a marked proliferation of intracellular membranes. The type of membranes observed varied with the expression of specific portions of p180. Rough membranes predominated when the ribosome binding domain of p180 was present, whereas expression constructs lacking this region resulted in smooth membranes. Northern analysis indicated that expression of the NH2-terminal 767 amino acids (CT), which include the ribosome binding domain, upregulated the transcription and translation of genes involved in exocytosis. The membranes that were proliferated were functional as these cells overcame a temperature-sensitive translocation defect. Most significantly, cells that overexpressed
CT and proliferated rough endoplasmic reticulum exhibited severalfold higher levels of secretion of an ectopically expressed secretory protein. We conclude that p180 expression triggers a cascade of events leading to an increase in secretory potential akin to the terminal differentiation of mammalian secretory cells and tissues.
Key Words: secretion, endoplasmic reticulum, membrane biogenesis, yeast, ribosome receptor
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Introduction |
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THE entry of proteins into the secretory pathway via the rough endoplasmic reticulum (ER) is essential for intracellular transport of proteins to the extracellular milieu as well as to a number of compartments within the cell (for review see
Pioneering studies by Palade, Rutter, and others documented a massive proliferation of membranes during the ontogeny of hepatocytes and cells of the pancreas (
The most obvious morphological change seen in liver, pancreas, and B cells is the proliferation of rough ER. Studies by Sabatini and others have shown that in such tissues the polysomes associated with the ER are predominantly synthesizing secretory products (for review see -helical double-stranded coiled-coil rod (
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A remarkable feature of p180 was first seen in studies in which the gene was expressed in yeast. When constructs encoding p180 were expressed under the control of a regulatable promoter, a rapid and significant proliferation of membranes occurred (
In this study we have ectopically expressed constructs encoding different forms of p180 in yeast. The transfected cells take on morphologies reminiscent of highly developed mammalian secretory cells. Our molecular and biochemical studies indicate that an upregulation of the secretory pathway has taken place with a striking increase in secretory capacity. These results support using yeast as a genetically manipulable model for the study of the differentiation of (mammalian) secretory cells.
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Materials and Methods |
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Construction of Expression Plasmids
Regulated expression of the constructs used in this study was achieved through the use of the vector pYEX-BX that utilizes the copper-inducible promoter from the CUP1 gene (Amrad Biotech). For the construction of the p180FL, CT, and
NT we cloned BamHI-SalI fragments of the vectors pRRFL-EW1, pBSRR
CT, and pBSRR
NT (
The fragment was cloned into pYEX-BX using BamHI and SalI sites. Plasmids were transfected into Escherichia coli XL1-Blue by the method of leu2-3,112 ura3-52 his3-
200 trp1-
901 lys2-801 suc2-
9) (
Cells were grown on SD or Sgal medium for experiments using galactose induction. SD medium, with or without 50 mM copper sulfate, was used for experiments in which proteins were expressed under control of the CUP1 promoter. SEY 6210 was the strain used for all experiments. After 5 h of growth, transformed cells were in logarithmic phase and were used for further study.
Electron Microscopy
Yeast cells were spheroplasted with oxalyticase in SD sorbitol buffer, fixed in 2% glutaraldehyde, and postfixed with 1% OsO4 in sodium cacodylate solution. Samples were dehydrated in ethanol and embedded in Spurr (Ted Pella, Inc.). Sections ~60 nm thick were made with an MT6000-XL ultamicrotome (RMC, Inc.) and stained with uranyl acetate and lead citrate. Sections were examined with a JEM-1200EX electron microscope (JEOL).
RNA Isolation and Northern Blot Analysis
RNA isolation was performed by the method of Hollingworth et al. (1990). Total RNA (10 µg) was separated on a 1.2% formamide containing agarose gel (
Northern blots were quantified using a PhosphorImager and Imagequant software (Molecular Dynamics). All lanes were corrected for loading inconsistencies by normalization to expression of PYK1. Values expressed are relative to vector-only controls.
Immunofluorescence Microscopy
The antiserum to RRp has been described previously (
Complementation of sec63
The temperature-sensitive strain ptl1/sec63 (
Rescue from BPTI Toxicity
SEY 6210 were cotransformed with a galactose-inducible high copy plasmid expressing secreted bovine pancreatic trypsin inhibitor (BPTI)1 (NT, or -
CT. Following induction of copper-dependent expression of the p180 constructs, 10-fold serial dilutions were plated on glucose or galactose containing media and incubated for 3 or 5 d at 30°C. For quantification of the secreted BPTI in the supernatant, aliquots were tested as described by
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Results |
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p180 Expression Results in Extensive Rough Membrane Proliferation in Yeast
Preliminary observations indicated that the expression of various domains of p180 led to membrane proliferation in yeast (
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This result was quite different from the types of membranes observed in cells where proliferation had been induced by the expression of the HMG1 gene (
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Expression of Different Parts of p180 Results in the Proliferation of Intracellular Membranes Having Different Morphologies
We constructed two versions of p180 harboring deletions in major domains (Figure 1). The first lacks the ribosome binding domain (the 54 tandem repeats of the decapeptide motif) and is referred to as NT. The second lacks the COOH-terminal coiled-coil forming domain and is referred to as
CT (see Materials and Methods and Wanker et al., 1995 for details). Expression of
NT resulted in the proliferation of smooth membranes, with consistent 80100 nm spacing, from the perinuclear region to the cell periphery (Figure 5). The smooth appearance would be expected, as the
NT construct lacks the ribosome binding domain. In fact, ribosomes are selectively excluded from areas of the cell where the smooth membranes are located, and are instead restricted to more peripheral areas of the cell. This is quite different from the random distribution of ribosomes throughout the cytosol of wild-type yeast (Figure 2). It is interesting to note, however, that the presence of the COOH-terminal domain on the
NT construct results in a definitely nonkarmellar type of membrane proliferation. In this case, the spacing between membranes is far greater than in that of karmellae, and could be a feature of the ability of
NT to interact through its putative coiled-coil domains with cytosolic components.
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A different, yet equally striking morphology was observed in the case of the CT construct. In this case, membrane spacing collapses to that closely resembling karmellae, yet there is no restriction to the perinuclear area, and several areas of proliferated membranes are observed at the cell periphery (Figure 6). In contrast to
NT, the membranes have attached ribosomes, as indicated by the dense staining in the intermembrane space, and a lower density of free cytosolic ribosomes compared with
NT.
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From these data, obtained through the expression of the various domains of p180, we can make the following provisional conclusions about the morphology of the induced membranes: The presence of the NH2-terminal 151 amino acids enables membrane proliferation in general, with the closely spaced perinuclear appearance of karmellae. The presence of the repeat region enables ribosome binding irrespective of membrane distribution or spacing, and the presence of the COOH-terminal domain enables a large intermembrane separation.
p180 Expression Results in the Coordinate Expression of Rough ERspecific Genes
These results beg the question as to the functionality of the proliferated membranes. Are they merely lipid bilayers produced to soak up an excess of ectopically expressed membrane protein, or do they represent bona fide rough and smooth ER? The first answer to this question comes from an examination of levels of expression of genes encoding resident proteins of these membrane systems. Northern blotting (Table 1) was carried out on RNA derived from p180FL, NT,
CT expressing strains, vector-only controls, and cells that overexpressed HMG1 and HMG2 genes. Overexpression of HMG1 induces karmellae, whereas HMG2 expression induces short karmellae, parallel membrane strips near the cell periphery, or membranous whorls in the cytoplasm (
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Overexpression of all of the constructs resulted in an upregulation of KAR2, ranging from a low of 2.2-fold in the case of NT to a high of 3.8-fold for
CT (Table 1). In contrast, transcripts encoding rough ER membrane proteins were upregulated to the greatest extent in strains in which the ectopically expressed versions of p180 contained the ribosome binding domain (p180FL and
CT). SEC61 was the most highly expressed of all, where close to 15-fold higher levels were achieved in cells expressing
CT. In the same strain, SEC72 and OST1 expression increased 3.6- and 6.9-fold, respectively. In contrast, the HMG1 HMG2 overexpressing strains showed significant increases only in KAR2 expression. Cells expressing the p180 construct lacking the ribosome binding domain (
NT) showed only a (comparatively) moderate upregulation of ER markers, despite high levels of membrane proliferation (see Figure 5).
Increased levels of transcription of membrane protein genes translates into increased levels of the proteins they encode (CT expression show a marked increase in the intensity of anti-Sec61p staining (Figure 7 D) comparable to the detection of p180 using anti-p180 antibodies (Figure 7 C). The localization of the fluorescence in the
CT strain overlaps nicely with the location of the proliferated membranes seen in the electron microscope (Figure 6). This result is a good predictor that functional membranes are being produced.
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Rough Membranes Produced by p180 Expression Are Functional
To establish the functionality of the proliferated rough ER, we turned to a genetic approach. We used a previously isolated strain that harbors a temperature-sensitive translocation defect. Originally isolated as ptl1 (CT expression, in which growth was virtually the same as vector-only at the permissive temperature. In contrast, membrane proliferation alone did not appear to be determinative in rescue, as expression of either HMG1 or
NT was not much better than vector-only. From these sets of experiments we conclude that the rough membranes produced in response to
CT expression are functional rough ER, and, from the previous data, that membrane composition is at least qualitatively similar to wild-type.
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p180 Expression Results in Coordinate Expression of Secretory Pathway Genes
The results presented to this point indicate that functional rough ER is being produced in response to CT expression. Are the more distal aspects of the secretory pathway being induced as well? We answered this question in two ways: by Northern analysis of marker genes for organelles that function post-ER in the secretory pathway, and morphologically using immunofluorescent detection of the Golgi complex. The following markers were examined: Sac1p, which is involved in nucleotide transport and has been localized to ER and to Golgi membranes; Gda1p, which encodes the guanosine diphosphatase required in the Golgi complex for oligosaccharide elaboration; and Sec1p, which is a cytosolic/peripheral membrane protein required in the exocytic fusion of secretory vesicles with the plasma membrane. The results shown in Table 1 demonstrate that transcripts encoding SAC1, GDA1, and SEC1 gene products were all produced at significantly (510-fold) higher levels in cells expressing
CT compared with controls. In contrast, inducers of smooth membranes, such as the HMG genes or
NT expressed these markers at levels close to those of the vector control. Although one cannot test for all genes involved in secretion, these threeas well as the ER markers assessed previouslywould likely participate in any process that would increase the secretory capacity of the cell.
Fluorescence microscopy on control and CT expressing cells was performed to further characterize the expression of post-ER markers. We used an anti-Gda1p antibody to reveal the Golgi complex in these cells. As can be seen in Figure 9 A, a single Golgi body can be visualized in vector-only cells. In contrast, numerous Golgi appear in
CT-expressing cells (Figure 9 B), some larger and some smaller. Taken together, these data suggest that the entire secretory pathway may be upregulated in response to
CT expression.
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p180 Expression Results in Increased Secretory Capacity
To get an approximation of the secretory capacity of wild-type yeast, we transfected control strains with a plasmid encoding BPTI. BPTI is a low molecular weight protein that can be easily measured colorimetrically when it appears in the growth medium. The assay is based on the ability of BPTI to inhibit trypsin hydrolysis of an artificial substrate. Accordingly, BPTI was expressed under GAL control in glucose or in galactose-containing medium. Interestingly, once levels of BPTI secretion approach 810 µg/ml in wild-type cells grown under standard culture conditions, cell growth is inhibited, ostensibly by blocking or overwhelming the secretory process (NT cells were unable to grow when BPTI was expressed through galactose induction for a period of 24 h. On the other hand, cells expressing BPTI were rescued by the expression of p180FL or
CT. Our preliminary conclusion was that an increase in secretory capacity enabled toxic quantities of BPTI to be removed from the cells. Recent studies by Wittrup and co-workers (
CT induces increases in levels of transcripts encoding these proteins (Table 1 and Becker, F., unpublished results).
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Measuring BPTI accumulation in the medium substantiated the hypothesis that the toxic effect of BPTI was overcome through increased secretion. In the case of cells grown on glucose, BPTI secretion was undetectable. In the case of cells expressing CT and grown on galactose, levels of BPTI that accumulated in the medium during the assay rose >400% compared with control cells (Figure 11). In contrast, membrane proliferation aloneas observed with
NT expressionaffected neither rescue from BPTI-mediated growth arrest (Figure 10), nor BPTI levels in the medium (data not shown). We take this to be direct proof of an increased secretory capacity mediated through the expression of
CT.
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Discussion |
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Our findings of an explosive proliferation of functional membranes coupled to an overall increase in secretory capacity in response to the heterologous expression of a single protein raise a number of intriguing questions about the regulation of membrane biogenesis. Previous reports, spanning more than three decades, have documented smooth membrane proliferation as a physiological response to the administration of xenobiotics (
In the case of p180, a strikingly different morphology was observed, depending upon which part of p180 was expressed. Expression of the full-length receptor resulted in a proliferation of widely spaced rough membranes. The 80100 nm spacing correlated with the presence of the COOH-terminal half of p180, as the NT construct induced smooth membranes with a considerable distance between them. In contrast, expression of
CT as well as p1-151 produced rough and smooth membranes, respectively, both possessing a very tightly packed appearance. Based on these data, one could make the prediction that the three separate domains induce three different aspects of rough membrane biogenesis. Production of membranes per se requires the expression of a membrane anchor and some minimum number of cytoplasmically exposed amino acids, e.g., p1-151. Adding on a ribosome binding domain, as in
CT, triggers the induction of functional rough ER, while addition of the COOH-terminal domain, as in p180FL and
NT, results in the typical uniformly spaced parallel arrays of membranes seen in mammalian secretory tissues (
The observation that p180 seems to reach its highest expression levels in tissues with high secretory capacity (
Why is there no p180 in yeast? The answer to this comes from the same data alluded to above. Yeast cells do not have the secretory capacity of mammalian pancreas or liver. Therefore, just as many mammalian cell types exist with minimal or no expression of p180 (
Equally if not more intriguing is the question of how the expression of p180 results in the proliferation of membranes in the secretory pathway in a cell in which it is not normally expressed. Hypotheses can be based on the elements of the protein mentioned previously, and their functional properties. It is clear that the membrane anchoring domain plus a few dozen amino acids of the cytosolic domain induces lipid bilayer proliferation. One can expect that the synthetic machinery needed for lipid biosynthesis is switched on in these cases via transcription of the requisite enzymes. This postulate is borne out by studies on other membrane proteins (NT did not induce SEC61 or any of the other ER resident proteins; their expression correlated with expression of the ribosome binding domain. How then does the addition of this domain make such a difference in the number of genes that are being upregulated, and is the process dependent upon its ability to bind ribosomes with high affinity? It is tempting to speculate that the cell erroneously mobilizes greater secretory capacity due to a loss of free ribosomes from the cytosol, and that sensing this loss is the key step in induction of the relevant genes. If this is true, one could expect that the high level of expression of any secretory or membrane protein would induce the upregulation of secretion as long as it removes ribosomes from the cytosolic pool and directs them to the membrane. This has not yet been systematically investigated. On the other hand,
Recent studies have elucidated an elaborate signaling pathway in rough ER. In response to a buildup of unfolded or misfolded proteins, a cascade is triggered that results in increased chaperone production (for review see
The work described here makes use of the canine p180 cDNA that was cloned in our laboratory. Extensive data are now available on the human p180 homologue (
Elucidation of the mechanism of membrane induction by p180 will benefit greatly from the observations made here in the yeast system. Through screening strategies it should be possible to identify genes whose products are capable of stimulating the upregulation of genes essential for membrane biogenesis. Moreover, similar schemes will enable mutant strains to be identified that lack the ability to upregulate membrane biogenesis in response to p180 expression. Identification and characterization of such genes and their products should provide the required toehold for further analysis of this interesting eukaryotic regulatory pathway.
It will also be interesting to analyze rough ER membrane induction by p180 in mammalian cells. Preliminary studies show that increasing cellular levels of p180 significantly stimulates rough membrane induction in nonsecretory cells (Castro-Vargas, E., F. Becker, and D.I. Meyer, unpublished observations). This implies that the expression of p180 may play a central role in the terminal differentiation of secretory tissues. The fact that a similar phenomenon is observed in mammalian cells makes the use of the genetically manipulable yeast model described here especially attractive.
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Footnotes |
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Frank Becker's present address is Genome Pharmaceutical Corporation AG, Lochhamerstrasse 29, D-82152 Martinsreid, Germany.
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Acknowledgements |
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We are grateful to Sergey Ryazantsev for his help with preparation of samples for electron microscopy, and to Ed Castro-Vargas and Chien-Min Chen for technical support. Thanks also to Peter Walter (University of California, San Francisco) for ire strains, Randy Schekman for anti-Sec61p, and Greg Payne for anti-Gda1p antibodies. We also thank Maureen Hyde, Ed Castro-Vargas, and members of the Payne group for helpful discussions and criticism.
Frank Becker was supported by a fellowship from the Deutsche Forschungsgemeinschaft (DFG). This work was supported by a grant from National Institutes of Health (GM 38538).
Submitted: December 3, 1998; Revised: June 15, 1999; Accepted: June 17, 1999.
1.used in this paper: BPTI, bovine pancreatic trypsin inhibitor; UPR, unfolded protein response
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