From the
Department of Medicine and the Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the ¶Departments of Physiology and Medicine and ||Departments of Anatomy, Biochemistry, and Biophysics, University of California, San Francisco, California 94143
Received for publication, December 26, 2002 , and in revised form, January 31, 2003.
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ABSTRACT |
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INTRODUCTION |
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Polarized epithelial cells have two distinct plasma membrane domains with very different protein compositions and functions: an apical domain comprising the luminal surface and a basolateral domain contacting adjoining and underlying cells. To establish and maintain their polarity, epithelial cells synthesize and then deliver specific proteins to the correct apical or basolateral plasma membrane (7). Madin-Darby canine kidney (MDCK) cells represent one of the best studied polarized epithelial cell lines and are derived from canine renal tubular epithelium (8). When grown on a permeable filter, MDCK cells form a well polarized epithelial monolayer, exhibiting apical and basolateral plasma membrane domains with unique compositions (9, 10), and well defined cell-cell junctional complexes containing tight junctions (11).
There are circumstances in which the cell might need to regulate the level of whole classes of proteins, such as those found in the plasma membrane or in particular in the basolateral plasma membrane of polarized epithelial cells. For example, cells must often alter their shape, polarization, and other characteristics during development and differentiation, and this plasticity could require the coordinate regulation of large classes of proteins. We have experience with two in vitro models where polarized epithelial cells coordinately regulate the abundance of all of the proteins at their basolateral plasma membrane. First, when these cells are dissociated by trypsin and EDTA and then plated in culture, the cells are initially relatively flat. As the cells establish contacts and confluency, they become columnar and their lateral surfaces increase greatly. This process resembles the polarization of epithelial cells that occurs during normal development. Second, when MDCK cells are grown in three-dimensional collagen gels, they form hollow cysts lined by a monolayer of cells with their apical surfaces facing the center (12). Addition of hepatocyte growth factor causes the cells to first send out long extensions of their basolateral plasma membrane, which requires an increase in basolateral surface area. Eventually, cells migrate out and reorganize into tubules in a process resembling tubulogenesis in vivo (13, 14).
One candidate for a component of the machinery in this process is the exocyst (or Sec6-Sec8) complex, first identified as being involved in the exocytosis of vesicles in yeast (15). The exocyst is a 750-kDa complex comprised of Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p (16, 17). In yeast (and probably mammals), Sec10p and Sec15p exist as a subcomplex within the greater exocyst complex. This Sec10-Sec15 subcomplex acts as a bridge between the Rab GTPase Sec4p, found on the surface of the secretory vesicles carrying polarized proteins, and the rest of the exocyst complex, which is in contact with the plasma membrane (18). In permeabilized MDCK cells, antibodies to the Sec6 and Sec8 subunits inhibit delivery of at least one basolateral protein to this surface but do not affect the delivery of at least one apical protein to the apical surface (19).
We have previously reported that overexpression of the human homologue of Sec10, hSec10, in MDCK cells resulted in multiple effects. (i) Polarized hSec10-overexpressing cells grown on a two-dimensional Transwell filters were significantly taller, but not wider, than control cells, suggesting an increased basolateral surface area and protein delivery. (ii) hSec10 overexpression specifically resulted in increased synthesis and delivery of endogenous basolateral plasma membrane proteins as well as secretory proteins, compared with control cells, as determined by pulse-chase experiments on filter-grown cells. (iii) hSec10-overexpressing cells in a three-dimensional collagen gel system formed cysts much more rapidly and efficiently than did control cells. When these cysts were stimulated with hepatocyte growth factor, they produced more tubules than did cysts made up of control cells (1).
These pleiotropic morphologic responses probably reflect an underlying increase in the synthesis of endogenous basolateral plasma membrane proteins and the consequent expansion of the basolateral surface area. We are less certain of the consequences of the increase in synthesis of the entire spectrum of major secretory proteins that are released into the apical and basolateral media. Our initial interpretation of these data was that, most likely, the increase in production of secretory and basolateral plasma membrane proteins was produced by the coordinated transcriptional up-regulation of all of these proteins. However, because these proteins have very different functions and are probably under the control of diverse transcriptional regulatory networks, this argues against a transcriptional model. For instance, the major secretory protein in MDCK cells is gp80 (20), a protein with 80% homology to rat-sulfated glycoprotein 2 (SGP-2). SGP-2 is the major protein secreted by rat Sertoli cells and is presumably under complex hormonal control (21, 22). What these basolateral plasma membrane and secretory proteins have in common, however, is they all transit through the secretory pathway. Regulation of their transit through the secretory pathway could be a facile means of regulating the abundance of these proteins.
The translocon, or protein-conducting pore in the RER through which newly made proteins are translocated into or across the RER membrane, consists of three main subunits, Sec61,-
, and -
(23, 24, 25). While the Sec61
subunit is primarily responsible for translocation, much less is known about the Sec61
subunit. It is not required for translocation of all proteins in reconstituted liposomes or for viability in yeast, but it does facilitate translocation (26). Seb1p is a yeast homologue of Sec61
. Intriguingly, SEB1 was isolated as a multicopy suppressor of the sec15-1 mutation, and, given that Sec15 is a component of the exocyst complex, this implies at least a genetic interaction of the exocyst and translocon (2).
Here we test the hypothesis that the exocyst is involved in the regulation of traffic through the secretory pathway, especially in coordination of events at the plasma membrane with events earlier in the secretory pathway, specifically those at the translocon. We present data showing that exocyst overexpression, despite unchanged mRNA levels, increases synthesis of basolateral and secretory, but not apical, proteins and biochemical evidence that the exocyst associates with Sec61. We propose that this association is one mechanism by which exocyst overexpression leads to an increase in secretory and basolateral protein synthesis.
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EXPERIMENTAL PROCEDURES |
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Antibodies/ReagentsMonoclonal mouse anti-rat Sec6 and Sec8 antibody was used at 1:500 for Western blot and 1:100 for immunofluorescence (StressGen). Type 3 inositol 1,4,5-triphosphate receptor (IP3R3) monoclonal antibody was used at 1:100 for immunofluorescence (Transduction Laboratories). Monoclonal anti-Myc antibodies were used at 1:1000 for Western blot. Sec61 antibodies were used at 1:500 for Western blot. For immunofluorescence Alexa 594 secondary antibody was used at 1:200 (Jackson ImmunoResearch Laboratories). Goat anti-mouse HRP (Jackson ImmunoResearch) was the secondary antibody used for Western blots (1:15,000) (Jackson ImmunoResearch).
Northern BlotMDCK cells overexpressing Sec10 and the polymeric immunoglobulin receptor (pIgR) or just pIgR were grown on Transwell filters for 67 days as described (27). RNA was then isolated using RNA STAT-50 (Tel-Test Inc.). The RNA was transferred to a nylon membrane and blotted in standard fashion (28) using 32P-labeled pIgR and glyceraldehyde-3-phosphate dehydrogenase cDNA probes generated with Prime-a-Gene (Promega). cDNA probes for rat concentrative nucleoside transporter 1 (CNT1) and gp80 (a kind gift from Dr. Claudia Koch-Brandt) were used in a similar fashion for their respective Northern blots.
Adenovirus ProductionThe tetracycline-regulated promoter has been subcloned from the tetracycline-inducible expression plasmid pUHD103 to pAdlox (29) 3' to the 5 packaging site and 5' to the poly(A) site, replacing the original cytomegalovirus (CMV) promoter with the regulated tetracycline promoter (30). Full-length hSec10 was then cloned into the adenovirus vector just downstream of the tetracycline/minimal CMV promoter. In the presence of doxycycline, a tetracycline derivative, expression of hSec10 is tightly repressed.
To examine the function of the adenoviral constructs, a Western blot was performed using antibodies against the Myc epitope tag, which confirmed hSec10-myc overexpression. As a further validation of both the function of our recombinant adenovirus and the experiment shown in Fig. 1a, increased pIgR protein synthesis was seen with an [35S]methionine pulse in cells infected with just hSec10-myc adenovirus and tetracycline transactivator (tta) adenovirus compared with control cells infected with hSec10-myc adenovirus, tta adenovirus, and doxycycline (data not shown).
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Generation of Sec10-GFP and TransfectionThe cDNA coding for hSec10 was cloned into the enhanced green fluorescent protein (EGFP) vector (Clontech) and then transfected into MDCK cells using a calcium phosphate precipitation method as described previously (1, 27). Selection was performed using medium containing G418 (Invitrogen).
Synthesis, Delivery, and Secretion AssaysMDCK type II cells constitutively expressing the basolateral protein pIgR and either overexpressing hSec10 or an empty vector from a confluent 100-mm plastic dish were trypsinized, and 5% of the cells in the dish were seeded/12 mm (0.4-µm pore size) Transwell filter (Costar). The cells were grown for 67 days with fresh medium added daily and allowed to grow to confluency as determined by hydrostatic pressure testing (27). After washing with phosphate-buffered saline, cells were starved for 20 min in minimum essential medium lacking cysteine and then labeled by exposing the basolateral surface to a 25-µl drop of starvation medium containing 4 µl of [35S]-cysteine (31.4 µCi/µl, PerkinElmer Life Sciences) for 20 min. After pulsing with 35S-cysteine for 20 min, cells were lysed in 0.5% SDS, equal volumes of 2.5% Triton X-100 were added, and immunoprecipitation was performed using antibody against pIgR. The immunoprecipitate was then run on an SDS-polyacrylamide gel and analyzed with a PhosphorImager (Amersham Biosciences).
For the synthesis and delivery assays of the apical plasma membrane protein CNT1, MDCK cells constitutively overexpressing pIgR and CNT1-GFP were grown on 12-mm Transwell filters for 6 days. 24 h before the pulse-chase was performed, all of the Transwells received 3 µl of tetracycline transactivator and 3 µl of Sec10 virus stock corresponding to 15 plaque-forming units/cell of recombinant adenovirus in 2 ml of binding medium (Hanks' salt solution containing 1 mM MgCl2, 1 mM CaCl2, and 10 mM Hepes, pH 7.2) for 1 h at 37 °C to enable viral attachment and infection as described (30). Half of the Transwell filters also received doxycyline at 20 ng/ml to repress expression of Sec10. The synthesis and delivery assays were then performed as described above, except [35S]methionine was substituted for 35S-cysteine, using anti-GFP antibody (Roche Applied Science) to immunoprecipitate CNT1-GFP.
For the secretion/proteosome inhibitor assays, half of the Transwell filters containing hSec10-overexpressing cells were treated with the proteosome inhibitor MG132 (Calbiochem) for 1 h at 25 µM as described previously (31). The cells were labeled as described above and washed extensively, and minimum essential medium was added (0.3 ml apically and 0.5 ml basolaterally) and collected at 60 min, run on an SDS-polyacrylamide gel, and analyzed with a PhosphorImager (Amersham Biosciences). All experiments were repeated at least three times and were reproducible.
Western blot/Co-immunoprecipitationCells were lysed in 1% N-octylglucosid (Roche Applied Science) and prepared in standard fashion (32). Immunoprecipitation was performed using 48 µg of antibody against Myc, Sec6, Sec8, or Sec61 per immunoprecipitation. The immunoprecipitate was then run on SDS-PAGE. The protein bands were detected by incubations with the same Myc antibody (1:500) or Sec61
(1:500) followed by goat anti-mouse horseradish peroxidase (Jackson ImmunoResearch) as the secondary antibody (1:15,000), and ECL (PerkinElmer Life Sciences).
Immunofluorescence and Confocal MicroscopyCells were rinsed in phosphate-buffered saline and fixed for 30 min with 4% paraformaldehyde as described previously (1, 27). Nonspecific binding sites were blocked and the cells permeabilized using 0.7% fish skin gelatin and 0.025% saponin. Samples were placed in medium containing antibody to IP3R3 (Transduction Laboratories). After extensive washing, the samples were incubated in blocking buffer containing Alexa 594-conjugated secondary antibody, 1:200 dilution (Molecular Probes). Cells were postfixed with 4% paraformaldehyde and mounted. Confocal images were collected using a krypton-argon laser (Bio-Rad 1024).
StatisticsUnitless PhosphorImager counts in Figs. 1 and 6 were significant within an experiment but not between experiments. For all statistics reported, the average and standard deviations were performed using Excel (Microsoft) software.
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RESULTS |
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As a first step to test whether the effect of hSec10 on synthesis of basolateral plasma membrane proteins was transcriptional or post-transcriptional, a pCB7 plasmid containing cDNA encoding an exogenous basolateral protein, pIgR, was transfected into the MDCK parent cell line prior to transfection of hSec10. We found increased synthesis of the exogenous basolateral pIgR in hSec10-overexpressing cells compared with control cells (Fig. 1a). We also found increased pIgR protein delivery to the basolateral plasma membrane at a degree corresponding to the increase in protein synthesis (data not shown). In the pCB7 plasmid, pIgR cDNA was driven off a CMV promoter. This cDNA was engineered to contain minimal 5'- and 3'-untranslated regions (no introns or likely regulatory elements) (35). The CMV promoter itself is a very strong and constitutively active promoter in MDCK cells (27). There is no a priori reason to suspect that the CMV promoter should be regulated by hSec10 or the exocyst.
We had previously transfected the same MDCK cells, stably overexpressing pIgR, with a pcDNA3 plasmid containing cDNA encoding CNT1, an apical protein. The pcDNA3 plasmid also utilizes the CMV promoter. Stable cell lines expressing CNT1 with a green fluorescence protein (GFP) tag were created, and it was confirmed that CNT1-GFP localized to the apical plasma membrane and that the GFP tag did not alter substrate selectivity (36). This cell line constitutively expressing the apical CNT1-GFP allowed us to perform an experiment similar to the one described above for the basolateral protein pIgR. However, to perform this experiment we used the approach of infecting the cells with a recombinant adenovirus encoding hSec10. The adenoviral approach was necessary because we were limited by the number of selection markers that could be used for transfection in this cell line (i.e. the CNT1-GFP cell line had already been transfected with plasmids encoding pIgR and CNT1-GFP). The recombinant adenovirus was constructed so that the hSec10 cDNA was driven off of a minimal CMV promoter, which itself was fused to a regulated tetracycline promoter. Expression of hSec10 was, therefore, repressed in the presence of doxycycline, a tetracycline derivative. Tests confirming the function of the recombinant adenoviral construct were performed (see "Experimental Procedures"). The cells were grown on Transwell filters and, 24 h prior to the [35S]methionine pulse experiments, were infected with recombinant adenovirus encoding hSec10, adenovirus encoding the tetracycline transactivator, and, in half of the cases, doxycycline to suppress hSec10 expression. We found no increase in CNT1 protein synthesis in cells that overexpressed hSec10 (Fig. 1b), consistent with an exocyst effect (1, 19, 33, 34). There was also no significant increase in CNT1-GFP delivery to the apical surface in cells overexpressing hSec10 (data not shown).
Assuming the pCB7 plasmid containing pIgR cDNA was constitutively active, pIgR mRNA should have been present in similar amounts in both control and hSec10-overexpressing cells. Northern blot analysis of pIgR mRNA from control and hSec10-overexpressing cells confirmed that mRNA levels were unchanged despite the increased protein synthesis (Fig. 2a). Similarly, we had previously shown that there was increased synthesis of the secretory protein gp80 in hSec10-overexpressing cells (1). Here we show by Northern blot that despite increased synthesis of gp80, there was no increase in gp80 mRNA levels (Fig. 2b). We also showed above that there was no increase in the synthesis of the apical protein CNT1 in cells overexpressing hSec10. There was also no significant change in CNT1 mRNA levels (Fig. 2c). This indicated to us that hSec10 overexpression did not alter the steady state level of pIgR and gp80 mRNA and, most likely, that it changed either the rate of synthesis or degradation of pIgR and gp80 by acting at a step downstream of the level of mRNA.
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Overexpressed hSec10-GFP Partially Localizes to the ER by Immunofluorescence and Co-immunoprecipitates with Sec61As noted, high level expression of the translocon subunit Sec61
suppressed a mutant of Sec15 in yeast (2). Knowing that Sec10p and Sec15p form a subcomplex within the greater exocyst complex (18), this suppression suggested an interaction of part of the exocyst, particularly the Sec10-Sec15 subcomplex, with the translocation machinery. If true, some portion of the hSec10 should localize to the RER. We cloned hSec10 into the EGFP vector (Clontech) and looked at expression of hSec10-GFP in polarized MDCK cells grown on Transwell filters. At least for cells containing the highest expression of hSec10-GFP, we saw partial co-localization of hSec10-GFP with the ER-resident protein, IP3R3 (Fig. 3) (possibly, in cells with lower levels of hSec10-GFP, the specific signal of hSec10 in the ER is not detectable above the diffuse cytosolic background, which is characteristic of transfected chimeras of GFP and exocyst subunits (37)). This placed the exocyst complex in an area where it could be involved in the process of protein translocation and/or translation and was similar to the results found by Shin and colleagues (38) who co-localized the endogenous exocyst with IP3R3 in pancreatic acinar cells.
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We therefore looked for a physical interaction between the transfected hSec10, containing a Myc epitope tag (hSec10-myc), and Sec61 proteins. We solubilized MDCK cells expressing both proteins, tested for co-immunoprecipitation of hSec10-myc with Sec61
, and found that hSec10-myc and Sec61
specifically co-immunoprecipitated. This co-immunoprecipitation could be observed in both directions, i.e. by immunoprecipitation of Sec61
and looking for co-immunoprecipitated hSec10-myc, and, conversely, by immunoprecipitation of hSec10-myc and looking for co-immunoprecipitated Sec61
(Fig. 4, a and d). Per 12-mm Transwell filter, there was an equivalent amount of Sec61
in hSec10-myc-overexpressing and control cells (Fig. 4c). By quantitating the intensity of the hSec10-myc band from a 10-µl aliquot of the hSec10-myc cell lysate (Western blot only; Fig. 4a, lane M), comparing that with the intensity of the hSec10-myc band that co-immunoprecipitated from 750 µl of total lysate, itself immunoprecipitated with Sec61
(Fig. 4a, lane S), and adjusting for inefficient or incomplete immunoprecipitation (Fig. 4b), we estimate that
35% of total cellular hSec10-myc co-immunoprecipitated with the Sec61
.
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We adjusted for inefficient or incomplete immunoprecipitation, i.e. the amount of protein that is lost during an immunoprecipitation, by immunoprecipitating hSec10-myc from 750 µl of total cell lysate using antibody against the Myc epitope tag and then blotting for Myc. This band was then compared with the band from 10 µl of lysate (Western blot only). We found that instead of the expected 75-fold (750 µl/10 µl) amount of hSec10-myc protein, assuming 100% of the hSec10-myc protein was immunoprecipitated, there was only 11-fold (Fig. 4b). This was very reproducible for hSec10-myc, although when a similar exercise was done with Sec61
(i.e. immunoprecipitate 750 µl of total lysate with Sec61
antibody and then blot for Sec61
; Fig. 4c), instead of the expected 75-fold excess there was only
2-fold. We attribute the
5-fold variation in efficiency of immunoprecipitation between hSec10-myc and Sec61
to differences in the respective antibodies. Immunoprecipitation was performed using 4 µg of antibody against Myc or Sec61
per immunoprecipitation. Increasing the amounts of Myc and Sec61
antibody up to 15 µg did not increase the amount of Myc and Sec61
protein co-immunoprecipitating beyond what was seen in Fig. 4, b and c. Using similar calculations, we estimate that
7.5% of the total Sec61
co-immunoprecipitated with hSec10-myc (Fig. 4, c and d), although it should be noted that endogenous Sec10 also bound Sec61
to a degree that we could not quantify. In addition, as controls we used a panel of antibodies to ER proteins such as SSR (signal sequence receptor) and GP94; all of these showed a lack of co-immunoprecipitation, corroborating the specificity of the interaction between hSec10-myc and Sec61
(Fig. 4e).
In hSec10-overexpressing cells, it is possible that hSec10 could be acting on the ER translation machinery on its own or in combination with Sec15, as the Sec10-Sec15 subcomplex has been shown to migrate separately from the rest of the exocyst complex (18). hSec10 could also be acting through the entire exocyst complex. To examine the role of the other exocyst complex members in this process, we used antibodies against endogenous Sec6 and Sec8 in co-immunoprecipitation experiments. We show that endogenous Sec6 and Sec8 also co-immunoprecipitate with Sec61
6 and
2%, respectively. Furthermore, using cell lysates from hSec10-overexpressing and control cells, we found that overexpression of hSec10 increased the amount of Sec61
that co-immunoprecipitated with the endogenous exocyst complex members Sec6 and Sec8, now
13 and
6%, respectively (Fig. 5). This could suggest a direct interaction between the entire exocyst complex and Sec61
or, alternatively, with increasing amounts of hSec10, an indirect association of Sec6 and Sec8 with Sec61
is strengthened, perhaps by simple mass action.
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Effect of Proteosome Inhibition on Secreted ProteinsGiven the striking increase in secretory and basolateral plasma membrane protein levels in cells that overexpress hSec10, one explanation could be decreased degradation of newly made proteins in addition to, or exclusive of, increased protein synthesis. Proteins are degraded inside a cell for many reasons including failure of the proteins to assemble into a complex or the existence of proteins in a damaged or misfolded state. Such proteins are exported from the ER back into the cytosol, where they are degraded. This retrotranslocation, also called dislocation, occurs via the same Sec61 translocon through which the proteins initially entered the ER, although additional proteins help the Sec61 complex act in reverse. Sec61 has been shown to play a major role in this process of retrotranslocation (39). The major pathway for protein degradation involves marking the proteins for destruction by covalent attachment of a small protein, ubiquitin, before transport to the proteosome complex where proteolysis occurs (40). Because a large fraction of all newly made proteins are immediately degraded, a reduction in this constitutive degradation would result in an "apparent" (but false) increase in synthesis. To test this possibility, we performed pulse-chase experiments using hSec10-overexpressing and control cells grown on Transwell filters. Half of the Transwell filters were treated with the cell-permeable proteosome inhibitor MG132 (31), and the apical and basolateral media were then collected following the pulse-chase. There was no difference detected in the secretion of gp80, the most abundant secretory protein in MDCK cells (20), or other secretory proteins in hSec10-overexpressing and control cells treated with proteosome inhibitor compared with the respective untreated cells (Fig. 6).
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DISCUSSION |
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As noted, several reports and experiments helped us narrow our search for a connection between the early and late stages of the secretory pathway. First, in yeast, high level expression of the Sec61 homologue, SEB1, suppressed an exocyst mutant, sec15-1 (2). Using co-immunoprecipitation, we showed that the mammalian exocyst complex and Sec61
specifically interacted. Second, the exocyst was initially found to localize to the ER where translocation occurs (38), which we confirmed by partial co-localization of overexpressed hSec10-GFP and the ER-resident protein IP3R3. Third, in addition to all major endogenous secretory proteins being increased, synthesis of an exogenous basolateral protein pIgR, but not an exogenous apical protein CNT1, was increased in hSec10-overexpressing compared with control cells. Finally, Northern blot analysis of mRNA from endogenous gp80 and transfected exogenous pIgR demonstrated that, despite the increase in gp80 and pIgR protein synthesis, there was no change in mRNA levels. Taken together, these data indicate that the exocyst is acting to affect protein translation and/or translocation, which are tightly coupled in the ER. This suggests that cells selectively control secretory and basolateral plasma membrane protein synthesis as a group not at the level of the nucleus but, rather, at the first specific stage of their synthetic pathway (i.e. at the level of the translational machinery and translocon of the ER).
Given that proteosome inhibition did not affect secretion in hSec10-overexpressing cells, it is unlikely that exocyst overexpression was simply decreasing the rate of protein degradation. It has previously been shown that the translocon is dynamic and can be regulated (43) and that Sec61 is not essential for co-translational translocation but does kinetically facilitate it (26). Gruss and colleagues (4) showed that calcium-dependent isoforms of protein kinase C are associated with the RER and phosphorylate essential components of the protein translocation machinery including Sec61
. Sec61
is extensively phosphorylated, and phosphorylation increases the translocation of preprolactin (4). This may be important given that the exocyst has recently been shown to govern the polarized expression of calcium signaling complexes and regulation of their activity in the ER of pancreatic acinar cells (38).
Another possible mode of interaction between the exocyst and Sec61 involves the ribosomal machinery of the cell. In mammalian cells, the import of proteins into the ER begins before the polypeptide chain is completely synthesized; that is, it is a co-translational process. The ribosome that is synthesizing the protein is tightly bound to the ER membrane. Because many ribosomes can bind to a single mRNA molecule, another mechanism for increasing protein synthesis in the face of a constant level of mRNA could be to increase the number of ribosomes binding to each mRNA molecule (44). Along similar lines, the Sec61 complex forms the core element of the translocon in the ER membrane. Ribosomes (both translating and nontranslating) bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61 complex (26). Therefore, the Sec61 complex is believed to serve as both a translocation pore and a ribosome-binding site, and it has been shown that the Sec61
subunit of the Sec61 complex makes contact with nontranslating ribosomes (45). Given our co-immunoprecipitation data showing a specific interaction between Sec61
and hSec10, it is possible that the exocyst complex modulates the interaction between Sec61
and nontranslating ribosomes.
In summary, we have demonstrated a link in mammalian cells between the membrane trafficking and protein synthetic machinery. This link could be significant for human diseases involving changes in protein synthesis and polarity such as autosomal dominant polycystic kidney disease, in which the exocyst has been shown to be misexpressed (34). Further studies are needed to determine the exact mechanism by which the exocyst/Sec61 interaction affects protein synthesis.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 215-573-1848; Fax: 215-898-0189; E-mail: jhlipsch{at}mail.med.upenn.edu.
1 The abbreviations used are: RER, rough endoplasmic reticulum; ER, endoplasmic reticulum; TGN, trans-Golgi network; MDCK, Madin-Darby canine kidney; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; hSec10, human Sec10; IP3R3, type 3 inositol 1,4,5-triphosphate receptor; pIgR, polymeric immunoglobulin receptor; CMV, cytomegalovirus; CNT1, concentrative nucleoside transporter 1; SSO, suppressor of Sec1.
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REFERENCES |
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