Journal of Cell Science 112, 1477-1486 (1999) 1477 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0213
The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment
Julia J. A. Greenfield and Stephen High* School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK *Author for correspondence (e-mail: [email protected])
Accepted 12 February; published on WWW 22 April 1999
SUMMARY
The heteromeric Sec61 complex is composed of α, β and γ in both the ER and the ERGIC/Golgi fractions of the subunits and forms the core of the mammalian ER gradient. The presence of the Sec61 subunits in a post-ER translocon. Oligomers of the Sec61 complex form a compartment suggests that these proteins can escape the transmembrane channel where proteins are translocated ER and be recycled back, despite the fact that none of them across and integrated into the ER membrane. We have contain any known membrane protein retrieval signals studied the subcellular localisation of the Sec61 complex such as cytosolic di-lysine or di-arginine motifs. We also using both wild-type COS1 cells and cells transfected found that another translocon component, the glycoprotein with GFP-tagged Sec61α. By double labelling TRAM, was present in post-ER compartments as immunofluorescence microscopy the GFP-tagged Sec61α demonstrated by subcellular fractionation. Our data was found in both the ER and the ER-Golgi intermediate indicate that the core components of the mammalian ER compartment (ERGIC) but not in the trans-Golgi network. translocon are not permanently resident in the ER, but Immunofluorescence studies of endogenous Sec61β and rather that they are maintained in the ER by a specific Sec61γ showed that these proteins are also located in both retrieval mechanism. the ER and the ERGIC. Using the alternative strategy of subcellular fractionation, we have shown that wild-type Key words: Endoplasmic reticulum, Translocation, GFP, ERGIC, ER Sec61α, β and γ, and GFP-tagged Sec61α, are all present retrieval
INTRODUCTION known ER retention or retrieval signals normally associated with resident ER proteins. ER resident proteins are generally The Sec61 complex is a core component of the mammalian believed to be retained in the ER either by their exclusion from translocon, forming a channel across the endoplasmic transport vesicles that exit the ER, or by their specific retrieval reticulum (ER) membrane through which newly synthesised from post ER compartments, or by a combination of both secretory proteins are translocated and via which membrane mechanisms (Harter and Wieland, 1996). proteins are integrated (High and Laird, 1997; Rapoport et al., Proteins that have been shown to be exclusively located in 1996). In addition to its central role in protein translocation and the ER, and that do not therefore apparently require any integration at the ER membrane, the Sec61 complex has also specific retrieval mechanism, include cytochrome p450 been shown to be capable of the retrograde transport, or (Szczesna-Skorupa and Kemper, 1993) and aldehyde dislocation, of misfolded proteins from the ER and into the dehydrogenase (Masaki et al., 1996). In the case of cytochrome cytosol prior to their degradation via the proteosome (Wiertz p450, it has been suggested that the large, bulky, cytoplasmic et al., 1996). domain may be responsible for its exclusion from transport The Sec61 complex is composed of three subunits, α, β and vesicles; and indeed ER retention information has been γ, which form a heterotrimer (Rapoport et al., 1996) and localised both in this region and in the N-terminal multiple copies of these heterotrimers surround a central transmembrane domain (Szczesna-Skorupa et al., 1995). It has aqueous pore which spans the ER membrane (Beckmann et al., also been proposed that some ER proteins, such as the 1997; Hanein et al., 1996). Whilst Sec61α is a polytopic ribophorins, form an interacting network of supramolecular protein with ten transmembrane spans, Sec61β and Sec61γ complexes that are excluded from transport vesicles (Ivessa et span the membrane only once (Rapoport et al., 1996). al., 1992). Although the Sec61 complex is known to play a crucial role in Specific motifs that mediate ER residency by retention have promoting translocation at the ER membrane, its exact not yet been described; in contrast, all retrieval mechanisms subcellular location has not been studied in any detail. appear to involve specific signals within the sequences of the Furthermore, none of the Sec61 subunits contain any of the resident ER proteins. The best characterised retrieval signal is 1478 J. J. A. Greenfield and S. High the KDEL sequence at the C terminus of ER lumenal proteins resident proteins that have escaped the ER (Bannykh et al., of mammalian cells (Pelham, 1990). Proteins with a KDEL 1998). In other systems, particularly the yeast Saccharomyces retrieval motif bind to the KDEL receptor (ERD2) and are then cerevisiae, it has been suggested that certain ER proteins, for recycled back to the ER (Lewis and Pelham, 1992a). The example some ER chaperones and components of the KDEL receptor is mainly localised in the first post-ER membrane translocation machinery, are efficiently sorted away compartment, the ERGIC (ER to Golgi intermediate from cargo destined for export at the ER. These components compartment) together with the cis-Golgi (Griffiths et al., are therefore largely excluded from the transport vesicles 1994). As a consequence of its retrieval function, the KDEL leaving the ER (reviewed by Aridor and Balch, 1996). receptor has also been localised to the ER, albeit to a lesser We have studied the localisation of the Sec61 complex in extent than to the ERGIC or cis-Golgi (Griffiths et al., 1994). mammalian cells using two approaches, namely fluorescence A number of single-spanning membrane proteins have a microscopy and subcellular fractionation. The wild-type carboxy-terminal di-lysine or an amino-terminal di-arginine Sec61α protein was tagged with the green fluorescent protein retrieval motif (reviewed by Teasdale and Jackson, 1996), and (GFP) in order to facilitate its visualisation in both fixed and these are found in type I and type II membrane proteins, live cells, and to establish its subcellular location. The respectively. COP1 subunits have been shown to bind directly subcellular location of the endogenous Sec61β and Sec61γ to proteins bearing the cytosolic di-lysine motif and are subunits were also studied by indirect immunofluorescence believed to function in the retrieval of the proteins to the ER microscopy, and in all cases double labelling studies were (Cosson and Letourneur, 1994). Very little is known about the carried out. Taken together with the results from the subcellular ER retention and retrieval of multiple spanning membrane fractionation studies we find that a proportion of all three proteins. subunits of the Sec61 complex are found beyond the ER of In order to ensure the minimum leakage of proteins from the mammalian cells and are clearly present in the ERGIC. We ER some proteins possess a combination of both retention and suggest that the Sec61 complex is transported between the ER retrieval signals. Hence, Sec12p, a yeast protein involved in the and the ERGIC, and that its ER location is dependent upon a formation of ER transport vesicles, has a transmembrane recycling mechanism. domain that acts as a retention signal and an N-terminal cytoplasmic domain which is involved in retrieval (Sato et al., 1996). Tang et al. (1997) later showed that the transmembrane MATERIALS AND METHODS domain was responsible for the formation of oligomers, and that this was the molecular basis for preventing the exit of Materials Sec12p from the ER. Clearly proteins which lack any COS1 African green monkey kidney SV40 transformed cell line retention/retrieval information in their sequence can be (ECACC no. 88031701) was obtained from the European collection retained in the ER if they are in a stable complex with other of animal cell cultures. proteins that do contain such information (Zhen et al., 1995). The Sec61α canine cDNA was a gift from T. Rapoport (Harvard The ER-Golgi intermediate compartment, or ERGIC, (also Medical School, Boston, USA). Restriction enzymes used for known as the intermediate compartment or ‘vesicular tubular recombinant DNA procedures were obtained from New England structures’), is the first recycling compartment of the Biolabs (Hitchin, UK) and Promega (Southampton, UK). T4 DNA ligase and alkaline phosphatase were from Boehringer Mannheim mammalian secretory pathway. It was originally identified as (Lewes, UK). PCR was carried out using QuikChangeTM site-directed the compartment where Semliki Forest virus-infected cells mutagenesis kit (Stratagene, Cambridge, UK). 35[S]methionine was accumulated envelope glycoproteins when intracellular from NEN-Dupont (Stevenage, UK). Bismaleimidohexane (BMH) transport was arrested by incubation at 15¡C (Saraste and cross-linking reagent was from Pierce and Warriner (Chester,UK). Kuismanen, 1984). It is usually defined by the presence of Rabbit reticulocyte lysate and the amino acid mixture minus resident marker proteins such as human ERGIC53 (Schweizer methionine were from Promega. The 7-methylguanosine 5′ et al., 1988), its rat homologue p58 (Saraste and Svensson, monophosphate was from Ambion (Austin Texas). Protein A and G 1991) and the KDEL receptor (Tang et al., 1993). These Sepharose came from Zymed (San Francisco, CA). Nycodenz was proteins continuously cycle between the ER the ERGIC and purchased from Nycomed Pharma (Oslo, Norway). Digitonin was the cis-Golgi. Whilst the role of the KDEL receptor (ERD2) obtained from Calbiochem (Nottingham, UK). Unless otherwise stated all other reagents were purchased from Sigma (Poole, UK) and has been established for some time (see above), until recently BDH-Merck (Lutterworth, UK). the role of ERGIC53/p58 was less clear. These proteins were shown to have lumenal ‘lectin’ like domains that recognise Antibodies high mannose oligosaccharides, and hence it was suggested The mouse monoclonal antibody against ERGIC53 was provided by that they may be involved in the sorting or recycling of H. P. Hauri (University of Basle, Switzerland), that against the KDEL glycoproteins during export from the ER (Fiedler and Simons, sequence (1D3) was from V. Allan (University of Manchester, UK), 1994; Itin et al., 1996). Recently, Nichols et al. (1998) found and the Myc epitope specific monoclonal antibody was purchased that mutations in the human ERGIC53 protein cause a from Invitrogen (Leek, Netherlands). All other antisera used were combined deficiency of the coagulation factors V and VIII. raised in rabbits and sourced as follows: ERD2 from H. D. Soling Thus, ERGIC53 appears to be responsible for the sorting and (University of Goettingen, Germany), TRAM from B. Dobberstein (ZMBH, Germany) Rab6 from B. Goud (Institute Curie, Paris, efficient secretion of a very specific subset of newly France), TGN46 from V. Ponnambalam (University of Dundee, UK). synthesised glycoproteins including coagulation factors V and The antibody specific for the 22/23 kDa signal peptidase subunit was VIII (Nichols et al., 1998) and cathepsin C (Vollenweider et provided by C. Nicchitta (Duke University, USA). The antisera al., 1998). To date, the main function that has been attributed against GFP and calnexin were purchased from Clontech (Palo Alto, to the ERGIC is that it acts as the site for the retrieval of ER CA) and StressGen (Victoria, Canada), respectively. The Sec61α Sec61 complex subcellular localisation 1479 antibody used for immunoblotting, and the Sec61β antibody, were Sepharose before recovery of the immunoprecipitates by both gifts from R. Zimmerman (University of Saarland, Hamburg, centrifugation. These were then washed extensively (Oliver et al., Germany). The Sec61α antibody used for immunoprecipitation of 1996) and analysed by SDS-PAGE. cross-linking products was raised against an N-terminal peptide (MAIKFLEVIKPF) by Research Genetics Inc. (Huntsville, AL,USA). Fluorescence microscopy The Sec61γ antibody was raised against a peptide close to the N Live cells transfected with the GFP-tagged Sec61α construct were terminus of the sequence (EPSRQFVKDSIR) by Research Genetics. transferred to Opti-MEM medium and visualised using an Olympus BX60 fluorescence microscope. Images were captured with a Constructs Princeton Instruments Pentamax integrated slow-scan cooled CCD The vector used for the expression of GFP-tagged Sec61α was a camera. For indirect immunofluorescence studies, wild-type or modified version of the mammalian expression vector pRcCMV transfected cells were fixed in methanol at −20¡C. The cells were (Invitrogen, Leek, Netherlands) in which a translation enhancer was rehydrated after fixing by incubation in PBS with 1% BSA for 15 introduced upstream of the Sec61α gene after the 5′ non coding region minutes, followed by incubation with various primary antibodies for had been removed. This translation enhancer constituted the UTK 1 hour. The cells were washed and then incubated with the appropriate region from pSPUTK translation vector (Stratagene Ltd, Cambridge, fluoroscein, rhodamine or Cy3 labelled second antibody for 1 hour. UK). PCR was used to introduce an NdeI site at the 3′ end of the After washing, the slides were mounted with mowiol and DABCO Sec61α just before the stop codon. This allowed the insertion of the antifade and the cells were observed with a Zeiss axiophot microscope GFP Ser65 mutant (Craven et al., 1998) into the NdeI site such that or a Leica TCS NT laser scanning confocal microscope with the it was in frame with the Sec61α coding region. appropriate filters. The vector used for the Myc/His tagged Sec61α construct was pcDNA3.1(-)Myc-His (Invitrogen). The translation enhancer Subcellular fractionation described above was again placed at the 5′ end of the Sec61α gene. Subcellular fractionation was carried out on Nycodenz gradients using PCR was used to introduce a HindIII site at the 3′ end of the Sec61α a modified protocol based on that of Hammond and Helenius (1994). coding region and at the same time remove the stop codon. The insert One 225 cm2 flask of approximately 80% confluent cells was was then cloned into the pcDNA3.1(-)Myc-His vector as a HindIII sufficient for one gradient and the resulting fractions could be used fragment. for two immunoblots. Cells were washed twice in homogenisation buffer (10 mM triethanolamine, 10 mM acetic acid, 250 mM sucrose, Cell culture and transfection 1 mM EDTA, and 1 mM DTT) then scraped into 2 ml/flask of COS1 cells were grown in DMEM with sodium pyruvate and homogenisation buffer containing 10 µg/ml each of PMSF, antipain, pyridoxine (Gibco BRL, Paisley, UK) supplemented with 10% FCS pepstatin A and leupeptin. Cells were homogenised by passing them and 1 mM L-glutamine. Cells were transfected with Sec61α-GFP, 5 times through 19, 23 and 25-gauge hypodermic needles Sec61αMyc/His or with the pRcCMV plasmid alone by lipofection. consecutively and then spun at 1500 g for 5 minutes at 4¡C. The DNA was mixed with the lipofection reagent Lipofectamine (Gibco postnuclear supernatants were then loaded directly onto preformed BRL) and OPTI-MEM medium for 30 minutes according to the Nycodenz gradients as described by Hammond and Helenius (1994) manufacturer’s recommendations. Cells were incubated at 37¡C in and nine fractions were collected. Equal volumes of the fractions were OPTI-MEM containing the DNA and lipofectamine for 5 hours after precipitated using trichloroacetic acid, separated by SDS-PAGE, and which the medium was replaced with the full medium described then analysed by western blotting using ECL detection (Amersham, above, and the cells grown for a further 43 hours. UK). For microscopy, 1×105 cells were placed in a 3 cm diameter dish with a glass coverslip, grown for 20 hours and then transfected using µ 1 g DNA. This was scaled up accordingly for the preparation of RESULTS semi-permeabilised cells or for subcellular fractionation experiments. Cross-linking analysis of recombinant Sec61α function Expression of the GFP-tagged Sec61α and Transfected cells were permeabilised using digitonin at a functionality assay concentration of 30 µg/ml and truncated preprolactin (PPL) was used The transfection efficiency of the GFP-tagged Sec61α in COS1 as a model secretory protein for translocation (Wilson et al., 1995). cells was relatively high and approximately 40% of the cells For the generation of a truncated RNA encoding the first 86 amino showed a high intensity GFP dependent signal when visualised acids of PPL, pGEM4 T7 PPL was linearised with PvuII and by fluorescence microscopy. Metabolic labelling and transcribed with T7 RNA polymerase (High et al., 1993). PPL RNA immunoprecipitation was used to determine the amount of the was then translated for 15 minutes at 30¡C. The translation reaction α contained 14 µl rabbit reticulocyte lysate, 0.4 µl of 1 mM amino acids GFP-tagged Sec61 protein produced by the COS1 cells. We minus methionine, 15 µCi of L-[S35]methionine, 2 µl RNA and 4-8 found that it was expressed at approximately 2.5 times the level µl of digitonin treated, semi-permeabilised, COS1 cells (equivalent to of the endogenous Sec61α protein present in both the same 1×105 cells). After 30 minutes 7-methylguanosine 5′ monophosphate cells and in cells transfected with the expression vector alone was added to 4 mM, incubation at 30¡C was continued for a further (data not shown). Since only 40% of the cells were actually 5 minutes and then cycloheximide was added to a final concentration transfected, the average expression level per GFP-tagged of 2 mM to prevent any further protein synthesis. The cells were Sec61α expressing cell was probably 4-5 times the level of washed and resuspended in 150 µl KHM buffer, and then incubated endogenous Sec61α. at 30¡C in the presence of 0.33 mM BMH cross-linker (cross-links To test whether the GFP-tagged Sec61α was functional we the -SH groups of cysteine residues). After a 10 minute incubation 2- used an established cross-linking assay (Wilson et al., 1995). mercaptoethanol was added to a final concentration of 7.33 mM to act as a quenching agent. Immunoprecipitation of the crosslinking Transiently transfected cells were semi-permeabilised using products was carried out for 16 hours at 4¡C using a Triton based digitonin and then added to a cell free translation reaction buffer with no SDS present (‘native conditions’; see Oliver et al., programmed with mRNA encoding a truncated version of the 1996). The samples were then incubated for 2 hours with 30 µl of secretory protein preprolactin (PPL). Since the truncated PPL Protein A Sepharose or a mixture of Protein A and Protein G lacked a stop codon it would be targeted to the ER membrane 1480 J. J. A. Greenfield and S. High
α Fig. 2. Fluorescence microscopy of live COS1 cells transfected with Fig. 1. Cross-linking analysis of tagged Sec61 proteins. Truncated, α radiolabelled, nascent chains of the secretory protein preprolactin GFP-tagged Sec61 . The GFP-dependent fluorescence emitted from (PPL) were synthesised in the presence of digitonin permeabilised a typical live cell is shown in A. Details from regions of the same COS1 cells expressing recombinant Sec61α tagged with either GFP cell at a higher magnification are shown in B and C. All transfected (lanes 1-5) or a Myc/His tag (lanes 6-10). The resulting translocation cells showed a similar intense perinuclear fluorescence (A) and a intermediates were incubated in the presence or absence of the fluorescent network pattern typical of the ER (A,B,C). bifunctional cross-linking reagent BMH as indicated. Total products were immunoprecipitated with antibodies specific for the PPL chains α (lanes 1, 2, 6 and 7). Specific cross-linking products were detected terminus of Sec61 could be modified without affecting by immunoprecipitation with antibodies recognising GFP (lane 3), function, a much smaller tag consisting of the Myc epitope and the Myc tag (lane 8), Sec61α (lanes 4 and 9) or a preimmune serum six histidine residues (Myc/His) was added in place of the C- (lanes 5 and 10). The samples were resolved by SDS-PAGE using a terminal GFP tag. In this case, the PPL translocation 12% gel and visualised using a Fuji Basis 2000 bioimager. intermediate could be cross-linked to both endogenous Sec61α (Fig. 1, lane 9) and the Myc/His tagged Sec61α (Fig. 1, lane 8). The cross-linking product with the Myc/His tagged protein and then become trapped in the ER translocation channel was noticeably larger than the wild-type product (the Sec61α because it remains attached to the ribosome (see High, 1995). -Myc/His construct contains an extra 23 amino acids), and was Upon the addition of the homobifunctional cross-linking specifically immunoprecipitated by an anti-Myc antibody reagent BMH, the ER translocon components adjacent to the which does not recognise the endogenous protein (Fig. 1, lane translocating PPL chain can be cross-linked to it (cf. Laird and 8 and data not shown). We therefore conclude that the addition High, 1997) and can be identified by immunoprecipitation. It of the 238 amino acid GFP tag to the C terminus of Sec61α had already been shown that endogenous Sec61α can be cross- specifically inhibits the function of the protein. linked to a PPL translocation intermediate (Wilson et al., 1995), and therefore this assay could be used to determine The subcellular localisation of GFP-tagged Sec61α whether the tagged Sec61α was also adjacent to secretory visualised by fluorescence microscopy proteins. The ability of tagged Sec61α proteins to cross-link Fluorescence microscopy was used to determine the PPL translocation intermediates would indicate that the tagged subcellular localisation of the GFP-tagged Sec61α following protein was functional in mediating ER membrane transient transfection of COS1 cells. Our initial studies were translocation. carried out using live cells in order to avoid any artefacts that We observed several BMH dependent cross-linking products might arise during the fixation process. The cells showed a with the PPL translocation intermediate (Fig. 1, lanes 2 and 7). large amount of perinuclear staining (Fig. 2A) together with a However, when cells transfected with GFP-tagged Sec61α clear reticular pattern that was particularly visible towards the were used, cross-linking to endogenous Sec61α (Fig. 1, lane periphery of the cell (Fig. 2B and C). This is the typical pattern 4) but not to GFP-tagged Sec61α (Fig. 1, lane 3) was detected. of staining obtained with a resident ER protein and indicates We had previously established that the anti-Sec61α antibody that much of the GFP-tagged Sec61α is correctly localised in used for this experiment could immunoprecipitate the GFP- the ER. No evidence of any labelling at the cell surface in live tagged Sec61α from metabolically labelled cells and that this cells was observed. In order to confirm the localisation of the product had a molecular weight of around 75 kDa on SDS- GFP-tagged Sec61α, double labelling experiments were PAGE (data not shown). In order to establish whether the C carried out using fixed cells and a monoclonal anti-‘KDEL’ Sec61 complex subcellular localisation 1481
Fig. 3. Co-localisation of GFP-tagged Sec61α with the ER marker 1D3. Peripheral region of methanol fixed COS1 cell showing detail of the reticular network labelled with GFP-tagged Sec61α (A) and the monoclonal antibody 1D3 visualised via a rhodamine conjugated secondary antibody (B). When these images are overlaid almost complete co-localisation is observed (C). antibody (1D3) recognising several soluble resident ER immunofluorescence studies using markers for other proteins, particularly protein disulphide isomerase (Vaux et al., subcellular compartments of the secretory pathway. The next 1990). During the course of these experiments both methanol subcellular location that we analysed was the ERGIC, using and paraformaldehyde/gluteraldehyde fixation methods were antibodies specific for the protein that defines this tested. Since both these approaches gave similar results compartment, namely ERGIC53 (Itin et al., 1995). In contrast (data not shown), methanol fixation was used for all the to the ER staining pattern described above, ERGIC53 was studies presented. The reticular network observed with the localised to a discrete perinuclear region of the cell that was GFP-tagged Sec61α (Fig. 3A) and using indirect visualised as a distinct punctate staining pattern (Fig. 5B). This immunofluorescence to visualise the 1D3 reactive proteins region partly overlapped with the structures labelled by GFP- (Fig. 3B) were very similar, and when the two images were tagged Sec61α (Fig. 5A) and specific co-localisation of a sub- merged a large degree of co-localisation was observed (Fig. population of GFP-tagged Sec61α with ERGIC53 was 3C, yellow areas). Hence the reticular network formed by the observed (Fig. 5C). Indeed, few if any of the ERGIC53 staining GFP-tagged Sec61α is largely co-localised with the 1D3 regions did not overlap with GFP-tagged Sec61α (Fig. 5C). reactive compartment of mammalian cells, ie: the endoplasmic The ERD2 protein (KDEL receptor) is also located primarily reticulum. We were unable to study the localisation of the in the ERGIC, and partly in the cis-Golgi (Tang et al., 1993) endogenous Sec61α protein by indirect immunofluorescence and double labelling studies of ERD2 and GFP-tagged Sec61α because none of our antibodies were suitable for such studies. gave similar results to those shown for ERGIC53 (data not We therefore continued to use the GFP-tag to visualise Sec61α shown). throughout this analysis. A useful tool that is often used in studies of the ERGIC (or Sec61α normally functions as one subunit of a heterotrimer, intermediate compartment) is a 15¡C temperature block. This we therefore compared its subcellular distribution to that of one is known to inhibit ER to Golgi transport (Saraste and of the other subunits of the Sec61 complex, Sec61β (Fig. 4). Kuismanen, 1984), and result in the accumulation of The subcellular distribution of GFP-tagged Sec61α (Fig. 4A) tubulovesicular intermediate compartment structures in and endogenous Sec61β (Fig. 4B) was very similar with a large peripheral cytoplasm (Saraste and Svensson, 1991). This degree of co-localisation extending to the periphery of the cell enables a more precise co-localisation of proteins with this (Fig. 4C). We therefore conclude that the subcellular compartment to be made. Following a 2 hour 15¡C temperature distribution of GFP-tagged Sec61α is similar to the distribution block we indeed observed a significant increase in the number of endogenous components of the Sec61 complex. of ERGIC53 labelled vesicular structures located towards the In order to determine whether GFP-tagged Sec61α was periphery of the cell (cf. Figs 5B and 6B). We observed a localised solely in the ER, we also carried out double labelling complete co-localisation of the ERGIC53 labelling and the
Fig. 4. Co-localisation of GFP-tagged Sec61α with Sec61β. The GFP signal from tagged Sec61α ( A) and the subcellular regions labelled by anti- Sec61β antibodies and a rhodamine conjugated secondary antibody (B) are shown. When these images are overlaid, the signal shows essentially complete co-localisation (C). 1482 J. J. A. Greenfield and S. High
Fig. 5. GFP-tagged Sec61α is present in the ERGIC. The GFP-tagged Sec61α signal (A) and the ERGIC detected via monoclonal anti-ERGIC53 and rhodamine conjugated secondary antibody (B) are shown. The image overlay shows that GFP-tagged Sec61α is present in all of the ERGIC53 labelled structures (C).
GFP labelling present in these scattered vesicular structures immunofluorescence imaging of TGN46 in red, whilst the (Fig. 6C). Scattered vesicular structures labelled with GFP- Sec61α-GFP labelling is in green and any overlapping regions tagged Sec61α are also clearly visible amongst the reticular are yellow. Fig. 7A and C show two cells where no co- ER network (Fig. 6A). In the example shown a complete co- localisation of GFP-tagged Sec61α and TGN46 was detected. localisation of the ERGIC53 compartment with structures In a few cases a small amount of overlap was still observed containing GFP-tagged Sec61α was observed after a 15¡C following brefeldin A treatment (Fig. 7B). However, this was temperature block. However, the amount of co-localisation was insignificant when compared to the co-localisation with the variable and some cells showed a proportion of ERGIC53 ERGIC53. labelled structures that did not co-localise with GFP-tagged Taking the results of these microscopy studies together, we Sec61α (data not shown). We believe that this reflects conclude that the GFP-tagged Sec61α is localised in the ER variations in the amount of GFP-tagged Sec61α expressed in and the ERGIC, but that it does not reach the TGN. individual cells. Furthermore, analysis of the GFP-labelling of live cells showed In order to determine whether the GFP tagged Sec61α was no detectable presence of the protein in the plasma membrane. found in compartments further along the secretory pathway, an On this basis we suggest that GFP-tagged Sec61α can be immunofluorescence study was carried out using the trans- transported out of the ER to the ERGIC, and perhaps the cis- Golgi network marker TGN46 (Prescott et al., 1997). By Golgi (cf. Tang et al., 1993) from where it is most likely standard indirect immunofluorescence microscopy, a recycled back to the ER. substantial portion of TGN46 labelling was distinct from the GFP-Sec61α labelled structures. Nevertheless, some degree of The subcellular localisation of endogenous Sec61β co-localisation was observed, albeit substantially less than that and Sec61γ seen with ERGIC53 (data not shown). Since several studies It was possible that the presence of GFP-tagged Sec61α in the have noted the potential difficulties in distinguishing between ERGIC that we observed was a result of either its moderate the ERGIC and the Golgi (Yang and Storrie, 1998), or the overexpression or its lack of functionality. In the absence of Golgi and the TGN (Banting and Ponnambalam, 1997) using any suitable antibodies with which we could study the immunofluorescence microscopy, we wanted to determine subcellular location of the endogenous Sec61α present in wild- whether the partially overlapping signals that we observed type cells, we investigated the distribution of endogenous indicated true co-localisation to the same subcellular Sec61β and Sec61γ, which form a heterotrimeric complex with structures. We therefore treated cells with brefeldin A, a drug Sec61α (Gorlich and Rapoport, 1993). Wild-type COS1 cells which has been useful in subcellular localisation studies were stained using antibodies specific for either Sec61β or because it redistributes Golgi proteins to the ER whilst those Sec61γ and ERGIC53, and the subcellular localisation of these of the TGN ‘collapse’ upon the microtubule organising centre proteins was determined by indirect immunofluorescence (Banting and Ponnambalam, 1997). Both conventional and microscopy. The Sec61β and Sec61γ antibodies both revealed confocal immunofluorescence microscopy were used to look at a large reticular network typical of the ER, together with the localisation of GFP tagged Sec61α and TGN46 following particularly intense staining in the perinuclear region (Fig. 8A Brefeldin A treatment of the cells. Confocal microscopy and D, respectively). The ERGIC53 antibody also resulted in images of the double labelling results for three different cells a distinct punctate staining located in the perinuclear region of are shown in Fig. 7, each image being an average of 18 optical the cell (Fig. 8B and E) and this area largely co-localised with sections (A to C). The collapsed TGN can be seen via Sec61β and Sec61γ (Fig. 8C and F, respectively, yellow
Fig. 6. GFP-tagged Sec61α redistributes with the ERGIC after a 15¡C temperature block. The effect of a 15¡C temperature block upon the co-localisation of GFP- tagged Sec61α (A) and ERGIC53 labelled structures (B) is shown. The 15¡C block causes scattering of some ERGIC53 labeled structures towards the cell periphery, and the image overlay reveals that these scattered structures also contain GFP-tagged Sec61α (C). Sec61 complex subcellular localisation 1483
conclude that Sec61β and Sec61γ are resident in both the ER and the ERGIC of wild-type COS1 cells, and perhaps recycle between these two locations. The localisation of the Sec61 complex using subcellular fractionation In order to investigate the subcellular distribution of the Sec61 complex by an alternative method, we used subcellular fractionation. Since we were particularly interested in testing our hypothesis that the Sec61 complex is located in both the ER and the ERGIC we used a Nycodenz gradient system developed specifically to separate these compartments (Hammond and Helenius, 1994). The proteins of interest could all be detected by immunoblotting therefore we were able to examine the subcellular distribution of all three subunits of the Sec61 complex using both wild-type (Fig. 9) and transfected cells (data not shown). The method we have used can determine whether or not a particular protein is present in the ER or the ERGIC fractions of the Nycodenz gradient. However, it is particularly important to stress that no estimate of the relative fraction of the protein present in each of these locations can be made. This is because a low speed centrifugation step is used to remove the nuclear material prior Fig. 7. GFP-tagged Sec61α does not co-localise with the TGN to fractionation, and this also results in the loss of a substantial following brefeldin A treatment. The distribution of GFP-tagged amount of the ER marker calnexin into the pellet. In contrast, Sec61α and the TGN marker TGN46 were compared after the very little of the ERGIC/cis-Golgi marker ERD2 is lost (data treatment of cells with Brefeldin A. Three different cells that are not shown). Hence, a significant amount of ER material is lost representative of the entire population. No significant overlap of the prior to fractionation, presumably as a consequence of the green GFP-based signal and the red are shown (A to C). TGN46 continuity of the ER and the nuclear membranes. derived signal is present. We used calnexin as an ER marker and found that it was restricted to the heaviest fraction of the gradient, i.e: fraction regions). When a similar experiment was carried out after a 1, in both wild-type (Fig. 9) and transfected cells (data not 15¡C temperature block the majority of the scattered vesicular shown). This region of the gradient had previously been structures derived from the ERGIC were also found to contain identified as containing the ER (Hammond and Helenius, Sec61β and Sec61γ (cf. Fig. 6, data not shown). We therefore 1994). In contrast, our marker for the ERGIC/cis-Golgi, ERD2,
Fig. 8. Endogenous Sec61β and Sec61γ are present in the ERGIC. Immunolocalisation of Sec61β and Sec61γ with the intermediate compartment marker ERGIC53. The panels show fluorescence derived from Sec61β and Sec61γ (A and D, respectively) and ERGIC53 (B and E). Image overlays of Sec61β or Sec61γ with ERGIC53 are shown in C and F, respectively. 1484 J. J. A. Greenfield and S. High
ERGIC/cis-Golgi (ERD2), (data not shown). We therefore only used subcellular fractionation to distinguish an ER location from a post ER, ERGIC/Golgi, location. From the results obtained we can conclude that the subunits of the Sec61 complex are present in both the ER and the ERGIC/Golgi compartments of mammalian cells. To test whether the presence of the ER translocon components in post ER compartments was a more widespread phenomenon amongst proteins involved in protein translocation we looked at the location of TRAM (translocating chain associated membrane protein) following fractionation. The TRAM protein was found in the same fractions as the Sec61 complex, namely, in both the ER and the ERGIC/Golgi fractions (Fig. 9).
DISCUSSION
In this paper we have investigated the localisation of the Sec61 complex in mammalian cells using two different approaches, Fig. 9. Subcellular fractionation studies of the ER and the ERGIC. namely microscopy and subcellular fractionation. All three The ER was separated from the ERGIC and the Golgi of wild-type subunits of the complex, α, β and γ, are known to function COS1 cells by subcellular fractionation using Nycodenz gradients. during protein translocation at the ER membrane, and all three The collected fractions were resolved by SDS-PAGE, transferred to subunits were indeed found to be present throughout the ER nitrocellulose, and incubated with antibodies recognising the ER network. marker (calnexin), the ERGIC/cis-Golgi marker (ERD2), and with α antibodies recognising Sec61α, Sec61β, Sec61γ and the TRAM When we expressed a GFP-tagged version of Sec61 in protein. When the same experiment was carried out using cells mammalian cells we found it to be present in both the ER and transfected with GFP-tagged Sec61α the distribution of these the ERGIC. The GFP-tagged protein was shown to be unable proteins was identical to that seen in wild-type cells, hence only the to mediate translocation on the basis of its inability to be cross- location of the GFP-tagged Sec61α, detected with a GFP specific linked to a secretory protein translocation intermediate. The antibody, is shown. addition of a 238 amino acid GFP tag to the C terminus of Sec61α, may interfere with its conformation, or with its ability to assemble into a functional translocation complex, both of was observed in the lightest fractions of the gradient (Fig. 9, which would be likely to inhibit its function. Alternatively, the fractions 8 and 9) and with a slightly increased mobility in the presence of the GFP tag on the cytoplasmic side of the ER ER fraction (Fig. 9, fraction 1). The presence of some ERD2 membrane may prevent the ribosome binding function of in the ER is consistent with previous work (Griffiths et al., Sec61α (Kalies et al., 1994) and hence block any co- 1994). The small difference in mobility that we observe translational protein translocation. Although in this study the between ERD2 in the ER and the ERGIC/cis-Golgi may reflect addition of a GFP tag to Sec61α resulted in its loss of function, a difference in the post-translational modifications of the ERD2 several studies have shown that both functional and non- present in the two compartments. Alternatively it may be due functional GFP chimeras can be appropriately localised to their to the presence of different isoforms of ERD2 in the two correct subcellular compartments (Cole et al., 1996; Shima et locations as two different ERD2 isoforms have been identified al., 1997). However, in view of the lack of function of GFP- (Lewis and Pelham, 1990, 1992b). tagged Sec61α we decided not to rely solely on its subcellular The location of the three subunits of the Sec61 complex on localisation to indicate the location of the wild-type Sec61 Nycodenz gradients were analysed using both wild-type and complex. We therefore studied the localisation of the two transfected cells. Similar results were obtained and therefore endogenous subunits of the Sec61 complex for which suitable only the results from the wild-type cells are shown (Fig. 9). We antibodies were available, namely Sec61β and Sec61γ. Both found that Sec61α, Sec61β and Sec61γ were detected in both these proteins were found to be localised to the ER and the the ER and the ERGIC/cis-Golgi fractions (Fig. 9). In the case ERGIC by immunofluorescence microscopy studies of wild- of the transfected cells expressing GFP-Sec61α, a GFP specific type cells. Since Sec61α is found in a heterotrimeric complex antiserum also detected the tagged protein in both the ER and with Sec61β and Sec61γ (Gorlich and Rapoport, 1993), these the ERGIC/cis-Golgi fractions (Fig. 9). We therefore conclude results indicate that the subcellular localisation of GFP-tagged that the Sec61 complex subunits, unlike calnexin, are not Sec61α faithfully reports the localisation of the wild-type restricted to the ER in their localisation. Although the Sec61 protein. complex proteins co-localise with fractions containing the The presence of GFP-tagged Sec61α further along the ERGIC/cis-Golgi marker ERD2, the Nycodenz-based secretory pathway was also investigated and we found that procedure that we used does not clearly separate other Golgi little, if any, of the protein reached the trans-Golgi network, compartments from the ERGIC/cis-Golgi. Hence we observed and none was visible at the plasma membrane of live cells. A a substantial overlap in the location of protein markers for the second approach was used to determine the subcellular Golgi (Rab6) and the trans-Golgi network (TGN46) and the localisation of the Sec61 complex, namely subcellular Sec61 complex subcellular localisation 1485 fractionation on Nycodenz gradients. Using this method GFP- performs a specific function. Hence, misfolded MHC class I tagged Sec61α, endogenous Sec61α, Sec61β and Sec61γ were molecules were observed to accumulate in the ERGIC (Raposo all found to be present in the heavy fraction containing the ER et al., 1995). Furthermore, the Sec61 complex is believed to and the lighter fractions where both the ERGIC and the Golgi participate in the retrograde translocation of misfolded MHC were located. Since microscopy studies showed that GFP- class I proteins prior to their degradation in the cytosol (Wiertz tagged Sec61α was localised to the ERGIC but not the TGN, et al., 1996). Thus it may be that such Sec61 dependent we conclude that the presence of the three Sec61 subunits in retrograde transport takes place at the ERGIC of mammalian the lighter fractions is primarily due to their localisation in the cells. This would enable the spatial separation of two functions ERGIC and perhaps the cis-Golgi. performed by the same protein complex, and thus allow both Our results show that all three components of the Sec61 forward and retrograde transport to occur at the same time but complex are localised both in the ER and in post ER in different locations. compartments, particularly the ERGIC. Τhis raises the Our results clearly show the presence of all three subunits question of why the Sec61 complex is present in the ERGIC. of the Sec61 complex, and the TRAM protein, in post ER We believe that the most likely explanation is that the Sec61 compartments. We therefore conclude that none of these complex escapes from the ER to the ERGIC from where it is proteins are permanent residents of the ER (cf. Jackson et al., recycled back to the ER. In contrast, studies in Saccharomyces 1993). Whilst we favour the idea that this reflects specific cerevisiae have shown that the yeast Sec61α homologue, recycling of these components via a retrieval pathway, the Sec61p, is excluded from transport vesicles carrying cargo precise biological basis for their subcellular distribution away from the ER (Barlowe et al., 1994). However, no remains to be established. equivalent of the ERGIC has been reported in Saccharomyces cerevisiae. This work was supported by funding from the Biotechnology and Our data indicate that the subunits of the Sec61 complex, Biological Sciences Research Council. We are very grateful to all our collegues who generously supplied antibodies. We thank Louise and the TRAM protein, are located in the ER by virtue of a α retention/retrieval mechanism akin to that which recycles Norbury for generating the original Sec61 expression vector, Iain Hagan for the use of the microscope and the modified GFP construct membrane proteins bearing a di-lysine motif (Jackson et al., and Viki Allan for help with the microscopy. We are also very grateful 1993). Although calnexin does have a di-lysine-like retrieval to Phil Woodman and Jaakko Saraste for valuable comments on the motif (Teasdale and Jackson, 1996), we could not detect the manuscript. protein in compartments beyond the ER during subcellular fractionation experiments. This result is in agreement with previous studies where calnexin was found to be excluded from REFERENCES ER derived transport vesicles (Rowe et al., 1996). The authors suggested that calnexin has a very low escape rate from the ER, Aridor, M. and Balch, W. E. (1996). 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